Effect of somatostatin on ion transport in the rat colon.

The effect of somatostatin (SRIF) on ion transport was determined in the rat colon in vitro. SRIF produced a sustained decrease in the short circuit current (Isc) (-0.8 +/- 0.1 mueq/h x cm2) and increased net Cl absorption (0.9 +/- 0.3 mueq/h x cm2). The threshold effect of SRIF on Isc was observed at 6 nM. 10 microM serotonin decreased net Na absorption (-2.6 +/- 0.4 mueq/h x cm2), net Cl absorption (-3.6 +/- 0.5 mueq/h x cm2) and increased Isc (0.7 +/- 0.1 mueq/h x cm2); these changes were totally blocked by 0.1 microM SRIF. SRIF completely blocked net Cl secretion induced by 10 mM theophylline (-2.5 +/- 0.7 to +4.1 +/- 2.0 muq/h x cm2) and partially blocked theophylline-induced inhibition of net Na absorption (0.7 +/- 0.5 to 2.1 +/- 0.4 mueq/h x cm2). SRIF also blocked prostaglandin E1 (PGE1) induced increase in potential difference and Isc (P < 0.001). Mucosal cyclic AMP levels were increased by theophylline and PGE1 but not by serotonin. SRIF had no effect on basal or theophylline- and PGE1-stimulated cyclic AMP levels. These results indicate that SRIF blocks both cyclic AMP and noncyclic AMP mediated changes in ion secretion and suggest that SRIF is acting at a step in the secretory process beyond the formation of cyclic AMP.

Oxalate transport by anion exchange across rabbit ileal brush border.

This study demonstrates the presence of oxalate transporters on the brush border membrane of rabbit ileum. We found that an inside alkaline (pH = 8.5 inside, 6.5 outside) pH gradient stimulated [14C]oxalate uptake 10-fold at 1 min with a fourfold accumulation above equilibrated uptake at 5 min. 1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (disodium salt; DIDS) profoundly inhibited the pH-gradient stimulated oxalate uptake. Using an inwardly directed K+ gradient and valinomycin, we found no evidence for potential sensitive oxalate uptake. In contrast to Cl:HCO3 exchange, HCO3 did not stimulate oxalate uptake more than was seen with a pH gradient in the absence of HCO3. An outwardly directed Cl gradient (50 mM inside, 5 mM outside) stimulated oxalate uptake 10-fold at 1 min with a fivefold accumulation above equilibrated uptake. Cl-stimulated oxalate uptake was largely inhibited by DIDS. Addition of K+ and nigericin only slightly decreased the Cl gradient-stimulated oxalate uptake, which indicates that this stimulation was not primarily due to the Cl gradient generating an inside alkaline pH gradient via Cl:OH exchange. Further, an outwardly directed oxalate gradient stimulated 36Cl uptake. These results suggested that both oxalate:OH and oxalate:Cl exchange occur on the brush border membrane. To determine if one or both of these exchanges were on contaminating basolateral membrane, the vesicle preparation was further fractionated into a brush border and basolateral component using sucrose density gradient centrifugation. Both exchangers localized to the brush border component. A number of organic anions were examined (outwardly directed gradient) to determine if they could stimulate oxalate and Cl uptake. Only formate and oxaloacetate were found to stimulate oxalate and Cl uptake. An inwardly directed Na gradient only slightly stimulated oxalate uptake, which was inhibited by DIDS.

Adenosine and adenosine analogues stimulate adenosine cyclic 3', 5'-monophosphate-dependent chloride secretion in the mammalian ileum.

Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. The purpose of these investigations was to determine the effect of adenosine on ion transport in rabbit ileum in vitro. Adenosine and some of its analogues were found to increase the short circuit current (Isc) and the order of potency was N-ethylcarboxamide-adenosine greater than or equal to 2-chloroadenosine greater than phenylisopropyladenosine greater than adenosine. Purine-intact adenosine analogues had no effect on Isc. The effect of adenosine on Isc was enhanced by deoxycoformycin, an adenosine deaminase inhibitor, and by dipyridamole, an adenosine uptake inhibitor. The increase in Isc induced by 2-chloroadenosine was partially reversed in a dose-dependent manner by 8-phenyltheophylline but not by theophylline or isobutylmethylxanthine. 2-Chloroadenosine increased cyclic AMP content, and stimulated net Cl secretion; these effects were partially blocked by 8-phenyltheophylline. These results suggest that there is an adenosine receptor on rabbit ileal mucosal cells that stimulates adenylate cyclase, which results in secondary active Cl secretion.

Membrane distribution of sodium-hydrogen and chloride-bicarbonate exchangers in crypt and villus cell membranes from rabbit ileum.

Present evidence suggests that in the small intestine, villus cells are primarily absorptive and crypt cells are primarily secretory. In order to further confirm that there are differences in transport properties between villus and crypt cells, we have separated villus from crypt cells, using calcium chelations techniques, and determined the distribution of Na:H and Cl:HCO3 exchange activity on brush border membrane and basolateral membrane preparations from these two cell populations. Separation of cells was determined utilizing alkaline phosphatase and maltase activity as a marker of villus cells and thymidine kinase activity as a marker of crypt cells. Utilizing these techniques, we were able to sequentially collect cells along the villus-crypt axis. Na-stimulated glucose and alanine uptake in brush border membrane vesicles diminished from the villus to the crypt region in the sequentially collected cells fractions, further suggesting separation of these cells. Brush border and basolateral membranes were then prepared from cells from the villus and crypt areas, utilizing a continuous sucrose gradient. In the villus cells, Na:H exchange activity was found associated with both the brush border and basolateral membrane, whereas, in crypt cells, Na:H exchange activity was only found on the basolateral membrane. Cl:HCO3 exchange activity was found only on the brush border membrane, in both villus and crypt cells. These studies suggest functional heterogeneity in ion transport between villus and crypt cells.

Mechanism of intestinal secretion. Effect of serotonin on rabbit ileal crypt and villus cells.

To determine the mechanism of action of an intestinal secretagogue, serotonin, we have isolated crypt and villus cells and demonstrated Na:H and Cl:HCO3 exchange activity using the intracellular pH-sensitive fluorescent dye, 2,7-bis (carboxy-ethyl)-5,6-carboxy-fluorescein. Serotonin alkalinized both crypt and villus cells. Alkalinization in villus cells was HCO3 dependent and Na independent. In contrast, alkalinization in crypt cells was HCO3 independent and Na dependent. In villus cells, recovery from an alkaline load induced by Cl removal, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or propionate pulse, known to occur via the Cl:HCO3 exchange, is inhibited by serotonin. In contrast, in crypt cells, recovery from an acid load induced by Na removal, amiloride and NH4Cl pulse, known to occur via Na:H exchange, is stimulated by serotonin. These data suggest that serotonin is inhibiting Cl:HCO3 exchange in villus cells and stimulating Na:H exchange in crypt cells. These effects of serotonin would be expected to inhibit coupled Na and Cl absorption by villus cells and stimulate HCO3 secretion by crypt cells in the intact ileum.

Augmentation of neutral sodium chloride absorption by increased flow rate in rat ileum in vivo.

Studies in intact animals have shown that intestinal solute absorption is enhanced with increasing flow rates; the mechanism of this phenomenon has not been explored in detail. We used single pass perfusions of rat ileum to study the effect of higher flow rate on electrolyte absorption. Augmenting perfusion rate from 0.5 to 5.0 ml/min resulted in increased rates of sodium (11.0 +/- 0.9 vs. 23.5 +/- 2.7 mueq/min X g) and chloride (12.1 +/- 0.8 vs. 25.0 +/- 2.2 mueq/min X g) absorption, reduction in the estimated unstirred layer thickness (668 +/- 31 vs. 433 +/- 28 micron), minimal changes in intraluminal pressure and transmural potential difference, and a small, though significant, increase in intraluminal volume (19.4 +/- 8.4%). Removal of sodium from the perfusion medium abolished the effect of increased flow rate on chloride absorption as did removal of chloride on sodium absorption; addition of furosemide or acetazolamide to Ringer's solution also inhibited this effect. In separate experiments, stepwise increases in intraluminal volume were induced by elevating the outflow tubing; no effect on electrolyte transport was observed. These studies demonstrate that neutral sodium chloride absorption is enhanced in rat ileum at higher flow rates, perhaps as a result of a decrease in the thickness of unstirred layers.

Evidence for carrier-mediated chloride/bicarbonate exchange in canalicular rat liver plasma membrane vesicles.

To determine whether anion exchangers might play a role in hepatic bile formation, we looked for the presence of Cl-:OH- and Cl-:HCO3- exchange in highly purified canalicular (c) and basolateral (bl) rat liver plasma membrane (LPM) vesicles. In cLPM vesicles, a pH gradient (7.7 in/6.0 out) stimulated 36Cl- uptake twofold above values obtained during pH-equilibrated conditions (7.7 in = out). When 50 mM HCO3- was also present inside the vesicles, the same pH gradient (7.7 in/6.0 out) resulted in Cl- uptake to levels fourfold above pH- and HCO3--equilibrated controls and two- to threefold above Cl- equilibrium (overshoot). Initial rates of both pH and HCO3- gradient-stimulated Cl- uptake were completely inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). A valinomycin-induced K+ diffusion potential (inside positive) also stimulated Cl- uptake in cLPM, but this conductive Cl- pathway was insensitive to DIDS. The DIDS-sensitive, pH and HCO3- gradient-stimulated Cl- uptake demonstrated: saturation with Cl- (Km approximately 6.3 mM; Vmax approximately 51 nmol X mg-1 X min-1); partial inhibition by bumetanide (26%), furosemide (33%), probenecid (37%), and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (49%); cis-inhibition by chloride and nitrate but not by sulfate and various organic anions, and independence from the membrane potential. These data demonstrate the presence of an electroneutral Cl-:OH- and Cl-:HCO3- exchanger in rat liver canalicular membranes that favors Cl-:HCO3- exchange. In contrast, no evidence was found for the presence of a Cl-:HCO3- (OH-) exchange system in blLPM vesicles. Furthermore, neither blLPM nor cLPM vesicles exhibited Na+-stimulatable Cl- uptake, indicating the absence of a NaCl co-transport system in either LPM subfraction. These findings are consistent with a functional role for a Cl-:HCO3- (OH-) exchanger in canalicular bile formation.

An experimental human model of 1,25-dihydroxyvitamin D-mediated hypercalciuria.

An increase in fasting calcium excretion occurs in hypercalciuric patients and has been interpreted by many investigators as evidence for a primary renal tubular leak of calcium. In a recent series of 50 patients with absorptive hypercalciuria, we found a mean increase in fasting fractional calcium excretion (calcium clearance) and provided data suggesting that this leak of calcium was secondary to intestinal hyperabsorption of calcium and suppression of parathyroid secretion. To examine this question, a model of 1,25-dihydroxyvitamin D [1,25-(OH)2D]-mediated hypercalciuria was created by administering a large dose of 1,25-(OH)2D to normal subjects. In addition to the expected features of absorptive hypercalciuria during 1,25-(OH)2D administration, the subjects had an increase in fasting calcium excretion and a marked increase in calcium excretion during a restricted calcium diet, points that might be interpreted as favoring a resorptive and/or renal component to the net hypercalciuria. However, total hydroxyproline excretion remained unchanged (18.0 vs. 19.0 mg/g creatinine), and the increase in fasting calcium excretion was found to reflect an increase in calcium clearance; the latter was inversely correlated with parathyroid function, as determined by fasting measurements of nephrogenous cAMP excretion (r = -0.77; P less than 0.01). We conclude that measurements of calcium excretion in the fasting state or on a restricted calcium diet do not represent valid criteria for differential diagnosis of the hypercalciurias.

The effect of somatostatin and enkephalin on ion transport in the intestine.

SRIF and enkephalin stimulate net Na and C1 absorption in the rabbit ileum and appear to do so primarily by stimulating the coupled influx of Na and C1 across the brush border membrane. The stimulatory effect of enkephalin on ion transport is blocked by verapamil and Ca-free solution, indirectly indicating that enkephalin may work as a Ca-channel blocker. The resultant lowering in cytosolic calcium level stimulates Na and C1 absorption. SRIF was able to block the effect of cyclic AMP-dependent and cyclic AMP-independent secretogogues in the rat colon without affecting cyclic AMP levels suggesting that it is inhibiting a distal step in the secretory pathway, probably involving a final common pathway. SRIF infusion blocked diarrhea in a patient with the carcinoid syndrome, indicating that it may be therapeutically useful in the treatment of secretory diarrheas. The antidiarrheal effect of opiates is probably due in part to their effect on electrolyte absorption, rather than an effect solely on intestinal motility.

Pulmonary manifestations of gastrointestinal disease.

The respiratory and gastrointestinal systems share several functional similarities. In health, the two systems remain structurally distinct and functionally integrated, so as to maintain physiologic homeostasis. However, in the setting of disease, pathophysiologic alterations in one system may be reflected in the other. This article focuses upon the nonmalignant gastrointestinal causes of respiratory illness. Using an anatomic approach, the pulmonary manifestations of selected gastrointestinal diseases are outlined. Emphasis is placed on the distinguishing features, pathophysiology, and therapy of the pulmonary sequelae of digestive diseases.

Nephrolithiasis and intestinal disease.

Kidney stones in patients with inflammatory bowel disease are usually composed of calcium oxalate. Two factors are important in the increased absorption of dietary oxalate which is responsible for those stones: 1) increased absorption of oxalate in the presence of steatorrhea, and 2) increased permeability of the colon to oxalate. Fortunately, some of the physiologic abnormalities can be corrected. A therapeutic approach is detailed.

Effect of bile salts and fatty acids on the colonic absorption of oxalate.

These studies were designed to evaluate the effect of bile salts and fatty acids on colonic oxalate absorption. Five millimolar deoxycholate significantly increased oxalate absorption from 34.2 +/- 9.4 nmoles per min per g dry weight to 330.4 +/- 47.3 (P less than 0.001) and changed water absorption to water secretion. Deoxycholate also increased the absorption of urea, decreased the electrical potential difference, and increased colonic clearance of oxalate, observations which are consistent with an increase in colonic mucosal permeability. In contrast, taurocholate did not increase oxalate absorption. Ricinoleic acid also significantly increased the absorption. These results suggest that bile salts and fatty acids increase colonic absorption of oxalate. Oleic acid had similar effects on oxalate absorption but was less effective than ricinoleic acid. Octanoic acid, a medium chain fatty acid, did not alter oxalate absorption of oxalate by a nonspecific alteration of mucosal permeability. These observations may further explain many of the clinical phenomena associated with enteric hyperoxaluria.

Evaluation of the patient with suspected malabsorption.

The term "malabsorption" is generally used to indicate any defect in absorption; strictly speaking, however, it is a defect in the mucosal phase of absorption. Defects in the intraluminal phase are termed "maldigestion." This distinction is essential when considering the pathophysiology to apply diagnostic tests in the evaluation of malabsorption. This article first discusses the signs and symptoms associated with malabsorption, the disorders associated with malabsorption, normal intestinal absorption, and, finally, the diagnostic test used to investigate absorption.

Sulfate and oxalate exchange for bicarbonate across the basolateral membrane of rabbit ileum.

The purpose of these studies was to further define the transport of SO4(2-) and oxalate across the basolateral membrane (BLM) of rabbit ileum. We previously found evidence for Cl-(-)SO4(2-) exchange but no evidence for carrier-mediated oxalate transport. A HCO3- gradient (but not a pH gradient) was found to stimulate SO4(2-) and oxalate uptake in BLM vesicles; uptake was inhibited by DIDS. Oxalate cis-inhibited HCO3- gradient SO4(2-) uptake and trans-stimulated SO4(2-) uptake, suggesting SO4(2-) and oxalate used the same carrier (SO4(2-)- and oxalate-HCO3-exchange). Cl- had no effect on HCO3- gradient-stimulated SO4(2-) uptake, indicating that Cl(-)-SO4(2-) exchange was a different carrier. Other substrates found to be transported on the HCO3- exchanger were oxaloacetate and S2O3(2-)-.SO4(2-), S2O3(2-). and oxalate were found to also use the SO4(2-)-Cl- exchanger, whereas oxalate was only transported on the HCO3- exchanger. SO4(2-)-HCO3- exchange was found on villus, but not crypt cell, BLM. These results indicate that there is a SO4(2-)-HCO3- exchanger on the BLM of villus cells that also transports oxalate, and along with our previous studies, the results provide evidence for the transepithelial transport of oxalate.

Importance of the colon in enteric hyperoxaluria.

To investigate the role of the colon in increased oxalate absorption, we measured urinary oxalate and fecal fat excretion in 26 patients with gastrointestinal disease. Eight patients with steatorrhea of various causes (Crohn's disease [two], chronic pancreatitis [four], jejunoileal bypass [one] and extrahepatic biliary obstruction [one]) had hyperoxaluria (greater than 45 mg per 24 hours). All these patients had intact colons. In contrast, none of five patients with ileostomies and steatorrhea secondary to ileal resection had hyperoxaluria. Absorption of 14C-oxalate was increased in three patients with steatorrhea and intact colons but not in three patients with steatorrhea and an ileostomy. Thus, the colon is both the site of and required for increased oxalate absorption in enteric hyperoxaluria. The lack of a direct relation between fecal fat excretion and urinary oxalate excretion in the patients with hyperoxaluria and steatorrhea suggests that steatorrhea, although important, is not the only determinant in the pathogenesis of hyperoxaluria.

Role of opiate receptors in regulation of enkephalin stimulation of active sodium and chloride absorption.

The mechanism of opioid peptide stimulation of active Na and Cl absorption in rabbit ileum is not known. Since vasoactive intestinal peptide (VIP) modulation of electrolyte transport is mediated by specific membrane receptors, these experiments sought to determine whether membrane receptors for opioid peptides are present on rabbit ileal enterocytes. Although we found specific binding both of radiolabeled opioid peptides to homogenates of cerebrum and ileal myenteric plexi and of 125I-VIP to ileal enterocytes, specific binding of radiolabeled opioid peptides to either ileal enterocytes or their homogenates was not identified. In parallel studies, tetrodotoxin, an inhibitor of neurotransmission, did not alter VIP-stimulation of Cl secretion but inhibited D-Ala2-methionine-enkephalinamide-induced electrolyte absorption. These studies suggest that opioid peptide stimulation of active Na and Cl absorption is mediated by an unidentified intermediary agonist.

Substrate and inhibitor specificity of anion exchangers on the brush border membrane of rabbit ileum.

In previous studies we have found that several anions can be transported by an exchange process in rabbit ileal brush border membranes. We demonstrated exchanges of C1 for OH or HCO3, SO4 for OH, oxalate for OH, and oxalate for C1. The purpose of these studies was to determine the number of distinct carriers mediating these exchanges. We utilized substrate and inhibitor specificity studies to distinguish between different anion exchange transporters. We conclude that SO4:OH and oxalate:OH exchange occur on the same carrier because: (i) pH-gradient stimulated transport of both 14C-oxalate and 35SO4 were equally sensitive to cis-inhibition by unlabeled SO4 or oxalate; and (ii) both were inhibited equally by K. We conclude that oxalate:OH and oxalate:C1 exchanges occur on different carriers because: (i) C1 or SO4 caused unequal cis-inhibition of these two exchanges; and (ii) as compared to oxalate:C1 exchange, oxalate:OH exchange was more sensitive to inhibition by probenecid and K and less sensitive to inhibition by bumetanide. Finally, we conclude that oxalate:C1 exchange and C1:HCO3 exchange occur on different carriers because: (i) C1:HCO3 exchange was almost completely insensitive to cis-inhibition by oxalate; and (ii) oxalate:C1 exchange was more sensitive to inhibition by DIDS and bumetanide than C1:HCO3 exchange. Thus we have found that there are at least three separate anion exchangers on rabbit ileal brush border: a Cl:HCO3 exchanger; a SO4:OH exchanger, which also transports oxalate; and an oxalate:C1 exchanger.

pH regulation in ileum: Na(+)-H+ and Cl(-)-HCO3- exchange in isolated crypt and villus cells.

Current evidence suggests that intestinal crypt and villus cells have different functions in electrolyte transport. To study the regulation of transporters, we isolated and separated these two cell types. This was accomplished by sequential collection of enterocytes from rabbit ileal loops incubated with buffered solutions of calcium chelators. Alkaline phosphatase and thymidine kinase activity, sodium-glucose cotransport, and morphological criteria were used to determine cell separation. Cell viability was evaluated with trypan blue exclusion, leucine incorporation into protein, and morphological features. The role of Na(+)-H+ and Cl(-)-HCO3- exchange in the regulation of intracellular pH was analyzed using an intracellular pH sensitive dye, BCECF. Removal of external Na+ or the addition of amiloride resulted in acidification of both crypt and villus cells. Removal of Cl- or the addition of DIDS resulted in alkalinization of both cell types. The cells could be acidified with NH4Cl, and recovery from this acid load was dependent on Na+ and inhibited by amiloride. Similarly, the cells could be alkalinized with propionate and recovery was Cl- dependent and DIDS sensitive. These data are consistent with the presence of Na(+)-H+ and Cl(-)-HCO3- exchange in both crypt and villus cells. Both exchanges appear to be involved in the regulation of basal pH as well as in recovery from alterations in intracellular pH. Having demonstrated the presence of Na(+)-H+ and Cl(-)-HCO3- exchange activity in both crypt and villus cells, we can now use these cells to determine the regulation of these exchangers by intracellular second messengers.

Mechanism of intestinal secretion: effect of cyclic AMP on rabbit ileal crypt and villus cells.

Cyclic AMP-dependent secretagogues such as cholera toxin inhibit the coupled absorption of Na+ and Cl- and stimulate the secretion of HCO3- and Cl- in the ileum. Aside from Cl- secretion, little is known about the mechanism of these cyclic AMP-mediated effects. We therefore determined the effect of forskolin, an agent known to increase intracellular cyclic AMP by stimulation of adenylyl cyclase, on Na+/H+ and Cl-/HCO3- exchange in isolated crypt and villus cells from rabbit ileum. Forskolin increased cyclic AMP in the villus cells and decreased intracellular pH. The effect of forskolin on pH in villus cells was HCO3- independent, Na+ dependent, and amiloride sensitive. Further, the rate of recovery from an acid load was decreased by forskolin. These data suggest that increasing cyclic AMP inhibits Na+/H+ exchange in villus cells. In crypt cells also, forskolin increased cyclic AMP; however, forskolin increased intracellular pH in these cells. The effect of forskolin in crypt cells was also HCO3- independent, Na+ dependent, and amiloride sensitive. However, the rate of recovery from an acid load was increased by forskolin, the opposite effect of that seen in villus cells. These data suggest that increasing cyclic AMP in crypt cells stimulates Na+/H+ exchange. Inhibition of Na+/H+ exchange on the brush border membrane in villus cells would be expected to inhibit coupled NaCl absorption (which occurs by coupling of Na+/H+ and Cl-/HCO3- exchange). Stimulation of Na+/H+ exchange in crypt cells, present only on the basolateral membrane, alkalinizes the cell, which would be expected to stimulate HCO3- secretion by stimulating the Cl-/HCO3- exchanger on the brush border membrane. Thus, these results provide a mechanism for some of the previously unexplained in vivo and in vitro effects of cyclic AMP on ileal electrolyte transport.

Characterization of Na(+)-H+ exchangers on villus cells in rabbit ileum.

The presence of Na(+)-H+ exchange activity is demonstrated on both the brush-border membrane (BBM) and the basolateral membrane (BLM) of villus cells from rabbit ileum. The possibility that the Na(+)-H+ exchange activity on the BLM represents HCO3- cotransport is excluded. The two Na(+)-H+ exchangers are then compared in terms of kinetics and substrate and inhibitor specificity. The most striking difference between the two exchangers was sensitivity to amiloride and K+. The IC50 for amiloride on the BLM was 10-fold lower than the BBM (11.2 +/- 2.1 vs. 103 +/- 20.9 microM; P less than 0.02). External K+, in concentrations as low as 10 mM, inhibited Na(+)-H+ exchange on the BBM but not on the BLM. The Na+ Km and proton Km were twice as high on the BLM exchanger (46.3 +/- 3.4 vs. 28.8 +/- 2.3 mM and 468 +/- 9 vs. 232 +/- 45 nM, respectively). Proton Vmax was similar, whereas Na+ Vmax was higher on the BLM. Inhibition by Li+ was similar on both membranes. These results indicate distinct differences between the two Na(+)-H+ exchangers. Whether these differences are due to the two different gene products or are the result of posttranslational modification of a single gene product remains to be determined.