Polyamines, vitamin D, and intestinal cells

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SELECTED SUMMARIES

POLYAMINES, VITAMIN INTESTINAL CELLS

GASTROENTEROLOGY Vol. 93, No. 2

D, AND

Shinki T, Takahashi N, Kadofuku T, Sato T, Suda T (Department of Biochemistry, School of Dentistry, and the Chemical Laboratory, School of Medicine, Showa University, Tokyo, Japan) Induction of spermidine Nl-acetyltransferase by la, .&dihydroxyvitamin D3 as an early event in the target tissues of vitamin D. J Biol Chem 1985;260: 2185-90 (February]. Earlier, this group of investigators reported that duodenal ornithine decarboxylase activity and the tissue content of putrescine was found to be increased markedly after a single intravenous injection of 1,25(QH)zD, into vitamin D-deficient chicks (Biochem J 1981;195:685-90). Of interest, however, in this regard was the subsequent finding by these investigators that prior administration of a-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, did not suppress the increase in putrescine levels in intestine by 1,25(OH),Ds. Thjs latter result suggested that the so-called “reverse pathway” for the synthesis of putrescine from spermine or spermidine, or both, might be involved in the 1,25(OH)zDs effect on putrescine levels. Therefore, in the present study, these investigators, using the same experimental system, examined the effect of 1,25(OH),D, on the activity of duodenal spermidine N’-acetyltransferase, a rate-limiting enzyme catalyzing the conversion of spermidine to putrescine. The latter enzymatic activity was found to increase within 30 min after a single injection of 625 ng of l,25(OH)2D3 apd attained a maximum level at 2 h. As little as 1.25 ng of 1,25(OH)2Ds induced a small but significant increase in enzymatic activity and the maximum response was seen with 125 ng of the vitamin. Furthermore, the dose of this hormone required to induce duodenal spermidine Nl-acetyltransferase activity was only one-tenth as much as that required to induce ornithine decarboxylase activity. Moreover, the former enzymatic activity was induced in several of the target tissues of vitamin D, whereas, ornithine decarboxylase activity was increased by the vitamin only in the intestine. Because certain target tissues do not appear to be involved in the regulation of mineral metabolism, these authors suggested that the increment of cellular putrescine induced by 1,25(OH)zDs was most likely related to cellular growth or differentiation, or both.

with their synthesis were found to be highest in mature cells along the villus axis of this organ. The findings of Shinki et al. now lend additional support to the contention that the polyamines may be involved in cellular differentiation in the intestine. The rgsults of their studies, however, raise another issue, which I believe is of interest to many gastroenterologists, i.e., are polyamines involved in the vitamin D-mediated effects on intestinal transcellular calcium transport? Transcellular calcium transport across intestinal epithelial cells appears to involve several different steps including (a) entry across the brush-border membrane into the cytoplasm, down a steep electrochemical gradient, via Ca’+ channels or carriers; (b) intracellular binding and transit through the cytoplasm, via Ca ‘+ binding proteins or intracellular organelles, or both; and (c) exit across the basolateral membrane, against a steep concentration gradient, via an adenosine triphosphate-dependent transport system, Ca2+-stimulated adenosine triphosphatase, and a Na+-Ca2+ exchanger (Am J Physiol 1986;250:G561-9). Based on studies from a number of laboratories (J Clin Invest 1985;76:2312-61, it would appear that 1,25(OH)*D3, the principal hormonal regulator of intestinal calcium transport, affects Ca2+ transport at each of these steps, but by different mechanisms. The studies by Shinki et al. are particularly interesting, as they demonstrate that the induction of spermidine N’-acetyltransferase activity by 1,25(OH),D3 occurs after only 30 min, i.e., is the earliest known example of a protein induced by this hormone, and temporally precedes the known action of 1,25(OH)ZD3 on intestinal Ca ‘+ transport. In this regard, recent studies by Lenzer et al. (J Biol Chem 1986;261:16478-83) have also demonstrated that certain polyamines, such as spermine, could act as physiologic activators of the mitochondrial Ca*+ uniporter in rat hepatic, heart, and brain cells. Taken together, these results suggest that polyamines might play a role in the action of 1,25(OH)2D3 on intestinal transcellular calcium transport. Further studies along these lines should prove interesting. T.A. BRASITUS,M.D.

Reply. We agree with the summary and comments of Dr. T. A. Brasitus. We would like to add the following points. Recently, we compared the relative contributions of ornjthine decarboxylase and spermidine N’-acetyltransferase in the 1,25(0H),D,-induced duodenal synthesis of putrescine (J Biol Chem 1986;261:11712-16). Prior administration of cu-difluoromethylornithine did not suppress the 1,25(0H),D,-induced increase in the duodenal content of putrescine, though it completely blocked ornithine decarboxylase activity. The enhancement of the duodenal content of putrescine coincided quantitatively with the amount of putrescine synthesized from spermidine but not from drnithine. This suggests that spermidine N’-acetyltransferase has a larger role than ornithine decarboxylase in the accumulation of duodenal putrescine induced by 1,25(OH)*D,. We also examined the distribution of spermidine N’-acetyltransferase and ornithine decarboxylase along the duodenal villus mucosa. The spermidine Comment. The aliphatic polyamines putrescine, spermidine, N’-acetyltransferase activity was distributed in a pattern opposite and spermine are natural constituents of many normal and tumor to that of the ornithine decarboxylase activity; the latter activity is cells (Ann Rev Biochem 1961;30:579-604). Although a large high in the crypt region, and the former activity is high in the number of studies have shown close correlations between the levels of the polyamines and cellular proliferation or differentiavillus region. These results suggest that putrescine, spermidine, tion, or both (Biochim Biophys Acta 1978;437:241-93), their exact and spermine appear to be sequentially synthesized from ornithine in the crypt cells. During the migration of the crypt cells physiologic role in these important processes has not been clearly toward the villus tip, putrescine is considered to be generated established (Ann Rev Biochem 1984;53:749-90). In the small intestine, for example, several groups of investigators have remainly from spermidine. At the present time, the biologic significance of cellular accucently questioned the role of ornithine decarboxylase or polymulation of putrescine in the vitamin D action is not known. amines, or both, in certain situations where cellular proliferation occurs (Biochim Biophys Acta 1978;541:415-19, 1982;716:439- Erwin et al. (Biochem Biophys Res Commun 1983;114:944) re42, Am J Physiol 1986;250:G709-13). The latter studies have ported that polyamine depletion inhibits in vitro differentiation of L-6 myoblast cells, and this inhibition is recovered almost comsuggested that the polyamines may, however, play a key role in pletely by adding putrescine. It is also possible that the decrease cellular differentiation in the intestine, as the enzymes associated

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in cellular spermidine is more critical than the increase in cellular putrescine in inducing vitamin D action. In fact, Casero et al. (J Cell Physiol 1984;121:476) have reported that the intracellular concentration of spermidine necessary for inducing proliferation of L-1210 cells is only 39% of its normal concentration. Therefore, it is likely that the increase in cellular putrescine or the decrease in cellular spermidine is related to growth or differentiation of intestinal cells, or both. How polyamines are involved in the 1,25-(OH),D,-induced stimulation of transcellular calcium transport across intestinal epithelial cells remains to be elucidated in future. T. SHINKI, Ph.D.

ON THE EXCITABILITY OF SMOOTH MUSCLE IN THE INFLAMED GUT Cohen ID, KCIO HW, Tan ST, Lechago J, Snape WJ Jr. (Departments of Medicine and Pathology, Harbor-University of California, Los Angeles Medical Center, Torrance, California) Effect of acute experimental colitis on rabbit colonic smooth muscle. Am J Physiol 1986; 251:G538-45 (October). Contractions of the colon are impaired in ulcerative colitis. Eating a meal, for instance, does not lead to an increase in spike potentials and of contractions as it does in healthy subjects. To determine whether inflammation affects the electrophysiologic properties of muscle cell membranes, the authors studied in vitro preparations from an experimental model of colitis. Colitis was produced in rabbits by the administration of formalin into the sigmoid colon and of serum albuminantigen complexes intravenously. The resulting immune complex colitis caused diarrhea in all animals. On histologic examination, there was edema and a dense infiltrate of granulocytes, many of which were eosinophilic. The inflammatory reaction involved the mucosa and submucosa but did not extend into the muscularis propria. Tissues were studied 4-6 days after the induction of colitis. The mechanical and electrophysiologic properties of the smooth muscle were studied in strips of muscle cut from the circular muscle layer and attached to force transducers in a tissue chamber. Strips were stretched in steps and their force development was measured. The tension generated by colonic muscle from animals with colitis was only about half that of normal controls. Membrane potential and membrane resistance were measured using the double sucrose gap and intracellular microelectrodes. The muscle membrane of animals with colitis generated a lower electrochemical potential than normal (-38 +- 2 mV vs. -52 rf: 1 mV in controls). Action potentials

occurred

more

frequently

mals with colitis, and potentials

in muscle

from

ani-

were of smaller amplitude

(48 * 5 cyclesimin of 8 t 0.2 mV spikes vs. 22 ? 2 cyclesimin of 11 t 0.7 mV spikes in controls). Membrane resistance was evaluated by exposing the muscle in the sucrose gap to pulses of depolarizing and hyperpolarizing current. Membrane potential for a given current was less in colitis, and so were the voltage response and the time constants. Depolarizing current

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pulses evoked spike potentials and contractions. The maximum rate of rise of evoked potentials was decreased in the strips from the animals with colitis, and no spike potentials occurred. These measurements suggest that colitis decreases the membrane resistance. The decrease in membrane resistance demonstrated here suggests an increased permeability of the cell membrane for ionic movements, but the present studies have not defined which specific ions are affected. The decreased amplitude of the spontaneous spike potentials and decreased rise rate of evoked potentials suggest that experimental colitis leads to a decrease in Ca2+ influx. Abnormalities of the membrane-bound Na+, K+-stimulated adenosine triphosphatase are unlikely to account for the alterations in membrane potential: smooth muscle from an animal with colitis was hyperpolarized after withdrawal of K+ and reexposure to K+ like normal smooth muscle. How inflammation changes the excitable characteristics of colonic smooth muscle without morphologic lesions involving the muscle proper remains obscure. One possibility is that autocoids released as part of the inflammatory response mediate the membrane abnormalities: prostaglandin synthesis in colonic smooth muscle is stimulated by colitis, and it is possible that the findings relate to the inhibitory action of prostaglandin E,. Comment. Motility disturbances are common with mucosal inflammation anywhere in the gut. Nausea, cramps, and diarrhea are as readily explained by alterations in muscular function as by alterations in epithelial function. Esophagitis lowers the lower esophageal sphincter pressure and the amplitude of contraction 1986;91: waves in the esophageal body (Gastroenteroiogy 897-904, 1975: 69:146-53, 1986;90:1566). Abnormal motility is a more sensitive index of esophagitis than are structural changes of the mucosa [Invest Radio1 1970;5:269-19). The radiographic diagnosis of chronic ulcerative colitis involves the disappearance of colonic haustra (i.e., a lack of contraction of circular muscle] in addition to the discovery of epithelial defects. Gastric motility and emptying are abnormal in gastritis. Experimental models of mucosal inflammation, whether the mucosal injury is caused by corrosion, radiation, or an immune response, are accompanied by profound alterations of gut motility. In experimental models and clinical disease alike, morphologic injury of the muscle coat is striking by its paucity: inflammatory cells and vascular changes, however prominent in the mucosa, do not prominently extend through the submucosa into the muscle coat (Fed Proc 1984;43:476). Accustomed to define disease states by the morphologic lesions they cause, most workers assume that changes in contractions in the inflamed gut must be mediated by injury elsewhere: healthy in themselves, muscle cells misbehave because of faulty signals. Accordingly, much work has focused on the release of autocoids by inflammation and their role in altered gut motility. Increased prostaglandin activity occurs in radiation esophagitis and enteritis, and the motility disturbances can be partially prevented by pretreatment with prostaglandin inhibitors. As pretreatment also decreases mucosal injury, it is not clear that the prostaglandins themselves are responsible for altered smooth muscle function (Gastroenterology 1980;78:833-97, Dig Dis Sci 1982; 27:923-8). The present paper demonstrates that what one sees depends on the methods with which one looks: profound changes in the excitable characteristics were demonstrated in a tissue that looked normal on routine histologic examination. How insensitive rou-