Search PubMed⌕ Search

Biomedical subjects

S S Rothman

Publications and source records attributed to S S Rothman.

At least 19 recordsLinked to original sources
PubMed1995

A trans-membrane pore can account for protein movement across zymogen granule membranes.

We have reported that the membrane of zymogen granules, secretion vesicles from the exocrine pancreas, is permeable to its contained proteins by measuring both the loss and accumulation of protein in response to mass action forces [1-3] However, the mechanism of transport has remained unknown. Here we consider evidence that this transport occurs through trans-membrane pores. Using freeze-fracture electron microscopic methods, Cabana et al. [4] have reported the presence of a 15 nm intramembrane particle in zymogen granule membrane which contains a 5 nm (+/- 0.1 nm, S.D.) diameter lucent center. In this article, we propose that this structure is a pore through which proteins can be transported, and test this hypothesis by comparing the predicted phenomenological permeability coefficient for transport by diffusion via this structure, to that calculated from protein flux measurements on granules using an X-ray microscope. The predicted and experimental values were essentially identical and hence support the hypothesis that this structure could be a protein transporting channel.

Biological Transport↗
PubMed1995

The protein content and morphogenesis of zymogen granules.

When zymogen granules, the secretion granules of pancreatic acinar cells, fill, secretory product is accumulated in immature granules, condensing vacuoles. Mature granules are formed when this product (protein) condenses into an osmotically inactive aggregate and, bulk water is expelled. This hypothesis for granule morphogenesis has two elements. The first is that immature granules are precursors to mature granules. The second is that a particular maturational event, condensation, which involves the aggregation of protein, takes place. These hypotheses lead to two straightforward predictions. One, that condensing vacuoles on average, should contain less protein than filled or mature granules. And two, that, due to condensation, mature granules should contain protein at a common concentration. In the current work, both of these predictions were tested using measurements of the protein content of individual granules acquired by X-ray microscopy. Neither prediction was affirmed by the experimental results. First, there was no distinguishable difference in the distribution of protein between immature and mature granules. Second, the protein concentration of mature granules varied widely between preparations, although granules from the same preparation had similar concentrations. From the data we conclude that: 1) mature granules and condensing vacuoles are different, though not necessarily unrelated, types of secretory vesicle, and not two forms of the same object; 2) as such, condensing vacuoles are not precursors to mature granules; 3) all granules do not contain protein at one particular concentration when "full," or mature; 4) granule maturation does not involve a condensation step; 5) concentration is not determined by such physical limits as the space available for protein packing or condensation; and 6) the amount of protein contained is physiologically regulated.

Animals↗
PubMed1992

Protein flux across the membrane of single secretion granules.

We have applied, for the first time to our knowledge, X-ray microscopy to measure the mass of protein contained in single sub-cellular membrane-bound structures and to make high resolution, time-resolved observations on them. Using this method we have been able to follow the flux of protein out of secretion (zymogen) granules isolated from the acinar cells of the exocrine pancreas. The results provide direct visual and quantitative confirmation of the hypothesis that the membrane enclosing this object is permeable to its various contained proteins, although the mechanism remains unknown.

Animals↗
PubMed1989

The interior of a whole and unmodified biological object--the zymogen granule--viewed with a high-resolution X-ray microscope.

We report the ability of focused soft X-rays to visualize at spatial resolution well beyond that of the optical microscope (less than 100 nm) the interior of a small, whole biological object without fixation, staining, dehydration or sectioning. Quantitative estimation of its protein content with unique femtogram sensitivity is also reported. The present results represent a significant step towards the goals of natural imaging and chemical mapping of biological structures with soft X-rays.

Animals↗
PubMed1986

Digestive end products release pancreatic enzymes from particulate cellular pools, particularly zymogen granules.

The effects of various amino acids and phosphorylated forms of glucose on the release of digestive enzymes from particulate cellular pools, particularly zymogen granules, were evaluated in rat pancreas. Whole tissue homogenates, as well as zymogen granules isolated either by differential centrifugation in 0.3 M sucrose or by preparation in buffered sucrose and subsequent centrifugation in a Percoll gradient, were studied. The basic amino acids L-arginine and L-lysine, sites of tryptic cleavage, caused the release of trypsinogen, but not chymotrypsinogen, whereas the aromatic amino acids L-phenylalanine and L-tryptophan, sites of chymotryptic cleavage, caused release of both trypsinogen and chymotrypsinogen. Neither led to the release of the starch-splitting enzyme amylase. All effects occurred within the range of normal plasma concentrations for these amino acids in the rat. Two amino acids, L-threonine and hydroxy-L-proline, that are not sites of cleavage by trypsin or chymotrypsin, and a nonmammalian amino acid, aminoadipic acid, did not lead to release of trypsinogen, chymotrypsinogen, or amylase. Two phosphorylated forms of glucose, glucose 1-phosphate and glucose 1,6-diphosphate, caused the release of amylase, but of neither trypsinogen nor chymotrypsinogen. Contrary to previous results, D-glucose was without effect, as was glucose 6-phosphate. We propose that certain digestive end products, by direct action on zymogen granules, cause the selective release of the enzymes involved in their evolution from polymeric substrates during digestion.

Amino Acids↗
PubMed1986

Zymogen granule size in pancreas of nursing rats.

Dramatic depression in granule volume density and size was measured in acinar cells of postnatal rat pancreas following the initiation of feeding. Volume density decreased about threefold from 45% at birth to 16% 2 days thereafter. Mean granule diameter decreased from 1.50 micron to 0.80 micron, an 85% decrease in corresponding granule volume. At the same time, numerical density approximately doubled. At 2 days after birth, cells with smaller granules had lower volume densities, and differences in mean granule volume between cells accounted for most of the differences in volume density. Although the distribution of granule diameter in newborns was lognormal, the distribution at 2 days was heavily skewed to larger sizes. This was the result of skewed distributions within individual cells and not an artifact of sampling. The results corroborate the central role of granule volume in determining changes in the volume density of zymogen granules in the pancreas and suggest that zymogen granules can act as capacitors that can change size as a function of the enzyme contained within.

Aging↗
PubMed1986

Characteristics of rat pancreatic zymogen granules prepared by different methods.

Zymogen granules isolated from tissue homogenates by differential centrifugation in isotonic sucrose solutions show substantial release of digestive enzyme when suspended in isotonic NaCl and in sucrose solutions at pH values above neutrality. A recent study reported a new method for isolating granules, involving the use of a complex homogenization medium and a Percoll gradient that was claimed to produce "stable" granules, i.e., granules that do not release their content in salt solutions and at pH values at or above neutrality. In the present study, we compare granules prepared in both ways, particularly in terms of their tendency to release amylase in isotonic ionic solutions and as a function of pH. The relative absence of amylase release from granules isolated by the new technique was found to be attributable to simple differences in the details of the experimental procedures that were used and not to actual differences in the characteristics of the two granule preparations. For example, previous studies with granules prepared in sucrose solutions reported substantial salt-induced release at 37 degrees C, whereas the recent study reporting the absence of salt-induced release from granules obtained from a Percoll gradient was done at 24 degrees C. Under the identical experimental conditions as used in the present study, little amylase release was seen at 24 degrees C for granules isolated by either technique, but substantial release was seen for both at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1985

Permeability of zymogen granule membrane to protein.

The evidence that the membrane of the pancreatic zymogen granule is permeable to its contained secretory proteins is outlined. Included is a discussion of the nature and characteristics of the equilibrium-dependent release of protein from isolated granules, the evidence for the permeability of the granule membrane to digestive enzyme protein in situ, and the seeming paradox that isolated granules release protein in medium similar to that thought to exist in the cell. The permeability hypothesis is reconsidered here in light of recent claims of stable nonpermeable granules.

Amylases↗
PubMed1985

Effects of proglumide on ductal and basolateral secretion of pancreatic digestive enzymes.

Previous studies have shown that proglumide acts as a cholecystokinin (CCK) receptor antagonist in isolated pancreatic acini. To establish the effect of proglumide in the intact organ, its effects on both CCK-stimulated ductal and basolateral secretion of digestive enzymes were studied in the in vitro rabbit pancreas. CCK-stimulated ductal secretion of chymotrypsinogen, amylase, and total protein, as well as basolateral secretion of amylase, was inhibited by proglumide in a dose-dependent manner. Regression lines comparing ductal chymotrypsinogen and amylase outputs also were altered significantly by proglumide in a dose-dependent manner, with amylase secretion inhibited to a lesser degree. Similarly, the relative rates of amylase secretion across the ductal versus the basolateral cell surface were altered, with basolateral secretion inhibited to a lesser degree. The effects seen with increasing concentrations of proglumide at a given concentration of CCK mirrored the effects seen with decreasing the concentration of CCK in the absence of proglumide. Proglumide did not affect ductal or basolateral secretion stimulated by a cholinergic agonist and did not affect unstimulated pancreatic secretion. Thus, proglumide appears to act as a selective antagonist of CCK-stimulated secretion in the intact organ. The results further indicate that interactions of an agonist and an antagonist at the CCK receptor can alter not only the overall amount of enzymes secreted but their relative proportions as well.

Amylases↗
PubMed1984

Regulation of digestion. I. Effects of glucose and lysine on pancreatic secretion.

Previous cell-free studies have shown that glucose selectively elicits the release of amylase from pancreatic zymogen granules, whereas lysine promotes the selective release of trypsinogen. To investigate the expression of these effects in situ, glucose or lysine was injected into the celiac artery of anesthetized rats, either alone or together with the pancreatic secretagogue cholecystokinin, to evaluate their effects on the secretion of amylase and trypsinogen by the pancreas. When given alone neither substance significantly changed the output of either enzyme. However, when given with cholecystokinin, each altered the effect observed with injection of cholecystokinin alone. The injection of glucose resulted in a twofold increase in both peak and total amylase output without significantly increasing trypsinogen secretion, whereas lysine increased both peak and total trypsinogen output by about 50%, leaving amylase output unchanged. These findings provide in situ confirmation for the selective enzyme release produced by glucose and lysine in cell-free studies and suggest that such end products of digestion can regulate the digestive process by modifying the secretory response of the pancreas to cholecystokinin.

Animals↗
PubMed1984

Regulation of digestion. II. Effects of insulin and glucagon on pancreatic secretion.

The endocrine islet-cell hormones insulin and glucagon are secreted at high concentrations into an intrapancreatic portal circulation and have been reported to affect the secretion of digestive enzyme by the exocrine pancreas. In the present experiments, insulin and glucagon were injected into the celiac artery of anesthetized rats to evaluate their effects on the secretion of amylase and trypsinogen by the pancreas. Neither hormone when given alone significantly changed the output of either enzyme. However, when given with the pancreatic secretagogue cholecystokinin, each altered the effect of injection of cholecystokinin. In a dose-dependent fashion insulin increased trypsinogen output without affecting amylase output, whereas glucagon inhibited amylase output and left trypsinogen output unchanged. Thus, both hormones produced a more trypsinogen-dominant pancreatic juice than that observed with cholecystokinin alone, although in different ways. These findings suggest that the endocrine hormones insulin and glucagon may regulate secretion of digestive enzymes by the pancreas by modulating the response to stimuli of overall protein secretion such as cholecystokinin.

Animals↗
PubMed1983

Distribution of three hexose derivatives across the pancreatic epithelium: paracellular shunts or cellular passage?

It has been proposed that the pancreatic epithelium is permeable to three presumably passively distributed non-electrolytes, namely sucrose, inulin and mannitol, via paracellular shunts, and that the increased flux of sucrose and inulin seen during augmented digestive enzyme secretion is due to an increase in the permeability of these shunts. The present study considers this hypothesis by comparing the permeability of the epithelium to three different hexose derivatives, mannitol, inositol and 3-O-methyl-glucose, in both the unstimulated state and after the augmentation of protein secretion with a cholinergic drug. The epithelium was found to be more permeable to mannitol than to either inositol or 3-O-methyl-glucose. In the unstimulated state, the concentration of mannitol in ductal fluid at the steady state was approx. 54% of its concentration in the interstitium, as compared to 12% for inositol and 8% for 3-O-methyl-glucose. Cholinergic stimulation substantially increased the concentration of inositol and 3-O-methyl-glucose in secretion, but did not increase that of mannitol. The increase in the concentration of inositol occurred in the absence of an increase in its rate of transepithelial movement. Taken together, the results suggest that: (1) there is a substantial passage of mannitol through the cells of the epithelial layer, and (2) the increased concentration of inositol and 3-O-methyl-glucose in ductal fluid that occurs with stimulation is due to an increase in their efflux from secretory cells.

3-O-Methylglucose↗
PubMed1983

Protein concentration in the pancreatic zymogen granule.

We have estimated the concentration of protein in the zymogen granule, a major storage depot for secretory protein in the pancreatic acinar cell, in four different ways. Each of these approaches yielded roughly similar values. The protein concentration in the granule is approx. 135-270 mg protein/ml granule volume in rat and rabbit, as compared to an average value for the protein concentration of tissue of 135 mg/g tissue for rabbits and 183 mg/g tissue for rats. This is equivalent to an average molarity for the contained proteins of between 4-9 mM based on an estimated average molecular weight for the mixture of 30 000-40 000. An upper limit for the concentration of protein in these granules can reasonably be set at about double the overall concentration of protein in the non-granule portion of the cell.

Amylases↗
PubMed1983

Increase in zymogen granule volume accounts for increase in volume density during prenatal development of pancreas.

The sudden increase in volume density of zymogen granules in acinar cells of the fetal rat pancreas was examined with particular attention to the respective roles of granule size and number in this event. Volume density increased some twelvefold, from about 3% of cytoplasmic volume at 17 days to about 45% at 20 days, following a sigmoidal pattern in which the greatest rate of increase occurred during day 18. This increase in volume density was primarily the result of an increase in granule volume. Zymogen granule diameter increased from 0.55 micron at 17 days to 1.20 micron at 20 days, an order of magnitude increase in average granule volume. The total number of granules in the tissue increased in proportion to the increase in organ weight (cell number and size), but changes in the number of granules per unit cytoplasmic volume were minor (+ 40%) in comparison to the increase in volume density. The distribution of granule diameter was roughly normal and unimodal at each time interval, and the increase in average diameter over time was marked by an increase in the upper limit of the size distribution and an increased percentage of large granules. The size of condensing vacuoles also increased during this period, and their distributions were roughly coextensive with those seen for zymogen granules at the same time. The potential origins of changes in granule size are discussed, as well as the important effect that size has on the number of granules observed in "two-dimensional" tissue sections viewed in the electron microscope. If size is not considered in our estimates, then we underestimate the numerical density in cells with small granules compared to those with large granules. The results indicate the central role of granule size, as opposed to number, in determining granule volume density in the embryonic pancreas.

Animals↗