Molecular mechanisms of membrane polarity in renal epithelial cells.

Exciting discoveries in the last decade have cast light onto the fundamental mechanisms that underlie polarized trafficking in epithelial cells. It is now clear that epithelial cell membrane asymmetry is achieved by a combination of intracellular sorting operations, vectorial delivery mechanisms and plasmalemma-specific fusion and retention processes. Several well-defined signals that specify polarized segregation, sorting, or retention processes have, now, been described in a number of proteins. The intracellular machineries that decode and act on these signals are beginning to be described. In addition, the nature of the molecules that associate with intracellular trafficking vesicles to coordinate polarized delivery, tethering, docking, and fusion are also becoming understood. Combined with direct visualization of polarized sorting processes with new technologies in live-cell fluorescent microscopy, new and surprising insights into these once-elusive trafficking processes are emerging. Here we provide a review of these recent advances within an historically relevant context.

pH heterogeneity at intracellular and extracellular plasma membrane sites in HT29-C1 cell monolayers.

In the colonic mucosa, short-chain fatty acids change intracellular pH (pH(i)) and extracellular pH (pH(e)). In this report, confocal microscopy and dual-emission ratio imaging of carboxyseminaphthorhodofluor-1 were used for direct evaluation of pH(i) and pH(e) in a simple model epithelium, HT29-C1 cells. Live cell imaging along the apical-to-basal axis of filter-grown cells allowed simultaneous measurement of pH in the aqueous environment near the apical membrane, the lateral membrane, and the basal membrane. Subapical cytoplasm reported the largest changes in pH(i) after isosmotic addition of 130 mM propionate or 30 mM NH(4)Cl. In resting cells and cells with an imposed acid load, lateral membranes had pH(i) values intermediate between the relatively acidic subapical region (pH 6.3-6.9) and the relatively alkaline basal pole of the cells (pH 7.4-7.1). Transcellular pH(i) gradients were diminished or eliminated during an induced alkaline load. Propionate differentially altered pH(e) near the apical membrane, in lateral intracellular spaces between adjacent cells, and near the basal membrane. Luminal or serosal propionate caused alkalinization of the cis compartment (where propionate was added) but acidification of the trans compartment only in response to luminal propionate. Addition of NH(4)Cl produced qualitatively opposite pH(e) excursions. The microscopic values of pH(i) and pH(e) can explain a portion of the selective activation of polarized Na/H exchangers observed in HT29-C1 cells in the presence of transepithelial propionate gradients.

Nuclear redistribution of tonicity-responsive enhancer binding protein requires proteasome activity.

Tonicity-responsive enhancer binding protein (TonEBP) is the transcription factor that regulates tonicity-responsive expression of the genes for the sodium-myo-inositol cotransporter (SMIT) and the sodium-chloride-betaine cotransporter (BGT1). Hypertonicity stimulates the activity of TonEBP due to a combination of increased protein abundance and increased nuclear distribution (proportion of TonEBP that is in the nucleus). We found that inhibitors of proteasome activity markedly reduce the induction of SMIT and BGT1 mRNA in response to hypertonicity. These inhibitors also reduce hypertonicity-induced stimulation of expression of a reporter gene controlled by the tonicity-responsive enhancer. Western and immunohistochemical analyses revealed that the proteasome inhibitors reduce the hypertonicity-induced increase of TonEBP in the nucleus by inhibiting its nuclear redistribution without affecting its abundance. Although the nuclear distribution of TonEBP is sensitive to inhibition of proteasome activity as is that of nuclear factor (NF)-kappaB, the signaling pathways appear to be different in that hypertonicity does not affect the nuclear distribution of NF-kappaB. Conversely, treatment with tumor necrosis factor-alpha increases the nuclear distribution of NF-kappaB but not TonEBP.

Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid.

Colonic luminal short-chain fatty acids (SCFA) stimulate electroneutral sodium absorption via activation of apical Na/H exchange. HT29-C1 cells were used previously to demonstrate that transepithelial SCFA gradients selectively activate polarized Na/H exchangers. Fluorometry and confocal microscopy (with BCECF and carboxy SNARF-1, respectively) are used to measure intracellular pH (pHi) in HT29-C1 cells, to find out which Na/H exchanger isoforms are expressed and if results are due to pHi gradients. Inhibition of Na/H exchange by HOE-694 identified 1) two inhibitory sites [50% inhibitory dose (ID50) = 1.6 and 0.05 microM] in suspended cells and 2) one inhibitory site each in the apical and basolateral membranes of filter-attached cells (apical ID50 = 1.4 microM, basolateral ID50 = 0.3 microM). RT-PCR detected mRNA of Na/H exchanger isoforms NHE1 and NHE2 but not of NHE3. Confocal microscopy of filter-attached cells reported HOE-694-sensitive pHi recovery in response to luminal or serosal 130 mM propionate. Confocal analysis along the apical-to-basal axis revealed that 1) luminal or serosal propionate establishes transcellular pHi gradients and 2) the predominant site of pHi acidification and pHi recovery is the apical portion of cells. Luminal propionate produced a significantly greater acidification of the apical vs. basal portion of the cell (compared with serosal propionate), but no other dependence on the orientation of the SCFA gradient was observed. Results provide direct evidence for a subcellular response that assures robust activation of apical NHE2 and dampening of basolateral NHE1 during pHi regulation.

Modulation of pancreatic secretion of individual digestive enzymes in octreotide (SMS 201-995)-infused rats.

We demonstrated previously that pancreatic secretion of individual enzymes is specifically regulated (1). In the present study, we investigated and defined contributing roles of cholinergic and cholecystokinin tones to the specific regulation of rat pancreatic secretion of digestive enzymes. Animals were provided with pancreatic, biliary, duodenal, and jugular vein cannulas allowing separate drainage of bile and pure pancreatic juice, as well as intravenous infusions of MK329 or atropine sulfate along with SMS 201-995 (SMS). Rats kept in restraint cages were divided into four groups. The first rat group was infused with 5 micrograms kg-1 h-1 SMS alone; the second group was infused with a mixture of SMS and MK329 (5 micrograms kg-1 h-1:0.5 mg kg-1 h-1); the third group received a mixture of SMS and atropine (5 micrograms kg-1 h-1); and rats in the fourth group were administrated a mixture of SMS, MK329, and atropine (5 micrograms kg-1 h-1:0.5 mg kg-1:100 micrograms kg-1 h-1). Food, but not water, was denied rats 10 h before the experiment and throughout the 6-h experimental period. During the experiment, pancreatic juice was continuously collected every 15 min from each rat, and a 15-microliter aliquot of the pancreatic juice sample was removed for total protein, amylase, lipase, trypsinogen, chymotrypsinogen, and proelastase assays. Pancreatic juice previously collected from a donor rat was mixed with the fresh bile and the mixture was recirculated into the duodenum. The secretory patterns over the 6-h experimental period showed that during the first hour of drug infusion, MK329 alone did not alter the SMS-induced inhibitory process of total protein and amylase, trypsinogen, and proelastase secretion, and there was no marked change in total protein and enzyme outputs. Adding atropine to SMS did not alter the secretory pattern during the first hour of drug infusion, but a significantly greater decrease in protein and enzymes outputs occurred. Correlations between paired enzyme outputs greatly increased with SMS alone, but some changed when either MK329 or atropine was infused along with SMS. When all drugs were infused together, enzyme outputs became strongly correlated. These results suggest that under fasting conditions, somatostatin and atropine can neutralize basal pancreatic enzyme outputs, leading to a constitutive type of secretion characterized by parallel secretion of the digestive enzymes. Furthermore, it is proposed that under basal secretion conditions, acetylcholine and cholecystokinin reaching the pancreatic acinar cells may act to dissociate pancreatic secretion of individual digestive enzymes originating from heterogeneous secretory granules.

Modulation of the relationship between amylase and chymotrypsinogen secretion in atropine- and MK329-infused rats.

We demonstrated previously in ad libitum fed and fasted rats that chymotrypsinogen and amylase secretions were weakly or not at all correlated (1). However, the mechanisms controlling these correlations remain undetermined. We investigated the influences of cholinergic and cholecystokinin-related systems on the relationship between amylase and chymotrypsinogen in rats. Animals provided with pancreatic, biliary, duodenal, and jugular vein cannulas were kept in restraint cages under controlled temperature and humidity, with a regular 12-h light cycle, and divided into five groups. The first group of fed rats was constantly infused with 200 micrograms kg-1 h-1 atropine, the second with 0.5 mg kg-1 h-1 MK329, and the third with both. In the group in which both drugs were simultaneously infused, 500 micrograms kg-1 h-1 atropine was intraperitoneally administered, whereas MK329 was infused by intravenous cannula. Two groups consisted of fasted rats, of which one was also atropinized (100 micrograms kg-1 h-1). Three-day experiments were performed separately with fed rats, and 2-day experiments with fasted rats; atropine and/or MK329 infusion was constant over 48 h, in both fed and fasted rats. Atropine alone did not alter the correlation between enzymes even though the total protein and amylase outputs decreased, whereas the chymotrypsinogen output increased; MK329, slowly but significantly, increased the correlation between enzymes, whereas it decreased the outputs for all secretory parameters. When both antagonists were simultaneously infused in fed rats, correlation coefficients between amylase and chymotrypsinogen rapidly and markedly increased. In fasted rats, atropine infusion induced a tremendous decrease in total protein and amylase mean outputs but a significant increase in chymotrypsinogen output, without any significant change in the correlation between both enzymes. These results indicate that the nonparallel secretion of amylase and chymotrypsinogen is strongly modulated by a cholecystokinin-dependent mechanism and that this modulatory process is potentiated by the parasympathetic system.

Amazing pancreas: specific regulation of pancreatic secretion of individual digestive enzymes in rats.

We investigated the effects of somatostatin (SMS)-201-995, atropine, and MK-329 on the role of cholinergic- and cholecystokinin-related systems and on the secretory relationship between five pancreatic digestive enzymes in rats. Animals kept in restraint cages and provided with pancreatic, biliary, duodenal, and jugular vein cannulas were treated as follows: 1) 0.25 micrograms.kg-1.h-1 caerulein alone, 2) both 0.25 micrograms.kg-1.h-1 caerulein and 100 micrograms.kg-1.h-1 atropine, 3) both caerulein and 5 micrograms.kg-1.h-1 SMS, 4) 91.3 micrograms.kg-1.h-1 carbachol alone, 5) both carbachol and 0.5 mg.kg-1.h-1 MK-329, and 6) both carbachol and 5 micrograms.kg-1.h-1 SMS, respectively. Food, but not water, was denied rats starting 10 h before the experiment and throughout the 6-h experimental period. The secretory patterns over the 6-h experimental period showed noticeably independent regulation of pancreatic secretion of individual digestive enzymes. The relationship between paired enzymes significantly varied according to the treatment. The correlation between chymotrypsinogen and the other enzymes was markedly modulated by MK-329. Our results suggest that SMS is a major "gate-keeper" in the regulation of exocrine pancreatic secretion and that the secretion of each digestive enzyme is individually regulated. Furthermore, they suggest that cholecystokinin and acetylcholine and their respective agonists are essentially initiators of secretory processes of the pancreas. Therefore, the paradigms of the regulation of pancreatic secretion heretofore accepted should be reexamined.

Stability of circadian and minor cycles of exocrine pancreatic secretion in atropine- and MK-329-infused rats.

We previously demonstrated the existence of a circadian rhythm of pancreatic secretion of fluid and digestive enzymes, which was superimposed on by a regular 1.84-h minor cycle of exocrine pancreatic secretion of fluid and total protein, amylase, and chymotrypsinogen (25). Direct control of these daily and hourly rhythms of pancreatic secretory function has not been addressed. Cholinergic and cholecystokinin (CCK)-associated influences on these two rhythms of exocrine pancreatic secretion were investigated in rats provided with pancreatic, biliary, duodenal, and jugular vein cannulas, allowing separate drainage of bile and pure pancreatic juice, as well as intravenous infusions of atropine sulfate and/or MK-329. Rats were kept in restraint cages under controlled temperature and humidity, with a regular 12-h light cycle, and divided into five groups. The first group of fed rats was constantly infused with 200 micrograms.kg-1.h-1 atropine, the second with 0.5 mg.kg-1.h-1 MK-329, and the third with both. In the group where both drugs were simultaneously infused, 500 micrograms.kg-1.h-1 atropine was intraperitoneally administered, whereas MK-329 was infused by intravenous cannula. Two groups consisted of fasted rats, of which one was also given atropine (100 micrograms.kg-1.h-1). Three-day experiments were performed separately with fed rats, and 2-day experiments were performed with fasted rats; atropine and/or MK-329 infusion, starting on day 2, was constant over 48 h in both fed and fasted rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic protein hypersecretion and elevated plasma CCK: prerequisites for increased pancreatic growth?

This study was undertaken to establish if a correlation exists between chronic elevated pancreatic secretion and growth of the pancreas. Rats provided with jugular, pancreatic, biliary, duodenal, or ileal cannulas were fed throughout the experiment with a liquid diet continuously infused into the duodenum. Four days after surgery, control rats and those infused with cerulein (CE) 0.45 microgram/kg/h had their pancreatic juice returned into the duodenum. Two other groups had their pancreatic juice either totally diverted outside (DO) or returned into the ileum (DI). In all groups, bile was returned into the duodenum. Pancreatic juice was collected every 4 h for 4 days with volume and protein determined. After 4 days, rats were killed and their pancreata were evaluated for weight and contents of DNA, RNA, protein, amylase, and chymotrypsinogen. The average volumes/4 h were significantly increased by 259, 241, and 270% in DO, DI, and CE rats, respectively. Protein output remained at control levels in DO rats, whereas increases of 200 and 90% above control values were observed in DI and CE rats, respectively, during the last periods of collection. Constant drainage of pancreatic juice outside (DO) had no effect on pancreatic growth; on the contrary, its reinfusion into the ileum and constant cerulein infusion were associated with impressive growth of the pancreas, with cerulein being the most potent stimulus. In conclusion these data support the hypothesis that increased protein output is associated with pancreatic growth, a phenomenon mediated by endogenous cholecystokinin.

Circadian rhythm of exocrine pancreatic secretion in rats: major and minor cycles.

The circadian variations of exocrine pancreatic secretion were studied in conscious rats provided with pancreatic, biliary, duodenal, and peritoneal cannulas and kept in restraint cages under controlled conditions, with a regular 12-h light cycle. Rats were divided into fed and fasted groups, and experiments were performed separately. During a 4-day post-surgical recovery period, rats were fed ad libitum. During the experiment, fed rats had free access to food and water. Food, but not water, was denied fasted rats 10 h before the experiment and for its 48-h duration. During the experiment, pancreatic juice was continuously collected for 4 and 2 days from fed and fasted rats, respectively. Every 30 min, a 20-microliters aliquot of sampled pancreatic juice was removed for total protein, amylase, and chymotrypsinogen assays. The remainder was mixed with bile collected simultaneously, and the mixture was recirculated into the duodenum. Over the 4- and 2-day periods there was a clear circadian rhythm of 24-h duration; for all measured parameters, secretory rates increased in the dark period and decreased during the light period. This major circadian rhythm was unexpectedly found to be superimposed on by a remarkably constant neurosecretory-like minor cycle of 2-h duration present in both fed and fasted states. The amplitude of the minor cycle was diminished by fasting. The outputs of fluid, total protein, and amylase were found to be only modestly correlated with each other, whereas chymotrypsinogen output was virtually completely independent of the others. The results suggest that the spontaneous major increase of exocrine pancreatic secretion in the dark was at least partially independent of food intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of cholecystokinin in peptide-YY release in response to pancreatic juice diversion.

It is generally accepted that peptide-YY (PYY), an ileocolonic peptide, is released by luminal nutrients and exogenous cholecystokinin (CCK). The importance of luminal nutrients as major PYY releasers was recently challenged when significant increases in plasma PYY were observed in response to pancreatic juice diversion in rats free of luminal nutrients. These recent data raise questions concerning the potential mediators of PYY release. This study was undertaken therefore to determine the role of endogenous CCK and the effects of exogenous caerulein, secretin and bombesin on PYY release. Male Wistar rats prepared with pancreatic, biliary, duodenal and jugular vein cannulae were used 4-7 days postoperatively. Pancreatic juice diversion significantly increased pancreatic volume and protein output with concomitant increases in plasma CCK and PYY concentrations. During pancreatic juice diversion, infusion of MK-329, a CCK-receptor antagonist, did not affect CCK release but completely abolished pancreatic volume and protein output and PYY release. The intraduodenal infusion of SBTI significantly stimulated pancreatic secretion and CCK release without any effect on PYY release. Furthermore, infusions of the CCK analog, caerulein, secretin and bombesin had no effect on PYY release. These results suggest the involvement of endogenous CCK in PYY release but this mediation seems to necessitate the participation of other as yet unknown factors since exogenous caerulein, secretin or bombesin cannot induce PYY release when given alone.

Endogenous cholecystokinin release responsible for pancreatic growth observed after pancreatic juice diversion.

This study was undertaken to determine whether intermittent pancreatic juice diversion (PJD) from the intestine can induce pancreatic and duodenal growth. Concomitant infusions of SMS 201-995, a somatostatin analog, and L-364,718, a cholecystokinin (CCK) receptor antagonist, were used to establish the involvement of endogenous CCK. Fed rats equipped with biliary, duodenal, and pancreatic cannulae had their pancreatic juice diverted 8 h/day for 4 days and were infused or not with either SMS 201-995 (5 micrograms/kg.h) or L-364,718 (0.5 mg/kg.h) during diversion. After 4 days, rats were killed, and their pancreas and duodenum were excised for measurements of parameters indicative of growth. In normally fed rats with pancreatic juice returned, SMS 201-995 inhibited daily pancreatic secretions of volume and protein, whereas L-364,718 inhibited only protein output. These two inhibitors had no effect on normal pancreatic and duodenal growth. PJD was associated with increased volume and protein output, increased plasma CCK level, and pancreatic growth. All of these effects were completely blocked by SMS 201-995 and L-364,718, with the exception of plasma CCK level by the CCK antagonist. None of these treatments affected duodenal growth. These results suggest that intermittent infusions of these two inhibitors had no effect on normal pancreatic and duodenal growth, but were successful in preventing pancreatic growth induced by PJD. They also indicate that endogenous CCK is involved in PJD-induced pancreatic growth.

Peptide-YY, a new partner in the negative feedback control of pancreatic secretion.

Peptide YY (PYY), a newly discovered ileocolonic peptide, is released by nutrients in the proximal and distal intestine and inhibits pancreatic secretion. However, it is not clear whether PYY can be released in the absence of nutrients in the intestine or whether a physiological role exists for endogenous PYY in negative feedback regulation of pancreatic secretion by pancreatic proteases. In the present study we measured plasma PYY concentrations and determined the effects of anti-PYY serum during stimulation of pancreatic secretion by pancreatic juice diversion (PJD). The effect of SMS 201-995 (SMS; an analog of somatostatin), another inhibitor of pancreatic secretion, on regulation of PYY release induced by PJD was also investigated. Male Wistar rats equipped with pancreatic, biliary, duodenal, and jugular venous cannulas were studied 4-6 days postoperatively. After 90 min of basal collection, pancreatic juice was diverted for 4 h with or without infusion of SMS (2 micrograms/kg.h), given either iv or intraduodenally (ID). Plasma PYY concentrations were significantly increased from a basal level of 177 +/- 15 pg/ml to a peak level of 328 +/- 43 pg/ml 2 h after PJD. These increases in PYY concentration paralleled those in pancreatic protein and fluid outputs. Both iv and ID infusion of SMS during the first 2 h of PJD markedly decreased the plasma PYY concentration to 134 +/- 27 pg/ml and 156 +/- 19 pg/ml, respectively; the total incremental PYY release during 4 h of PJD was inhibited by 100% and 84% by iv and ID SMS, respectively. One milliliter of anti-PYY serum given iv significantly augmented the increment in protein and fluid output during PJD. These results suggest that endogenous PYY released by PJD may play a physiological role in negative feedback regulation of pancreatic secretion in rats.

Effects of SMS 201-995 on basal and stimulated pancreatic secretion in rats.

Somatostatin (SRIF) is a potent inhibitor of most gastrointestinal and pancreatic functions. Recently, we showed that SRIF given either iv or intraduodenally (id) strongly inhibited stimulated pancreatic secretion induced by pancreatic juice diversion (PJD) from the duodenum. In this study we evaluate the effects of iv and id infusion of a long acting analog of SRIF, SMS 201-995 (SMS), on pancreatic secretion during basal conditions (pancreatic juice returned) and PJD. Conscious rats prepared with bile, pancreatic, duodenal, and jugular cannulae were studied 3-8 days postoperatively. Protein and fluid outputs were evaluated, and plasma cholecystokinin (CCK) was measured by bioassay. iv SMS infusion (5 micrograms kg-1 h-1) inhibited basal pancreatic protein and fluid secretion by 84 and 64%, respectively. Addition of atropine (500 micrograms kg-1 h-1 ip) did not cause further inhibition. During PJD, SMS iv from 0.005-1.28 micrograms kg-1 h-1 for 3 h caused a dose-dependent inhibition with maximal 90% and 75% reductions of protein and fluid, respectively, at 1.28 micrograms SMS. Plasma CCK was also reduced by 83% from 3.01 +/- 1.15 to 0.51 +/- 0.22 pM. SMS, id at 1.7 micrograms kg-1 h-1 for 1.5 h before and 2 h after PJD, caused inhibition of basal secretion by 25% and that induced by PJD by 60%. Plasma CCK, measured 1.5 h after diversion, increased from 1.55 +/- 0.06 to 5.9 +/- 1.14 pM in the presence of SMS. Intravenous SMS was 20 times more potent than SRIF in inhibiting pancreatic protein and volume secretion stimulated by PJD. Iv SMS inhibited basal and stimulated fluid and protein pancreatic secretion as well as plasma CCK levels. SMS was also effective when given id in inhibiting fluid and protein pancreatic secretion, but id SMS increased plasma CCK levels. This effect on plasma CCK may be due to the inhibition of hormonal inhibitors of CCK release.