1 Brandeis University, Waltham, Massachusetts 02254-9110; 2 State University of New York Buffalo, Buffalo, New York 14214-3008; and 3 University of California San Francisco, San Francisco, California 94143-0444
![]() |
ABSTRACT |
---|
The exocrine pancreas and certain salivary glands of mammals secrete a variety of enzymes into the gastrointestinal tract, where they digest food. The same glands also release these enzymes into the bloodstream. This latter process has commonly been assumed to occur solely as the result of a pathological condition or as an inadvertent by-product of exocrine secretion due to the leakage of trace quantities of the enzymes into blood. However, a variety of evidence suggests that the endocrine secretion of digestive enzymes is a normal occurrence that can be of substantial magnitude in healthy individuals, is responsive to various physiological stimuli, and is distinct from exocrine secretion. Recent research has focused attention on this process as a promising means for the delivery of engineered proteins into the systemic circulation for pharmaceutical purposes. In this review, we survey research in this area and consider the evidence for the existence of an endocrine secretion of digestive enzymes, the cause of enzyme release into the bloodstream, its source within the tissue, and, finally, the physiological purposes that this secretion process might serve.
pancreas; salivary glands; secretion
![]() |
INTRODUCTION |
---|
THE CLASSICAL VIEW is that there are two distinct types of secretory glands, exocrine and endocrine. Exocrine glands secrete the products they manufacture into the external environment, such as the lumen of the gastrointestinal tract, via the gland's duct system. In contrast, endocrine glands release their products internally, initially into interstitial fluid and then into the bloodstream. The movement of secretory products within the cells of these two different types of glands is thought to reflect this polarity. Thus, in exocrine cells, transport occurs from the site of synthesis to the duct-facing or apical membrane of the cell, whereas in endocrine cells, transport is to its blood-facing or basolateral membrane surface. In this way the cells are organized to release their products in either one direction or the other, but not both.
In this article we discuss a process that runs counter to this understanding: the endocrine secretion of digestive enzymes by the exocrine pancreas and certain salivary glands of mammals. In addition to secreting large amounts of digestive enzymes into the intestinal tract, these glands also secrete the same products into the bloodstream. As such they are not strictly exocrine glands but "duacrine" organs, capable of secreting their products into both the external environment and the circulatory system.
![]() |
DIGESTIVE ENZYMES IN BLOOD |
---|
It has been known for a long time that digestive enzymes, such as
trypsin, amylase, and lipase, can be detected in human blood. Traditionally their appearance in the bloodstream has been viewed as
the result of pathological insult or as an accidental by-product of
exocrine secretion. This reflects a strong inclination to assume by
definition that exocrine glands only secrete in an exocrine direction.
Consistent with this view, elevated levels of digestive enzymes in
blood are common symptoms of, and important diagnostic criteria for,
inflammatory disease of the digestive glands, such as pancreatitis.
Although their levels in blood are elevated in certain disease states,
these increases occur relative to normal values. That is to say, many,
if not all, digestive enzymes are normal constituents of
blood and found routinely in the plasma of apparently healthy
disease-free individuals (Table 1) (3, 4,
6, 7, 9, 11, 12, 14, 27-30, 46, 51, 56, 57, 60). Furthermore,
active forms of digestive proteases (6, 7, 57), as well as their
inactive precursors, and a variety of other active digestive enzymes
are present in blood without precipitating disease or tissue
destruction. This is not a novel observation in the sense that active
proteolytic, lipolytic, and amylolytic enzymes derived from
nondigestive sources are normal constituents of the plasma and
extracellular spaces of healthy individuals. In these circumstances,
potentially dangerous enzymes are held in check in a variety of ways
that include the regulation of their production, secretion, and
metabolism as well as the presence of specific enzyme inhibitors, such
as 1-anti-trypsin and
2-macroglobulin.
|
If the endocrine secretion of digestive enzymes need not be a pathological phenomenon, their presence in blood may nonetheless be the result of nonphysiological causes. It may be perfectly normal and harmless enough, but the result of the accidental, functionally meaningless leakage of trace quantities of these substances during the process of ductal secretion. At first glance, such a conclusion may seem warranted by the low concentration of the various digestive enzymes in plasma compared with their concentrations in glandular tissue (millimolar concentrations). But this does not in and of itself justify such a conclusion. It should not be surprising that the pancreas contains its digestive enzymes at far higher concentrations than plasma, because the gland stores large quantities of these substances before their release. Many physiologically active substances are found in blood at low concentrations relative to their source compartment, such as peptide hormones (Table 1) and various plasma proteases. Nor can the concentrations of digestive enzymes found in blood, in the nanogram per milliliter range, be considered trace quantities. They are sufficient to allow measurable enzymatic hydrolysis, and many enzymes are active at substantially lower concentrations.
Nor does their concentration in blood alone establish the amount present. That is a function of the volume of distribution as well, that is, the size of the pool in which the substance is suspended. In a 70-kg man, the volume of blood is ~5 liters, and overall extracellular fluid volume is ~15 liters. This is far greater than the volume of the pancreas, for example, which is only 90-100 ml. In the case of such a large discrepancy in the relative volumes of two compartments, the larger pool would be found in blood and extracellular fluid at <1 in 100 concentration. Although the concentration of digestive enzymes in blood is substantially less than this, some three to four orders of magnitude lower than their concentration in the glands, this concentration still represents a substantial amount, in the range of 1-10% of tissue levels. Values as high as 13% have been reported after prolonged cholinergic stimulation (40). Clearly, these are not trace quantities.
Finally, and importantly, even if the amount of a substance in a pool is small, it does not necessarily mean that its rate of transport into that pool is insubstantial. The amount entering a compartment is also a function of its rate of turnover; that is, how quickly it leaves as well as how quickly it enters that compartment. Taken together, these facts constrain us from concluding, based solely on the understanding that their concentration in blood is low compared with their tissue of origin, that the presence of digestive enzyzmes in blood is simply a reflection of their quantitatively trivial leakage from the glands as an accidental by-product of ductal secretion. To justify the conclusion that it is accidental, we need additional information, most importantly an assessment of the properties of the processes that produce endocrine secretion.
![]() |
THE ENDOCRINE SECRETION OF DIGESTIVE ENZYMES |
---|
Evidence for the endocrine secretion of digestive enzymes
by exocrine glands dates back to the 1930s. Studies in dogs and rabbits
revealed large increases in serum amylase levels attributable to the
pancreas after the administration of cholinergic agonists (acetylcholine, physostigmine, and acetyl--methylcholine) that activate exocrine secretion by this and other gastrointestinal glands
during the digestive process (2, 54, 58). Some thirty years later, a
similar effect was reported in rats (53), but in this case it was also
found that plasma amylase levels more than doubled after feeding.
Subsequently, a variety of studies (39, 42, 44, 52) confirmed that
amylase in blood is due to its release from the salivary glands, the
pancreas, and probably the liver.
Not only was endocrine secretion responsive to digestive stimuli, but the responses could be quantitatively significant. In rabbits, for example (16), the pancreatic secretagogue cholecystokinin (CCK)-pancreozymin, at a dose within the normal range of postprandial serum concentrations (18, 25, 33, 45), increased the amylase concentration of plasma by an amount equal to ~15% of duct-directed secretion, or ~42 nmol, during the same period of time. Similar values were reported in response to CCK administration in anesthetized dogs (15%) (42) and in rabbits in vitro (18%) (39).
![]() |
THE SOURCE OF ENDOCRINE SECRETION WITHIN THE GLANDS |
---|
Release of digestive enzyme into blood has most commonly been envisioned as being the result of some fraction of the enzyme secreted into the duct finding its way into interstitial fluid through paracellular channels and from there into the bloodstream. It has been proposed that channels large enough to act as shunts for digestive enzymes penetrate apically placed tight junctions connecting adjacent glandular epithelial cells. In this case, endocrine secretion would be a passive, obligatory by-product of exocrine secretion. If such a mechanism were operative, we would anticipate that all stimulants as well as all inhibitors of exocrine secretion would dependently and proportionately affect endocrine release. In this situation, the fact that endocrine secretion is responsive to physiological stimuli would be inconsequential, the result of an inadvertent or accidental occurrence, not a meaningful physiological event.
The Pancreas
The paracellular model can be tested simply by comparing the effect of stimulants on secretion in the two directions. Do stimulants produce changes in endocrine secretion proportional to those that occur in the exocrine direction? Given a passive paracellular route, we would expect that when the concentration of an enzyme in the duct system is elevated by a stimulant, its endocrine secretion would be increased in proportion to the increase in the diffusion (concentration) gradient. Or, if the stimulant produced an increase in intraductal pressure generated by an increase in the volume of fluid held in the duct system, this would increase the flow of the enzyme-containing fluid through paracellular channels. In this case, endocrine secretion would be increased in proportion to the increase in flow rate. In either situation or as a consequence of both, some fraction or proportion of ductal secretion would find its way into the interstitial spaces of the gland.If such proportional responses were not observed, or at least were not observed on all occasions, then a different mechanism would have to be responsible for the endocrine secretion of digestive enzymes. This is not to say that we could conclude in this eventuality that paracellular shunts permeable to digestive enzymes did not exist at all, but rather that if they did, passage through them would not account, that is not be quantitatively responsible, for endocrine secretion.
This was the case. Endocrine secretion does not necessarily, or even
commonly, parallel the exocrine process. For example, in the pancreas,
CCK can produce large (magnitudinal) increases in exocrine secretion
without any noticeable increase in the endocrine direction at all (22).
On the other hand, at higher physiological doses, the same hormone has
a substantial effect on endocrine secretion (52), whereas secretion in
the ductal direction, already at its maximal rate, is unaffected. This
dose-dependent distinction between secretion in the two directions is
reflected in fully separable dose-response curves (Fig.
1) (39). This is consistent with the
reported presence of low- and high-affinity CCK receptors on exocrine
cells in the pancreas (41, 59).
|
Similarly, the hormone secretin, a potent stimulant of salt and water secretion by the exocrine pancreas, does not enhance exocrine protein secretion in humans, even though at high concentrations it elevates plasma amylase levels substantially (10). Also in humans, the hormone bombesin, a general stimulant of enzyme secretion in the exocrine direction, elevates plasma trypsin but not amylase or lipase (16). If paracellular shunts were responsible for endocrine secretion, we would expect secretion of the various enzymes to be enhanced in a parallel or proportionate fashion.
The Rabbit Pancreas in Organ Culture
Similar indications came from in vitro studies on the pancreas. Direct measurements of amylase transport across the blood-facing or basolateral surface of digestive glands were first attempted in the 1970s (16, 22, 39) by use of an isolated organ preparation of rabbit pancreas (47). Because of its diffuse nature, the rabbit pancreas could be incubated whole in vitro with its natural exocrine-endocrine polarities intact (47). As a consequence, protein transport across the basolateral surface of the gland could be measured directly and separately from exocrine secretion. Endocrine secretion is measured by tracking release into the incubation medium, whereas exocrine secretion is monitored by collecting fluid from the cannulated pancreatic duct (22, 47).When this method was used, an efflux of amylase into the incubation
fluid was not only measurable, but quite large, at least at the initial
rate (22). Indeed, it was some three times the concurrent flux into the
duct system in the unstimulated state (Fig.
2). When secretion was stimulated with a
cholinergic agonist, ductal secretion was greatly enhanced as expected,
and, consistent with prior in vivo evidence, endocrine secretion was
increased as well. However, the cholinergic agent had a relatively
greater effect on exocrine than endocrine secretion and produced fluxes of about equal magnitude in both directions (22).
|
Most striking was the fact that unstimulated endocrine secretion, presumed to be a by-product of ductal secretion, was three times the simultaneous rate of secretion in the exocrine direction (Fig. 2). How could the by-product be three times the size of the "product" itself? In the same experiments it was also found that the flux of amylase into the medium declined in a roughly exponential fashion over its several-hour course, until amylase concentration there reached a stationary value and release into the medium ceased (Fig. 2). This occurred even though exocrine secretion continued unabated over this period of time in regard to both its protein concentration and volume. This difference between the two processes, one ceasing while the other was maintained unchanged, seemed to rule out the possibility of paracellular shunts.
Still, the falloff in endocrine secretion might be explained by a paracellular shunt if a diffusional equilibrium had been achieved between amylase in the duct and interstitium. This could account for the diminution and eventual disappearance of the net flux in the endocrine direction. If this was what happened, then the concentration of amylase in the medium at the stationary state would be, if not identical (due to Donnan effects and the like), very similar to that in the duct system. When the two concentrations were compared, the differences were very large. The concentration of amylase in the medium at the stationary state was far lower, some three orders of magnitude lower, than in the duct. Clearly the two compartments were not in diffusional equilibrium.
This view was confirmed by adding exogenous amylase to the medium (23). Its addition would be expected to reduce the concentration gradient between duct and medium if a duct-to-interstitium equilibrium were involved. Not only was this not observed, but addition of amylase actually increased the gradient. This was because the elevated concentration in the incubation medium was more than matched by the subsequent increase in ductal concentration that it produced. In competition experiments in which the exocrine secretions of endogenous and exogenous (added to the medium bathing the gland) sources of the enzyme were compared, it was shown that the increase seen in the concentration gradient was the result of amylase transport "uphill" from the medium to the duct across the exocrine cells (23).
Finally, endocrine and exocrine secretion did not display identical time courses in the pancreas. If endocrine secretion were simply a dependent outcome of the exocrine process, then both processes should parallel each other over time, rising and falling in concert with some defined delay between them. At least in some circumstances this was not the case. For example, in rabbits in situ after stimulation with CCK, endocrine secretion rose more slowly than exocrine secretion and returned to control values even while exocrine secretion remained highly elevated (16).
![]() |
A PARACELLULAR SHUNT IN THE PANCREAS? |
---|
Although all of this argued against the paracellular shunt hypothesis, there was one significant line of evidence in the pancreas that appeared to lend support to it. In another study with the rabbit pancreas in organ culture (24), the passage of the traditional extracellular space markers mannitol, sucrose, and inulin was measured across the epithelial layer from bathing medium to ductal fluid. Because these substances do not enter cells, their appearance in ductal fluid would presumably demonstrate their paracellular passage. And because they are larger than the sorts of substances that commonly pass between epithelial cells, such as water and small ions, this would confirm the presence of unusually leaky junctions between adjacent exocrine cells. Although the presence of channels large enough to accommodate these still comparatively small molecules would not prove that much larger proteins could traverse the same channels, such a result would at least raise, or be consistent with, that possibility. Surely, if the extracellular space markers could not cross the epithelium paracellularly, it would be hard to imagine how the far larger digestive enzymes could.
When these measurements were made, the extracellular space markers added to the bathing medium appeared in ductal fluid in short order, and in fact were present at such high concentrations that the authors suggested that the rabbit pancreas was the most permeable epithelial barrier that had yet been described (24). If the pancreas was permeable to these extracellular markers, then perhaps the channels they traveled through were sufficiently broad to also accept digestive enzymes moving in the opposite direction.
But how could we resolve the contradiction between such a conclusion and conclusions drawn from the various observations described above that seemed to exclude a paracellular explanation? Of course, as we have said, a paracellular permeability to these extracellular space markers only raised the possibility that the larger digestive enzymes crossed the epithelium by the same route. This extrapolation might not be warranted. The channels might be large enough for mannitol, sucrose, and inulin but not for the digestive enzymes. But there was also another possibility. The conclusion that paracellular channels were responsible for the passage of the extracellular space markers was based on the reasonable but untested assumption that the substances used definitively marked the extracellular space of the gland and that, as such, they did not enter or cross the exocrine cells themselves.
When this proposition was tested (36-38, 50), it was found not to be the case. Not only did the data indicate that the extracellular space markers entered and passed through the cells of the pancreatic epithelium, they did not seem to pass between them to any significant extent. For example, the concentration of mannitol in ductal fluid was found to be quite high at the steady state, 54% of its concentration in the medium bathing the gland, but the concentration of two other molecules of similiar size and mass, inositol and 3-O-methylglucose, was far lower (12 and 8%, respectively) (38). If paracellular shunts had accounted for the transport of mannitol, then these other substances should likewise have been accommodated by these shunts and consequently been found in ductal fluid at approximately the same concentration as mannitol, not several times less. Indeed, we would have anticipated that they would be present at somewhat higher concentrations because, unlike mannitol, there are transport systems in the cell membrane for inositol and 3-O-methylglucose that allow them to enter the cells of this tissue and, hence, cross them in addition to passing across the epithelium via paracellular channels.
It was also found that the tissue spaces for sucrose and inulin were some three times (54 and 44%, respectively) the albumin space (16%). This suggested a substantial intracellular distribution for both molecules (37), as did other observations. For example, when either sucrose or inulin was added to the medium bathing the rabbit pancreas in organ culture, the molecules reached steady-state values in ductal fluid in ~30-60 min. If a paracellular model applies, when they are removed from the medium (by changing the bathing solution), we would expect them to disappear from ductal fluid, passing down their respective concentration gradients into the sucrose- or inulin-poor medium more rapidly than they had initially equilibrated (37). Although their concentrations in ductal fluid fell in a roughly exponential fashion when this was done, they fell far more slowly than they had risen during their initial equilibration. Even after 2 h, they remained highly elevated relative to their respective concentrations in the bathing medium. There appeared to be a renewable source that replaced the inulin- and sucrose-rich fluid as it was excreted via the duct system. That source could not be the low concentrations of these substances in the bathing medium, and absent that possibility, it would have to have been previously accumulated sucrose and inulin released from the cells of the gland.
This conclusion was confirmed by the response to hormonal stimulants. The addition of cholinergic agonists or CCK increased severalfold the levels of both substances in ductal fluid (37). That is, their secretion was stimulated by hormones that act on cells when these substances were virtually absent from the medium bathing the gland. Hormonal stimulation also increased the concentrations of inositol and 3-O-methylglucose in exocrine secretion (38), as well as the concentration of the bulky inorganic phosphate ion (36), all coming from within the cell.
Thus it seemed that nonelectrolytes of modest size, such as mannitol, sucrose, and inulin did not cross the epithelium paracellularly. Suprisingly, all of the molecules seemed readily able to enter and cross exocrine cells. The pancreatic epithelium was leaky all right, but its leakiness seemed to be a property of cells, not paracellular channels. And if these relatively small molecules did not pass between cells to enter the pancreatic duct, then certainly the far larger digestive enzymes did not. In the end, observations that at first glance seemed to support the notion of large paracellular channels, and at least raised the possibility of paracellular passage for the digestive enzymes, excluded it. The endocrine secretion of digestive enzyme by the pancreas did not appear to involve its passage out of the duct via a system of paracellular shunts.
The Salivary Glands
Another striking dissociation between endocrine and exocrine secretion similar to that found in the pancreas was reported for the rat parotid glands (43). Sympathetic nerve stimulation results in a large mobilization of amylase into saliva but little or no change in amylase activity in serum. In contrast, parasympathetic nerve stimulation produces a saliva with a relatively low amylase concentration (although significant amounts are secreted), but yields a substantial increase in serum amylase levels.On the face of it, this observation once again argues against transport via paracellular shunts and for an independent secretory mechanism for endocrine secretion. However, the responses to parasympathetic and sympathetic stimulation differed in another way. Parasympathetic stimulation, but not sympathetic stimulation, increased fluid flow through the duct system (43). It was proposed that this difference could account for the release of amylase into blood with parasympathetic stimulation by means of paracellular shunts. Release would occur as a consequence of the increase in the flow of saliva through pressure-driven paracellular shunts. Because this increase was only seen with parasympathetic stimulation, plasma levels would only be increased in this case. But this explanation is not satisfactory, unless the flow of material through the putative shunts only occurs in the presence of parasympathetic stimulation, that is, none occurs in its absence. If, on the other hand, paracellular transit were proportional to flow, being driven by the extant pressure gradient, then the large increase in the amylase concentration of saliva seen with sympathetic stimulation, even in the absence of an increase in flow, should lead to elevated blood levels of the enzyme in simple direct proportion to the elevated concentration in the duct system. And if we propose that the shunts only open as a consequence of parasympathetic stimulation, then how does salivary amylase enter blood in the absence of parasympathetic stimulation? Does release stop during sleep, for example? Whatever plausibility such a model may have for the salivary glands, it can be ruled out for the pancreas, where differential rates of release in exocrine and endocrine directions occur without alterations in fluid secretion.
Nonetheless, other observations appeared to reinforce the notion of paracellular passage in the parotid gland. When various marker proteins were instilled into the duct system of the gland in a retrograde fashion under particular stimulated conditions (19, 34, 35), some were found subsequently in the lateral intercellular spaces of glandular tissue. From this, the authors of these studies concluded that the proteins had traveled through paracellular shunts to reach these destinations. However, their observations did not in and of themselves prove this. The mere presence of marker proteins in intercellular spaces does not tell us how they got there. In particular, it does not allow us to distinguish between movement via paracellular channels and through the glandular cells themselves. Evidence that bore directly on the route of transit did not bolster the idea of passage through paracellular channels. For example, in freeze-fracture images, the authors were unable to demonstrate any correlation between junctional structure and duct permeability (35). Perhaps more significantly, uptake into cells being a prerequisite for passage across them, the cytoplasm of both ductal and acinar cells showed a diffuse distribution of marker proteins [in particular lactoperoxidase (82 kDa) and horseradish peroxidase (40 kDa)] (35).
A variety of other observations also seemed to be inconsistent with paracellular channels. As we have discussed in regard to extracellular space markers, such channels are generally viewed as sieves that sort molecules primarily as a function of size. Although shape and charge can also play an important role, size is determinative. Whatever the charge or shape, if the molecule is too large to fit, it cannot gain entry to the channel. The results of experiments with marker proteins do not agree with such a model. For example, in resting glands, lactoperoxidase (82 kDa) and horseradish peroxidase (40 kDa) were found in intercellular spaces, whereas other proteins of either similar or smaller size were restricted to the ductal space [in particular, microperoxidase (1.8 kDa), cytochrome c (12 kDa), myoglobin (17.8 kDa), tyrosinase (34.5 kDa), and hemoglobin (66 kDa)]. These discrepancies cannot be accounted for on the basis of distinctions in either the overall charge or gross shape of the molecules. More specific differences must have been involved.
But perhaps the most curious observation, and one that is hard to explain by the paracellular shunt model, is what occurs to such markers with stimulation. With myoglobin used as the tracer, either a sympathomimetic (isoproterenol) or parasympathomimetic (methacholine) drug was given to the rat to elicit salivary secretion (34). Remember that parasympathetic stimulation to this gland increases plasma amylase levels, whereas the more potent exocrine stimulant isoproterenol has no effect on plasma levels. If the effect of the parasympathetic drug was due to the passage of amylase through a paracellular shunt, then the relatively small tracer protein myoglobin (17.8 kDa) should appear in interstitial spaces in the presence of this stimulant, following the same path as the larger amylase (55 kDa). Also, if this model is correct, because sympathetic stimulation has no effect on the endocrine secretion of amylase, there is no reason to expect the tracer protein to pass into the interstitial spaces in its presence. Not only was this not observed, but exactly the opposite was seen (34). With myoglobin as the tracer molecule, isoproterenol, but not the cholinergic agonist methacholine, led to its appearance in the intercellular spaces of the gland.
In another study, amylase (bacterial) was instilled into the duct system as the marker protein (19). In this case, unlike the experiment just discussed and unlike the situation for the endogenous proteins, both sympathetic and parasympathetic stimulation enhanced plasma amylase levels relative to saline-instilled controls. Studies on the secretion of kallikrein, a serine protease produced by ductal cells of the submandibular gland that is secreted into both blood and the duct, are also hard to explain in terms of paracellular shunts (13). In this case, sympathetic stimulation increases kallikrein secretion into the duct some 100-fold more than parasympathetic stimulation. Yet the effect on blood levels was comparable for the two agents. As the authors concluded, it is "unlikely that the (kallikrein) had reached the blood via intercellular leakage or permeation from luminal contents" given this large disparity. In other work, the same group (55) provided evidence that kallikrein is released into saliva, and perhaps blood as well, from a "nongranular" rather than a granular cellular pool.
![]() |
THE RELEASE OF DIGESTIVE ENZYMES FROM EXOCRINE CELLS |
---|
To account for all of the evidence on the endocrine secretion of digestive enzymes that we have discussed, whether in the pancreas or salivary glands, in terms of the passage of these molecules through paracellular channels from ductal contents is a formidable task. We believe that if such a model could be devised on the basis of the evidence in its entirety, and it is not clear how readily this could be done, it would require complexities of a sort and character for which no documentation exists, not only in these glands but in epithelia more generally. This is not to say that the paracellular channels in these glands are absolutely impermeable to digestive enzymes, or to extracellular space markers for that matter, but that if such a permeability exists, it does not account in a substantive quantitative way for the transport of these molecules in the endocrine direction.
If we can properly exclude such channels, then the endocrine secretion of digestive enzymes must be of cellular origin, most likely the product of the same cells that manufacture and secrete the digestive enzymes in an exocrine direction. In this case, if we still wish to consider this secretion an accidental event, we have to look at the basolateral surface of these cells for the accident. Such accidental release has been proposed in terms of the exocytosis model of secretion (1). Every so often an errant vesicle would find its way to the basolateral membrane, fuse with it, and release its contents into the interstitium. When elevated levels of enzyme are seen in blood in inflammatory disease of these glands, it would be due to an increase in the frequency of exocytosis at the basolateral surface of the cell, perhaps at the expense of release in the exocrine direction.
At least in the absence of pathological causes, we would anticipate that such an accidental effect would occur in some fixed proportion to ductal secretion. Conditions that increase the rate of exocytosis at the apical surface of the cell would at the same time proportionately increase the likelihood of exocytosis at the basolateral surface. As we have discussed for a variety of cases, the alterations that occur in the two processes in response to one or another specific condition do not show such a proportionality. In fact, not only are disproportionate changes observed, but to the best of our knowledge proportionate ones have not been reported. But putting aside the occurrence of disproportionate change, how in this fashion would we explain the fact that the initial rate of amylase release into the medium incubating the rabbit pancreas in organ culture is some three times its rate of secretion in the exocrine direction and about equal to that rate in the presence of a cholinergic agonist? We would have to propose that, in the unstimulated state, exocytosis, inadvertent or not, is far more likely to occur at the basolateral surface of the secretion cell than at the apical membrane, presumably the intended location. Or that, in the presence of a cholinergic stimulant, a major natural stimulant of exocrine secretion, exocytosis occurs with about equal frequency at endocrine and exocrine sites.
In addition to these peculiarities, the falloff of amylase release into the incubation medium over time in vitro (Fig. 2) can be wholly prevented simply by periodically replacing the fluid bathing the gland. When this is done, the initial rate of release, the highest rate, can be reinstated at will (22). If inadvertent exocytosis at the basolateral membrane of the cell accounted for this effect, we would have to propose that changing the medium bathing the gland, at whatever frequency we choose to do so, greatly and equivalently increases the rate of exocytosis at this surface. How can simply changing the external medium with an identical solution affect a process presumably controlled by intracellular events, while leaving its companion exocrine process at the apical cell membrane, which does not display this falloff but is controlled by the same events, unaffected? Whether the exocytosis model can explain such results, or whether membrane transport mechanisms are required, as we believe to be the case (48, 49), such evidence does not support the notion that endocrine secretion is merely an inadvertent outcome of cellular exocrine secretion.
![]() |
THE PHYSIOLOGICAL FUNCTION OF THE ENDOCRINE SECRETION OF DIGESTIVE ENZYMES |
---|
We believe that the evidence we have discussed, taken together, argues strongly that the endocrine secretion of digestive enzymes is a normal, that is, a physiological, cellular process. It has properties that we would expect for a distinct physiological occurrence: 1) it is mechanistically separate (or at least separable) from exocrine release; 2) it can be of substantial magnitude and is not a trace phenomenon; 3) it is responsive to various hormones involved in the digestive process; and 4) it is regulated separately from exocrine secretion.
Regulation of Plasma Amylase Levels
This conclusion would be greatly bolstered if it could be shown that plasma levels of the secreted substances are regulated. Even though the regulation of blood levels is in and of itself not a requirement for a transport process to serve a physiological function, regulation would certainly suggest such a purpose. Although this has not been directly assessed, there is significant evidence that suggests that blood levels of amylase are regulated. For example, animal-to-animal variance in the amylase concentration (as % of the mean value) of pancreatic tissue and the small intestines, whose contents are known to be physiologically regulated, is some 3.5-4 times greater than in blood (calculated from Ref. 40). That is to say, amylase levels in blood are far more constrained than in either the pancreas or gut. Also consistent with regulation is the fact that, when amylase is added to medium that bathes slices of pancreatic tissue in vitro, it inhibits de novo amylase synthesis but not the synthesis of other digestive enzymes (32). Regulation is also suggested by the fact that the concentration of amylase in the medium bathing the rabbit pancreas in organ culture at the stationary state is essentially the same as the plasma concentration of this enzyme in the fasted rabbit (16). Moreover, this stationary-state amylase concentration is increased in a dose-dependent fashion by hormonal stimulants. Finally, when the parotid glands, a major source of plasma amylase in rats (44), are extirpated, plasma amylase levels, although depressed initially, rise again to normal values over time despite absence of the glands. Other sources of the enzyme compensate their loss, and such compensation attests to the fact that the level of amylase in blood is a regulated parameter (44).Some Possibilities
If the endocrine secretion of digestive enzymes is a physiological process, and not the result of either pathology or inadvertence, then we might anticipate that it plays some role in the economy of the body. If such a role exists, it remains unknown, although we can speculate about some nonexclusionary possibilities. Broadly speaking, these proteins can either serve enzymatic or nonenzymatic purposes in blood. A nonenzymatic purpose, for example, would be for immunological self-monitoring. Enzymatic purposes might be related to either digestive or nondigestive functions. For example, digestion in vertebrates is thought to be restricted to the lumen of the gastrointestinal tract. However, it is known today that a major site for the final reduction of small peptides into their constituent amino acids is the absorptive cells of the small intestine, not the gut lumen. Taking this one step further, the presence of digestive enzymes in blood could serve as a fail-safe mechanism to deal with the unavoidable absorption of small amounts of incompletely digested food. The relatively low level of enzyme activity and the presence of inhibitors for proteolytic enzymes could be advantageous, preventing this activity from getting out of control.It is also possible that the circulation of digestive enzymes through the bloodstream provides a means of transporting these proteins from one part of the digestive tract to another via the capillary beds of various gastrointestinal tissues. For example, there is some suggestion that this could be the case for the salivary glands. In the rat, the submandibular, sublingual, and parotid salivary glands take up substantial quantities of amylase from blood (magnitudinal increases in their tissue concentrations) subsequent to elevating plasma levels with cholinergic agonists (20, 21, 40, 53).
Finally, when released into blood, these enzymes might serve purposes quite unrelated to the digestion of food. They might act much like other serine proteases, nucleases, amylolytic and lipolytic enzymes of intracellular metabolism, or various extracellular processes. Indeed, the original observation of elevated blood amylase levels came from a study undertaken to determine whether blood amylase levels could explain the increase in glycogen metabolism that results from vagal stimulation (2). In such a scenario, the pancreas and salivary glands would serve as the source of these enzymes, from whence they would be transported to (sorted to) cells and extracellular spaces in distant organs. This possibility is consistent with the observation that prolonged cholinergic stimulation increases the amylase contents of various, although not all, solid nondigestive tract tissues, including muscle and brain (40).
![]() |
NEW DIRECTIONS |
---|
Endocrine secretion from exocrine glands has become a matter of
practical interest recently. This process has been employed as a
delivery system for gene-based systemic protein therapy. In addition to
correcting various genetic defects in somatic cells, gene therapy
offers the possibility of delivering a broad range of protein
pharmaceuticals, such as hormones, growth factors, clotting proteins,
antibiotics, antibodies, antigens, and the like into the systemic
circulation (5, 8, 12, 31). The development of practical methods for
achieving this goal requires the identification of effective in vivo
procedures to administer recombinant DNA vectors, to manufacture
desired proteins, and to secrete them into the bloodstream (5, 8, 12,
31). The endocrine secretion process of exocrine glands, such as the pancreas and the various salivary glands, has many ideal features for
this type of therapy (15, 26). In significant proof-of-principle studies (15), recombinant DNA for human growth hormone and insulin was
administered to the exocrine cells of the pancreas and salivary glands
by retrograde instillation into the gland's duct system. Subsequently,
the cells manufactured the engineered proteins and secreted them into
the bloodstream. By use of this method it was possible to attain
physiological levels of the exogenous peptides in blood, demonstrate
their regulated secretion, and even treat systemic disease. In regard
to this last achievement, it was possible to correct elevated blood
glucose levels in diabetic animals (Fig. 3). A single injection of a plasmid
encoding human insulin was sufficient to attain near normal glucose
homeostasis in rats whose -cells had been destroyed by the
administration of a toxin.
|
![]() |
A FINAL COMMENT |
---|
Wherever the evidence points, it must be said that there is no clear consensus in the research literature as to the exact pathway(s) by which the exocrine pancreas and salivary glands secrete protein into blood. However, there does at least seem to be broad acknowledgment today that, in one way or another, many of the proteins synthesized by the acinar tissues of both the pancreas and salivary glands do normally reach the blood.
In the past, the absence of known functions for the endocrine secretion of digestive enzymes has reinforced the traditional view that this process is inadvertent or pathological, not physiological. No doubt there would have been less skepticism about it being a natural process if it had been studied in the context of some known or potential physiological function. It is certainly easier to accept the reality of a biological process, that is, to accept that it is not somehow artifactual or inadvertent, if we know what it does, or think we do. But, however desirable, this was not the historical path of discovery in this case.
Instead, over the years researchers found themselves trying to rule in or out the possibility that the phenomenon was an artifact of some sort, or otherwise the outcome of nonphysiological causes. In fact, attention has been focused almost exclusively on this problem since the first report of digestive enzymes in blood. Nonetheless, research spanning some 75 years has provided substantial evidence for a natural endocrine secretion of digestive enzymes. Given this evidence, there is good reason to seek presently unknown function(s) for this unexpected process. Such an effort may be greatly aided by technologies that are now readily available, such as the tools of molecular biology and methods for gene delivery. This only seems appropriate, because it was the realization that a natural endocrine secretion of protein exists from certain exocrine glands that gave rise to the insight that this route can be used for, and is well suited to, gene-based approaches to systemic protein therapy.
![]() |
FOOTNOTES |
---|
Address for reprint requests: S. Rothman, Depts. of Physiology and Stomatology, Schools of Medicine and Dentistry, UCSF, 3rd St. and Parnassus Ave., San Francisco, CA 94143-0444.
![]() |
REFERENCES |
---|
1.
Adler, G.,
G. Rohr,
and
H. F. Kern.
Alteration of membrane fusion as a cause of acute pancreatitis in the rat.
Dig. Dis. Sci.
27:
993-1002,
1982[Medline].
2.
Antopol, W.,
A. Schifrin,
and
L. Tuchman.
Blood amylase response to acetylcholine and its modification by physostigmine and atropine.
Proc. Soc. Exp. Biol. Med.
32:
383-385,
1934.
3.
Basso, D.,
C. Farbis,
A. Meani,
G. Del Favero,
A. Panucci,
D. Vianello,
A. Piccoli,
and
R. Naccarato.
Serum deoxyribonuclease and ribonuclease in pancreatic cancer and chronic pancreatitis.
Tumori
71:
529-532,
1985[Medline].
4.
Buchler, M.,
P. Malfertheiner,
H. Shadlich,
T. J. Nevalainen,
H. Freiss,
and
H. G. Beger.
Role of phospholipase A2 in human acute pancreatitis.
Gastroenterology
97:
1521-1526,
1989[Medline].
5.
Buckel, P.
Recombinant proteins for therapy.
Trends Pharmacol. Sci.
17:
450-456,
1996[Medline].
6.
Borgstrom, A.,
J. Kukora,
and
K. Ohlsson.
Studies on immunoreactive pancreatic elastase 2 in human serum.
Hoppe-Seyler's Z. Physiol. Chem.
361:
633-640,
1980[Medline].
7.
Carrere, J.,
C. Figarella,
O. Guy,
and
J. P. Thouvenot.
Human pancreatic chymotrypsinogen A: a non-competitive enzyme immunoassay, and molecular forms in serum and amniotic fluid.
Biochim. Biophys. Acta
883:
46-53,
1986[Medline].
8.
Crystal, R. G.
Transfer of genes to humans: early lessons and obstacles to success.
Science
270:
404-410,
1995[Abstract].
9.
Duane, W. C.,
R. Freirichs,
and
M. D. Levitt.
Distribution, turnover, and mechanism of renal excretion of amylase in the baboon.
J. Clin. Invest.
50:
156-165,
1971[Medline].
10.
Florholmen, J.,
P. G. Burhol,
R. Jorde,
and
H. L. Waldum.
The effect of graded doses of secretin on serum trypsin, serum pancreatic amylase, serum insulin, plasma somatostatin, and plasma pancreatic polypeptide in man.
Scand. J. Gastroenterol.
19:
24-30,
1984[Medline].
11.
Fridhandler, L.,
J. E. Berk,
K. A. Montgomery,
and
D. Wong.
Column chromatographic studies of isoamylases in human serum.
Clin. Chem.
20:
547-552,
1974
12.
Friedmann, T.
Human gene therapyan immature genie, but certainly out of the bottle.
Nat. Med.
2:
144-147,
1996[Medline].
13.
Garrett, J. R.,
J. Chao,
G. B. Proctor,
C. Wang,
X. S. Zhang,
K.-M. Chan,
and
D. K. Shori.
Influences of secretory activities in rat submandibular glands on tissue kallikrein circulating in the blood.
Exp. Physiol.
80:
429-440,
1995[Abstract].
14.
Geokas, M. C.,
C. Largman,
J. W. Brodrick,
and
M. Fassett.
Molecular forms of immunoreactive pancreatic elastase in canine pancreatic and peripheral blood.
Am. J. Physiol.
238 (Gastrointest. Liver Physiol. 1):
G238-G246,
1980
15.
Goldfine, I. D.,
M. S. German,
H. C. Tseng,
J. Wang,
J. L. Bolaffi,
J. W. Chen,
D. C. Olson,
and
S. S. Rothman.
The endocrine secretion of human insulin and growth hormone by exocrine glands of the gastrointestinal tract.
Nat. Biotechnol.
15:
1378-1382,
1997[Medline].
16.
Grendell, J. H.,
and
S. S. Rothman.
Effect of changes in circulating amylase levels on amylase output in bile.
Am. J. Physiol.
243 (Gastrointest. Liver Physiol. 6):
G54-G59,
1982
17.
Hafkenscheid, J. C. M.,
M. Hessels,
J. B. M. J. Jansen,
and
C. B. H. W. Lamers.
Serum trypsin, a-amylase and lipase during bombesin stimulation in normal subjects and patients with pancreatic insufficiency.
Clin. Chim. Acta
136:
235-240,
1984[Medline].
18.
Harvey, R. F.,
L. Dowsett,
M. Hartog,
and
A. E. Read.
Radioimmunoassay of cholecystokinin-pancreozymin.
Gut
15:
690-699,
1974[Medline].
19.
Ikeno, T.,
K. Ikeno,
and
H. Kuzuya.
Transport to the bloodstream of amylase following retrograde infusion of amylase into the parotid glands in the rat.
Archs. Oral Biol.
29:
587-589,
1984.
20.
Ikeno, T.,
J. Nasu,
S. Hashimoto,
and
H. Kuzuya.
Mechanisms of increase in amylase activity in rat submandibular and sublingual glands after administration of pilocarpine.
Arch. Oral Biol.
27:
597-601,
1982[Medline].
21.
Ikeno, T.,
J. Nasu,
and
H. Kuzuya.
Quantitative changes in amylase activity in the salivary glands, pancreas, saliva, and serum after administration of isoproterenol, pilocarpine, and acetylcholine.
J. Dent. Res.
62:
56-57,
1983[Abstract].
22.
Isenman, L. D.,
and
S. S. Rothman.
Transport of a-amylase across the basolateral membrane of the pancreatic acinar cell.
Proc. Natl. Acad. Sci. USA
74:
4068-4072,
1977[Abstract].
23.
Isenman, L. D.,
and
S. S. Rothman.
Transpancreatic transport of digestive enzyme.
Biochim. Biophys. Acta
585:
321-332,
1979[Medline].
24.
Jansen, J. W. C. M.,
J. J. H. H. M. DePont,
and
S. L. Bonting.
Transepithelial permeability in the rabbit pancreas.
Biochim. Biophys. Acta
551:
95-108,
1979[Medline].
25.
Johnson, A. G.,
and
S. J. McDermott.
Sensitive bioassay of cholecystokinin in human serum.
Lancet
2:
589-591,
1973[Medline].
26.
Kagami, H.,
B. C. O'Connell,
and
B. J. Baum.
Evidence for the systemic delivery of a transgene product from salivary glands.
Hum. Gene Ther.
7:
2177-2184,
1996[Medline].
27.
Korotko, G. F.,
and
A. N. Kurzanov.
Secretion of amylase and lipase in composition of bile.
Fiziol. Zh. SSSR Im. I. M. Sechenova
64:
81-89,
1978[Medline].
28.
Largman, C.,
J. W. Brodrick,
M. C. Geokas,
and
J. H. Johnson.
Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay.
Biochim. Biophys. Acta
543:
450-454,
1978[Medline].
29.
Largman, C.,
S. B. Ray,
J. W. Brodrick,
and
M. C. Geokas.
Clearance and metabolism of circulating pancreatic proelastase in the rat.
Am. J. Physiol.
239 (Gastrointest. Liver Physiol. 2):
G504-G510,
1980
30.
Levitt, M. D.,
C. Ellis,
and
R. R. Engel.
Isoelectric focusing studies of human serum and tissue amylases.
J. Lab. Clin. Med.
90:
141-152,
1977[Medline].
31.
Lever, A. M. L.,
and
P. Goodfellow.
Gene Therapy. New York: Pearson Professional, 1995, p. 1-91.
32.
Liebow, C.
Specific end-product feedback regulation of pancreatic protein synthesis.
Pancreas
2:
136-140,
1987[Medline].
33.
Marshall, C. E.,
E. H. Egberts,
and
A. G. Johnson.
An improved method for estimating cholecystokinin in human serum.
J. Endocrinol.
79:
17-27,
1978[Abstract].
34.
Mazariegos, M. R.,
and
A. R. Hand.
Regulation of tight junctional permeability in the rat parotid gland by autonomic agonists.
J. Dent. Res.
63:
1102-1107,
1984[Abstract].
35.
Mazariegos, M. R.,
L. W. Tice,
and
A. R. Hand.
Alteration of tight junctional permeability in the rat parotid gland after isoproterenol stimulation.
J. Cell Biol.
98:
1865-1877,
1984[Abstract].
36.
Melese, T.,
and
S. S. Rothman.
Increased phosphate efflux from acinar cell during protein secretion.
Am. J. Physiol.
245 (Cell Physiol. 14):
C121-C124,
1983
37.
Melese, T.,
and
S. S. Rothman.
The pancreatic epithelium is permeable to sucrose and insulin across secretory cells.
Proc. Natl. Acad. Sci. USA
80:
4870-4874,
1983[Abstract].
38.
Melese, T.,
and
S. S. Rothman.
Distribution of three hexose derivatives across the pancreatic epithelium: paracellular shunts or cellular passage?
Biochim. Biophys. Acta
763:
212-219,
1983[Medline].
39.
Miyasaka, K.,
and
S. S. Rothman.
Endocrine secretion of -amylase by the pancreas.
Am. J. Physiol.
241 (Gastrointest. Liver Physiol. 4):
G170-G175,
1981
40.
Miyakasa, K.,
and
S. S. Rothman.
Redistribution of amylase activity accompanying its secretion by the pancreas.
Proc. Natl. Acad. Sci. USA
73:
5438-5442,
1982.
41.
Pandya, P. K.,
S. C. Huang,
V. D. Talkad,
S. A. Wank,
and
J. D. Gardner.
Biochemical regulation of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.
Biochim. Biophys. Acta
1224:
117-126,
1994[Medline].
42.
Papp, M.,
S. Feher,
E. P. Nemeth,
V. Somogyi,
and
G. Folly.
Exit routes for secretory proteins from the dog pancreas.
Acta Physiol. Acad. Sci. Hung.
56:
401-410,
1980[Medline].
43.
Proctor, G. B.,
B. Asking,
and
J. R. Garrett.
Effects of nerve stimulation on the movement of rat parotid amylase into the circulation.
Arch. Oral Biol.
34:
609-613,
1989[Medline].
44.
Proctor, G. B.,
B. Asking,
and
J. R. Garrett.
Serum amylase of non-parotid and non-pancreatic origin increases on feeding in rats and may originate from the liver.
Comp. Biochem. Physiol.
98B:
631-635,
1991.
45.
Rayford, P. L.,
H. R. Fender,
N. K. Ramus,
D. D. Reeder,
and
J. C. Thompson.
Release and half-life of CCK in man.
In: Symposium on Gastrointestinal Hormones, edited by J. C. Thompson. Austin: Univ. of Texas Press, 1974, p. 301-318.
46.
Reidelberger, R. D.,
M. O'Rourke,
P. R. Durie,
M. C. Geokas,
and
C. Largman.
Effects of food intake and cholecystokinin on plasma trypsinogen levels in dogs.
Am. J. Physiol.
246 (Gastrointest. Liver Physiol 9):
G543-G549,
1984
47.
Rothman, S. S.
Exocrine secretion from the isolated rabbit pancreas.
Nature
204:
84-85,
1964.
48.
Rothman, S. S.
Protein transport by the pancreas.
Science
190:
747-763,
1975[Medline].
49.
Rothman, S. S.
Protein Secretion: A Critical Analysis of the Vesicle Model. New York: Wiley, 1985.
50.
Rothman, S. S.,
and
T. Melese.
"Leaky" cells of glandular epithelia.
Int. Rev. Cytol.
112:
225-244,
1988[Medline].
51.
Ryan, J.,
and
H. Appert.
Circulatory turnover of pancreatic amylase.
Proc. Soc. Exp. Biol. Med.
149:
921-925,
1975[Abstract].
52.
Saito, A.,
and
T. Kanno.
Concentration of pancreozymin as a determinant of the exocrine-endocrine partition of pancreatic enzymes.
Jpn. J. Physiol.
23:
477-495,
1973[Medline].
53.
Schneyer, C. A.,
and
L. H. Schneyer.
Amylase in rat serum, submaxillary gland and liver following pilocarpine administration or normal feeding.
Am. J. Physiol.
198:
771-773,
1960.
54.
Schrifin, A.,
L. Tuchman,
and
W. Antopol.
Blood amylase response to acetyl--methylcholine chloride in rabbits.
Proc. Soc. Exp. Biol. Med.
34:
539-540,
1936.
55.
Shori, D. K.,
G. B. Proctor,
J. Chao,
K.-M. Chan,
and
J. R. Garrett.
New specific assays for tonin and tissue kallikrein activities in rat submandibular gland. Assays reveal differences in the effects of sympathetic and parasympathetic stimulation on proteinases in saliva.
Biochem. Pharmacol.
43:
1209-1217,
1992[Medline].
56.
Stiefel, D. J.,
and
D. J. Keller.
Preparation and some properties of human pancreatic amylase including a comparison with human parotid amylase.
Biochim. Biophys. Acta
302:
345-361,
1973[Medline].
57.
Temler, R. S.,
and
J. P. Felber.
Radioimmunoassay of human plasma trypsin.
Biochim. Biophys. Acta
445:
720-728,
1976[Medline].
58.
Tuchman, L.,
A. Schifrin,
and
W. Antopol.
Blood amylase response to acetyl--methylcholine chloride in pancreatectomized dogs.
Proc. Soc. Exp. Biol. Med.
33:
142-144,
1936.
59.
Weatherford, S. C.,
W. B. Laughton,
J. Salabarria,
W. Danho,
J. W. Tilley,
L. A. Netterville,
G. J. Schwartz,
and
T. H. Moran.
CCK satiety is differentially mediated by high- and low-affinity CCK receptors in mice and rats.
Am. J. Physiol.
264 (Regulatory Integrative Comp. Physiol. 33):
R244-R249,
1993
60.
Weickmann, J. L.,
and
D. G. Glitz.
Human ribonucleases.
J. Biol. Chem.
257:
8705-8710,
1982