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Experimental diabetic diarrhea in rats
GASTROENTEROLOGY 1986;91:564-9
Experimental Diabetic Diarrhea in Rats Intestinal Mucosal Denervation Hypersensitivity and Treatment With Clonidine EUGENE B. CHANG, RICHARD N. FEDORAK, and MICHAEL FIELD Division of Gastroenterolonv, Department of Medicine, Physicians & Surgeons, New York, New York
Diarrhea in streptozocin-induced chronically diabetic rats is caused by an impaired adrenergic regulation of intestinal fluid and electrolyte transport. Stimulation of az-adrenergic receptors on enterocytes normally promotes NoCi absorption and inhibits HC03 secretion. The purpose of this study was to determine (a) if adrenergic denervation of intestinal mucosa in chronically diabetic rats alters postsynaptic receptor response, and (b) if the az-adrenergic agonist clonidine could correct observed fluid malabsorption. Mucosal norepinephrine stores, a measure of adrenergic tone, were markedly reduced in diabetic rats compared with nondiabetic littermates. In vitro, short-circuit current changes to exogenously hdded I-epinephrine were significantly greater in dicibetics, suggesting that denervation hypersensitivity was due to increased numbers of postsynaptic a,-adrenergic receptors. In vivo loop studies demonstrated net fluid secretion in the ileum and colon of diabetics. In diabetics, clonidine reversed the secretion to absorption, but it had no effect on fluid absorption in controls. We conclude that diabetic diarrhea in streptozocin-induced chronically diabetic rats is [a) due to impaired adrenergic regulation of mucosal ion transport, [b) accompanied by a postsynaptic denervation hypersensitivity that can be reversed by clonidine, and (c) accompanied by net intestinal fluid secretion that can be effectively reversed with clonidine. Received August 28, 1985. Accepted February 22, 1988. Address requests for reprints to: Eugene B. Chang, M.D., Columbia University College of Physicians & Surgeons, 630 West 188th Street, New York, New York 10032. Dr. Chang is a recipient of a New Investigator Research Award, the John A. and George L. Hartford Fellowship, and the AGA/ Hoffman-La Roche Research Scholar’s Award. Dr. Fedorak is a recipient of the Alberta Heritage Foundation for Medical Research Fellowship. This work was supported by grants AM-35382 and AM-35096. 0 1986 by the American Gastroenterological Association 0016-5085I861S3.50
Columbia University
College of
Diarrhea can be one of the most troublesome gastrointestinal cpmplications in diabetes mellitus. Patients with this problem are usually insulin-dependent, have autonomic neuropathy, and have little or no evidence of nutrient malabsorption or enteropathy. Frequently, the diarrhea is intermittent, sometimes alternating with constipation, but it can be persistent and severe. Nocturnal fecal incontinence is common, and treatment with conventional antidiarrheal agents and antibiotics is of limited use. Recently, impaired adrenergic regulation of intestinal fluid and electrolyte transport has been suggested as the pathophysiologic basis for diabetic diarrhea (1). Streptozocin-induced, chronically diabetic rats were found to have intestinal adrenergic neuropathy and impaired electrolyte absorption in the ileum and colon, a finding also present in nondiabetic rats sympathectomized with 6-hydroxydopamine. Morphologic studies have also shown impairment of adrenergic innervation of the intestine in chronically diabetic rats (2,3). Clonidine, a specific crz-adrenergic receptor agonist, enhances mucosal absorption of fluid and electrolytes and inhibits anion secretion (4). Its potential usefulness in correcting the pathogenic defect in diabetic diarrhea was recently investigated in 3 diabetic patients with severe diabetic diarrhea that was refractory to conventional antidiarrheal therapies. They experienced a significant decrease in stool volume and frequency when using clonidine, which worsened again when the medication was withdrawn and which diminished again when clonidine was reinstituted (5). Although clonidine was effective in these patients, explanations for both the diarrhea and its response to clonidine remain somewhat speculative. SimilarAbbreviation current.
used in this paper: I,,, transmural
short-circuit
September
CLONIDINE IN DIABETIC DIARRHEA
1986
ly, in streptozocin-induced diabetic rats, which may be a good model for diabetic diarrhea in humans, the effectiveness of clonidine in enhancing fluid absorption in vivo had not been tested. In the present study we have examined the effect of clonidine on in vivo fluid absorption in streptozocin-treated and control rats. Through parallel in vitro experiments, we examined evidence of intestinal denervation hypersensitivity.
Materials and Methods Materials Clonidine [2-(2,6-dichloroanilino)-2-imidazoline] was purchased from Sigma Chemical Co., St. Louis, MO.;
streptozocin was purchased from the Upjohn Company, Kalamazoo, Mich.; L-epinephrine was purchased froin Parke-Davis, Morris Plains, N.J.; and ketamin&% acepromazine was purchased from Veterinary Products Laboratories, Syracuse, N.Y. Remaining chemicals were reagent grade, from Sigma Chemical Co.
isolating the loops, care was taken not to compromise mesepteric, vascular, or neuronal continuity. A 27-gauge needle was inserted obliquely through the outer muscle layer along the antimesenteric border, and 2 ml of 0.9% NaCl, prewarmed to 37”C, was instilled into each empty loop. No fluid leakage was detected and loops were only mildly distended. The viscera were returned to the abdominal cavity and the incision was closed. The rats were allowed to recover from the anesthesia. Sixty minutes after abdominal closure, animals were killed by ether overdose, and gut loops were removed. The length of each loop was measured, and the loops were weighed full and emptied to determine the residual intraluminal volume. The loops were then dried in a 50°C oven for 24 h. Results were expressed as the difference between initial and residual luminal loop volume per gram dry weight or length [in centimeters) of bowel. Treatment with clonidine. Clonidine was dissolved in water (20 mgiL) on the day of injection and administered by subcutaneous injection (0.02 mg/kg) daily for 3 successive days. In vivo and in vitro measurements were carried out 12 h after the last injection of clonidine. In Vitro
Induction
of Diabetes
Diabetes was induced in adult male Lewis rats (250-275 g) by injection of streptozocin (50 mgikg), dissolved in buffered saline (pH 7.4), into the dorsal tail vein. Diabetic and age-matched controls were housed in a lightcycled room with free access to standard pelleted rat diet and water. The presence of diabetes was established by blood glucose determinations both 4 days after administration of streptozocin and on the day animals were killed. A reflectance photometer and glucose reagent strips (Ames Division, Miles Laboratories Inc., Elkhart, Ind.) were used to make these determinations. Chronic diabetes was defined as hyperglycemia levels >300 mg/dl for 6 mo.
In Vivo Studies Measurement of intestinal fluid absorption. Nonfasting rats were anesthetized with ketamine-4% acepromazine (30 mgikg, subcutaneously) and kept warm with a thermostatic heat lamp. The intestinal tract was exposed through a midline abdominal incision. An occluding ligature was placed at the ligament of Treitz and ileocecal valve. Incisions through the gut wall were made distal and proximal to the ligatures, respectively. A cannula was inserted through the proximal incision, and the luminal contents were flushed through the distal incision with warm 0.9% NaCl. The colon was handled in a similar manner: the cecal-colonic junction was ligated and the luminal contents were flushed out the rectum by a cannula placed through an incision just distal to the proximal occluding ligature; residual saline was emptied by gentle manual expression. Three intestinal loops of -15-cm length were created with ligatures in the jejunum (beginning 2 cm distal to the ligament of Treitz), in the ileum (beginning 2 cm proximal to the ileocecal valve), and in the colon (beginning 2 cm below the cecal-colonic junction and extending distally to the peritoneal reflection]. In
565
Studies
In vitro studies were performed on ileum that was directly proximal to the ileal loop. These tissues were remdved immediately after the animal was killed, cut open along the mesenteric border, and incubated with a solution of the following ionic composition (mM): Na, 143; K, 5: Mg, 1.1; Ca, 1.25; Cl, 124; HC03, 25; HPO,, 1.65; HzP04, 0.3; and fructose, 40. The pH was 7.4 when gassed with 5% CO, in 95% O2 at 37°C. Ileal segments were placed serosal-side-facing-up on a plexiglass dissecting board, and were stripped of their serosa and underlying longitudinal muscle layer using a flat-edged dissecting forceps. Mucosal strips were then mounted in Ussing chambers, and transmural electric potential difference, resistance, and short-circuit current (I,,) were determined as previously described (6). Response to tyramine. Norepinephrine stores in ileal mucosa were estimated by I,, response to the serosal addition of tyramine (10 PM). Tyramine effectively releases 80% of all stored norepinephrine (7). Theophylline (1 mM) was added to the serosal reservoir 15 min before the addition of tyramine to stimulate the tissues to secrete, thus maximizing the I,, response to tyramine. Pargyline (50 ELM),a monoamine oxidase inhibitor, was added to the serosal reservoir 10 min before tyramine to prevent tyramine metabolism (7). After the response to tyramine, o-glucose was added to both reservoirs, and the resulting I,, was determined as a measure of tissue viability. In no instance was this ~20 PA/cm”. Epinephrine dose-response relationship. Transmural short-circuit current responses to accumulative additions of I-epinephrine were determined in ileal mucosa of clonidine-treated and untreated chronic diabetic and control rats. The concentration of epinephrine producing a response that was 50% of maximal was estimated in each set of tissues in which I,, responses to accumulative additions were determined. Results were then subjected to Student’s t-test analysis for unpaired variables.
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GASTROENTEROLOGY Vol. 91. No. 3
CHANGET AL.
Results Animal
Groups
Two groups of chronically diabetic rats were studied: one group received no treatment and the other was treated with clonidine. Littermates of each group served as controls. All chronically diabetic rats had persistent hyperglycemia, failure to gain weight (see Table l), polyuria, and persistent watery diarrhea. Although stool and urine could not be separated for independent quantitations, the diarrhea in the clonidine-treated chronically diabetic rat group appeared to improve, as assessed by the stool consistency and degree of fecal soilage in each cage. Intestinal
Fluid Absorption
In Vivo
As shown in Figure 1, control rats had a net fluid absorption in jejunum, ileum, and colon. Untreated chronically diabetic rats, however, exhibited net fluid secretion in ileum and colon, but not in jejunum. This was the case whether results were expressed as microliters per gram dry weight (data not shown) or as microliters per length (in centimeters) of bowel (Figure 1).These results are in agreement with our previous observations (l), with the exception that net secretion had not previously been noted in the colon of chronically diabetic rats. Treatment of chronically diabetic rats with clonidine reversed the net fluid secretion in ileum and colon to net fluid absorption (Figure 1). This dose of clonidine did not, however, completely correct fluid malabsorption in chronically diabetic rats, and clonidine at this dose did not alter basal fluid absorptive rates in control rats or increase fluid absorption in the jejunum of chronically diabetic rats. Ileal Transmural Measurements
Short-Circuit
Current
; Figure
1.
rcolO”{
1. Effect of clonidine treatment (closed bars) on in vivo intestinal fluid absorption per centimeter in control and diabetic animals. Location of jejunum, ileum, and colon is as described in Materials and Methods. Values are mean f 1 SEM for 5 rats.
follow I,, alone rather than attempt to measure changes in net fluxes of individual ions. Transmural short-circuit current changes to catecholamines reflect an inhibition of electrogenic anion secretion (8). In the presence of theophylline, decreases in I,, stimulated by these agents are caused by an inhibition of active chloride secretion (8). Transmural short-circuit current responses to theophylline (1 mM) were the same in chronically diabetic rats and littermate, nondiabetic controls (59 + 5 vs. 69 + 7 pAlcm2). The I,, response to tyramine in chronically diabetic rats was 29 2 6 pAlcm2, significantly less than the 48 + 6 PA/cm2 response in controls (n = 5, p < 0.05). The response relationship between the concentration of exogenously added I-epinephrine and I,, is shown in Figure 2. There was no apparent difference in the EDso for I-epinephrine, i.e., the concentration stimulating 50% of the maximal effect, in the ileum of control and treated and untreated chronically diabetic rats. The EDso values for each group were as follows (in nanomoles per liter): control, 12.6 + 2.6 (n = 6); control (clonidine treated), 26.8 t 9.6 (n = 8); chronically diabetic rats, 10.4 k 1.2 (n = 10); and chronically diabetic rats
As Na, Cl, and HC03 transport responses to theophylline and I-epinephrine have been well characterized in vitro and correlate well with oppositely directed I,, reponses to these agents, we elected to
Table
{}
8o/
100 ??
Dlab&c/untreated
A Control/untreated
0 Dmbetlc treated ~Control treated
Animal Group Profile
Group Chronic diabetes Clonidine-treated Untreated Control Clonidine-treated Untreated
Body weight
Blood glucose
(g)
(mg/dl)
5 5
255 2 16 235 " 5
483 2 17 417 t 36
5 5
440 f 14 445 k 7
79 +- 10 85 2 8
n
n, number of rats. Values are mean T SEM.
0
’6*T?“’
9
1
6
5
Epmephrlne HogM)
Figure
2. Effect of varying doses of I-epinephrine on in vitro intestinal short-circuit current (1.J determinations. Diabetic and control untreated groups (closed circles, closed triangles, respectively) are compared with the diabetic and control clonidine-treated groups (open circles, open triangles, respectively). Values are for n 2 6 animals for all groups.
September 1986
(clonidine treated), 11.3 _+ 2.5 (n = 12).There was, however, a significant increase (p < 0.05) in the maximal I,, response to lop5 M I-epinephrine in untreated chronically diabetic rats compared with controls (68 -+ 8 vs. 45 + 10 PA/cm’, respectively). As we had previously noted (l), the I,, response to I-epinephrine was inhibited by the az-antagonist yohimbine (10U5 M) but not by the P-receptor antagonist propanalol (10m5 M) or the al-antagonist prazosin (lob5 M). Treatment of chronically diabetic rats with clonidine decreased the maximal I,, response so that it was no different from controls. Clonidine did not alter basal I,, in untreated or treated chronically diabetic rats (6.9 5 1.2 vs. 11 + 2.1 pAlcm2, respectively) or in untreated or treated control rats (13.8 +- 2.9 vs. 19.2 2 4.7 PA/cm’, respectively). Although not shown in Figure 2, clonidine treatment had no effect on the I,, responses to exogenously added I-epinephrine in nondiabetic controls.
Discussion Adrenergic innervation has a major role in the regulation of fluid and electrolyte transport in the intestinal tract. In vitro stimulation of postsynaptic cu,-adrenergic receptors on ileal enterocytes causes an active absorption of Na and Cl and inhibits active secretion of Cl and HCO, (8). These effects have been shown by Tapper et al. (7) to also occur when norepinephrine is released from endogenous stores in rabbit ileal mucosa by tyramine. Wu and Gaginella (9) demonstrated the presence in rat colonic mucosa of a functional noradrenergic neural network associated with the epithelium. Newsome et al. (10) have shown electron-micrographic evidence of actual synapses between putative noradrenergic fibers and basal granulated epithelial (crypt) cells. Autonomic neuropathy and, in particular, adrenergic neuropathy, is a frequent complication of chronic diabetes mellitus. Hensley and Soergel (11) reported lesions in the dendritic processes of diabetic prevertebral and paravertebral sympathetic ganglia. Schmidt et al. (2) have demonstrated, in rats made chronically diabetic with streptozocin, histologic evidence of visceral nerve destruction and biochemical evidence of cholinergic as well as adrenergic denervation. More recently, Lincoln et al. (3) suggested that there might be a selective destruction of adrenergic nerves of the myenteric plexus of streptozocin-treated diabetic rats. Impaired adrenergic regulation of mucosal fluid and electrolyte transport can, therefore, alter the homeostatic balance exerted by neuroendocrine elements to favor net secretion. This increased secretory tone could also be due to marked changes in the
CLONIDINEIN DIABETICDIARRHEA 567
pattern of innervation by nerve fibers to the intestinal mucosa. For example, cholinergic and vasoactive intestinal polypeptide-like immunoreaction innervation to the submucosa and myenteric plexus appear to be increased in the ileum and proximal colon of streptozocin-treated, chronically diabetic rats (3,12). Basal secretion would be increased because of the release of these neurosecretagogues, in the absence of adrenergic tone, to the intestinal mucosa. In support of this contention, recent observations in chronically diabetic rats showed impaired absorption of fluid by the ileum and colon (1). These chronically diabetic rats also showed depletion of mucosal norepinephrine stores as estimated in vitro by the I,, response to tyramine. After sympathetic denervation with 6-hydroxydopamine, the pattern and magnitude of impaired fluid absorption in nondiabetic rats was the same as that observed in chronically diabetic rats. It was concluded that impaired intestinal mucosal absorption of fluid and electrolytes develops slowly in rats made diabetic with streptozocin, and that this absorptive impairment is due to a loss of normally present noradrenergic innervation of enterocytes. The present studies confirm our previous study, except that frank net secretion was observed in both the ileum and colon of chronically diabetic rats. A possible explanation for this difference is that the chronically diabetic rats in this study were anesthetized with ketamine, a dissociative anesthetic, rather than ether. As a consequence, rats (particularly chronically diabetic rats) were better able to tolerate abdominal surgery and were hemodynamically more stable. In our previous study we noted a significant increase in the I,, response to exogenously added I-epinephrine in jejunum of chronically diabetic rats, suggesting the possibility of postsynaptic denervation hypersensitivity. This was not noted in ileum, where the I,, responses were more variable. To explore this further, we reexamined the effects of exogenously added I-epinephrine on I,, in the ileum. The dose-response relationship shown in Figure 2 suggests that postsynaptic hypersensitivity is also present in ileum of chronically diabetic rats. There was no change in the EDs0 to I-epinephrine, suggesting that receptor affinity was not altered, but a significant increase in the I,, response to maximal concentrations of I-epinephrine was noted. It is unlikely that this increase was due to mucosal hypertrophy, as I,, responses to theophylline were no different in chronically diabetic rats than in controls. Basal I,, was unaffected by clonidine treatment. Therefore, it is very likely that any residual clonidine bound to receptors was washed out during the preparation and preincubation periods. This
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GASTROENTEROLOGY Vol. 91. No. 3
CHANG ET AL.
assumption is supported by the finding that yohimbine had no effect on basal I,, in tissues from treated or untreated chronically diabetic rats. These results suggest that postsynaptic denervation hypersensitivity may be a consequence of increased numbers of cr2-adrenergic receptors on enterocytes. Similar findings have been reported in other tissues. Denervation produces a moderate increase in postsynaptic cu2-adrenergic receptor density in membranes prepared from whole rat submandibular glands (IS), without a significant increase in binding affinity. Denervation with intraventricular injection of 6-hydroxydopamine also increased postsynaptic a2-receptor density, but not binding affinity, in rat cerebral cortex (l&15). The denervation hypersensitivity of the ileum of chronically diabetic rats can be corrected by treatment with the a2-adrenergic agonist clonidine. The implications of these findings are that postsynaptic denervation hypersensitivity is a reversible adaptation to diminished adrenergic tone. Clonidine may therefore be potentially useful in the treatment of diabetic diarrhea. In normal subjects, clonidine appears to enhance intestinal fluid and electrolyte absorption by an enteropooling effect and by a direct action on mucosal function (5,16). We previously reported 3 cases in which patients with severe diabetic diarrhea responded to oral therapy with clonidine (5). Goff (17) has reported a similar experience using the a2-adrenergic agonist lidamidine. In chronically diabetic rats, clonidine, at a dose nearly equivalent in milligrams per kilogram body weight to that used in patients with diabetic diarrhea, reversed net fluid secretion in ileum and colon to net absorption. Clonidine treatment was continued for 3 days in order to reach a steady-state serum concentration. This dose of clonidine did not, however, alter basal absorptive rates in control rats. In rats, this dose appears to be well below the EDS0 (0.06-0.12 mg/kg) for clonidine to have an effect on stimulated intestinal secretion (17-19). However, with the techniques employed in this study to measure intestinal fluid absorption, small changes of
cal sympathectomy with 6-hydroxydopamine (1). Furthermore, the decrease in I,, response to the in vitro addition of I-epinephrine was smaller in the jejunum than in the colon and ileum. Diarrhea in streptozocin-induced, chronically diabetic rats appears to be a good model for the study of diabetic diarrhea in humans. Although these animals uniformly develop diarrhea, wherereas most patients have intermittent diarrhea, we think the difference may be attributable to the fact that chronically diabetic rats have diabetes for prolonged periods without the benefit of treatment with insulin. This circumstance is rarely encountered in a patient population. Intractable diarrhea in these rats most likely represents a predominantly adrenergic rather than cholinergic neuropathy. Hence, the impairment of adrenergic innervation of the intestinal mucosa shifts neuroendocrine regulation of ion transport in favor of net secretion. The present studies demonstrate that this impairment of adrenergic innervation of the intestinal epithelium causes a denervation hypersensitivity of postsynaptic a2-adrenergic receptors. This alteration in receptor responses appears to be a result of increased receptor numbers rather than of any change in receptor-binding affinity for catecholamines. CQAdrenergic agonists in this model also appear to have a role in reversing intestinal secretion and the observed a2-receptor denervation hypersensitivity, suggesting potential clinical utility of these agents for the treatment of diabetic diarrhea.
References 1.
Chang EB, Bergenstal RM, Field M. Diarrhea in streptozocintreated rats. J Clin Invest 1985;75:1666-70. 2. Schmidt RE, Nelson JS, Johnson EM. Experimental diabetic autonomic neuropathy. Am J Path01 1981;103:210-25. 3. Lincoln J, Bokor JT, Crowe R, Griffith SG, Haven AJ, Burnstock G. Myenteric plexus in streptozocin-treated rats. Neurochemical and histochemical evidence for diabetic neuropathy in the gut. Gastroenterology 1984;86:654-61. 4. Durbin T, Rosenthal L, McArthur K, Anderson D, Dharmsathaphorn K. Clonidine and lidamidine stimulate sodium and chloride absorption in the rabbit intestine. Gastroenterology 1982;82:1352-8. 5. Fedorak RN, Field M, Chang EB. Treatment of diabetic diarrhea with clonidine. Ann Intern Med 1985;102:197-9. 6. Field M, Fromm D, McCall I. Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current. Am J Physiol 1971;220:1388-94. 7. Tapper EJ. Bloom JS, Lewand DL. Endogenous norepinephrine release induced by tyramine modulates intestinal ion transport. Am J Physiol 1981;229:86-92. a. Field M, McCall I. Ion transport in rabbit ileal mucosa. III. Effects of catecholamines. Am J Physiol 1973;225:852-7. 9. Wu ZC, Gaginella TS. Functional properties of noradrenergic nervous system in rat colonic mucosa: uptake of [3H] norepinephrine. Am J Physiol 1981;241:G13742. 10. Newsome B, Ahlman H, Dahlstrom A, Das Gupta TK, Nyhus
September
11. 12.
13.
14.
1986
LM. On the innervation of the ileal mucosa in the rat - a synapse. Acta Physiol Stand 1979;105:387-9. Hensley GT, Soergel KH. Neuropathological findings in diabetic diarrhea. Arch Path01 1968;85:587-97. Belai A, Lincoln J, Mulner P, Crow R, Loesch A, Burnstock G. Enteric nerves in diabetic rats: increase in vasoactive intestinal polypeptide but not substance P. Gastroenterology 1985;89:967-76. Arnett CD, Davis JN. Denervation-induced changes in alpha and beta adrenergic receptors of the rat submandibular gland. J Pharmacol Exp Ther 1979;211:394-400. U’Prichard CD, Bechtel WD, Ronuot BM, Snyder SH. Multiple apparent alpha-noradrenergic receptor binding sites in rat brain: effect of 6-hydroxydopamine. Mol Pharmacol 1979; 16:47-60.
CLONIDINE IN DIABETIC DIARRHEA
569
15. Dausse JP, LeQuan-bui KH, Meyer P. Alpha-l and alpha-2 adrenoreceptors in rat cerebral cortex: effects of neonatal treatment with 6-hydroxydopamine. Eur J Pharmacol 1982; 78:15-20. 16. Schiller LR, Santa Ana CA, Morawski SG, Fordtran JS. Effect of clonidine on experimental diarrhea in humans (abstr]. Gastroenterology 1984;86:1235. 17. Goff JS. Diabetic diarrhea and lidamidine. Ann Intern Med 1984;101:874-5. 18. Nakaki T, Chang PC, Tokunaga Y, Kato R. Alpha,adrenoceptors modulating diarrhoea in morphine-dependent rats. J Pharm Pharmacol 1981;33:397-9. 19. Spraggs, CF, Bunce KT. Alpha,-adrenoceptors and the delay of castor oil-induced diarrhoea by clonidine in rats. J Pharm Pharmacol 1983;35:321-2.
Experimental Diabetic Diarrhea in Rats Intestinal Mucosal Denervation Hypersensitivity and Treatment With Clonidine EUGENE B. CHANG, RICHARD N. FEDORAK, and MICHAEL FIELD Division of Gastroenterolonv, Department of Medicine, Physicians & Surgeons, New York, New York
Diarrhea in streptozocin-induced chronically diabetic rats is caused by an impaired adrenergic regulation of intestinal fluid and electrolyte transport. Stimulation of az-adrenergic receptors on enterocytes normally promotes NoCi absorption and inhibits HC03 secretion. The purpose of this study was to determine (a) if adrenergic denervation of intestinal mucosa in chronically diabetic rats alters postsynaptic receptor response, and (b) if the az-adrenergic agonist clonidine could correct observed fluid malabsorption. Mucosal norepinephrine stores, a measure of adrenergic tone, were markedly reduced in diabetic rats compared with nondiabetic littermates. In vitro, short-circuit current changes to exogenously hdded I-epinephrine were significantly greater in dicibetics, suggesting that denervation hypersensitivity was due to increased numbers of postsynaptic a,-adrenergic receptors. In vivo loop studies demonstrated net fluid secretion in the ileum and colon of diabetics. In diabetics, clonidine reversed the secretion to absorption, but it had no effect on fluid absorption in controls. We conclude that diabetic diarrhea in streptozocin-induced chronically diabetic rats is [a) due to impaired adrenergic regulation of mucosal ion transport, [b) accompanied by a postsynaptic denervation hypersensitivity that can be reversed by clonidine, and (c) accompanied by net intestinal fluid secretion that can be effectively reversed with clonidine. Received August 28, 1985. Accepted February 22, 1988. Address requests for reprints to: Eugene B. Chang, M.D., Columbia University College of Physicians & Surgeons, 630 West 188th Street, New York, New York 10032. Dr. Chang is a recipient of a New Investigator Research Award, the John A. and George L. Hartford Fellowship, and the AGA/ Hoffman-La Roche Research Scholar’s Award. Dr. Fedorak is a recipient of the Alberta Heritage Foundation for Medical Research Fellowship. This work was supported by grants AM-35382 and AM-35096. 0 1986 by the American Gastroenterological Association 0016-5085I861S3.50
Columbia University
College of
Diarrhea can be one of the most troublesome gastrointestinal cpmplications in diabetes mellitus. Patients with this problem are usually insulin-dependent, have autonomic neuropathy, and have little or no evidence of nutrient malabsorption or enteropathy. Frequently, the diarrhea is intermittent, sometimes alternating with constipation, but it can be persistent and severe. Nocturnal fecal incontinence is common, and treatment with conventional antidiarrheal agents and antibiotics is of limited use. Recently, impaired adrenergic regulation of intestinal fluid and electrolyte transport has been suggested as the pathophysiologic basis for diabetic diarrhea (1). Streptozocin-induced, chronically diabetic rats were found to have intestinal adrenergic neuropathy and impaired electrolyte absorption in the ileum and colon, a finding also present in nondiabetic rats sympathectomized with 6-hydroxydopamine. Morphologic studies have also shown impairment of adrenergic innervation of the intestine in chronically diabetic rats (2,3). Clonidine, a specific crz-adrenergic receptor agonist, enhances mucosal absorption of fluid and electrolytes and inhibits anion secretion (4). Its potential usefulness in correcting the pathogenic defect in diabetic diarrhea was recently investigated in 3 diabetic patients with severe diabetic diarrhea that was refractory to conventional antidiarrheal therapies. They experienced a significant decrease in stool volume and frequency when using clonidine, which worsened again when the medication was withdrawn and which diminished again when clonidine was reinstituted (5). Although clonidine was effective in these patients, explanations for both the diarrhea and its response to clonidine remain somewhat speculative. SimilarAbbreviation current.
used in this paper: I,,, transmural
short-circuit
September
CLONIDINE IN DIABETIC DIARRHEA
1986
ly, in streptozocin-induced diabetic rats, which may be a good model for diabetic diarrhea in humans, the effectiveness of clonidine in enhancing fluid absorption in vivo had not been tested. In the present study we have examined the effect of clonidine on in vivo fluid absorption in streptozocin-treated and control rats. Through parallel in vitro experiments, we examined evidence of intestinal denervation hypersensitivity.
Materials and Methods Materials Clonidine [2-(2,6-dichloroanilino)-2-imidazoline] was purchased from Sigma Chemical Co., St. Louis, MO.;
streptozocin was purchased from the Upjohn Company, Kalamazoo, Mich.; L-epinephrine was purchased froin Parke-Davis, Morris Plains, N.J.; and ketamin&% acepromazine was purchased from Veterinary Products Laboratories, Syracuse, N.Y. Remaining chemicals were reagent grade, from Sigma Chemical Co.
isolating the loops, care was taken not to compromise mesepteric, vascular, or neuronal continuity. A 27-gauge needle was inserted obliquely through the outer muscle layer along the antimesenteric border, and 2 ml of 0.9% NaCl, prewarmed to 37”C, was instilled into each empty loop. No fluid leakage was detected and loops were only mildly distended. The viscera were returned to the abdominal cavity and the incision was closed. The rats were allowed to recover from the anesthesia. Sixty minutes after abdominal closure, animals were killed by ether overdose, and gut loops were removed. The length of each loop was measured, and the loops were weighed full and emptied to determine the residual intraluminal volume. The loops were then dried in a 50°C oven for 24 h. Results were expressed as the difference between initial and residual luminal loop volume per gram dry weight or length [in centimeters) of bowel. Treatment with clonidine. Clonidine was dissolved in water (20 mgiL) on the day of injection and administered by subcutaneous injection (0.02 mg/kg) daily for 3 successive days. In vivo and in vitro measurements were carried out 12 h after the last injection of clonidine. In Vitro
Induction
of Diabetes
Diabetes was induced in adult male Lewis rats (250-275 g) by injection of streptozocin (50 mgikg), dissolved in buffered saline (pH 7.4), into the dorsal tail vein. Diabetic and age-matched controls were housed in a lightcycled room with free access to standard pelleted rat diet and water. The presence of diabetes was established by blood glucose determinations both 4 days after administration of streptozocin and on the day animals were killed. A reflectance photometer and glucose reagent strips (Ames Division, Miles Laboratories Inc., Elkhart, Ind.) were used to make these determinations. Chronic diabetes was defined as hyperglycemia levels >300 mg/dl for 6 mo.
In Vivo Studies Measurement of intestinal fluid absorption. Nonfasting rats were anesthetized with ketamine-4% acepromazine (30 mgikg, subcutaneously) and kept warm with a thermostatic heat lamp. The intestinal tract was exposed through a midline abdominal incision. An occluding ligature was placed at the ligament of Treitz and ileocecal valve. Incisions through the gut wall were made distal and proximal to the ligatures, respectively. A cannula was inserted through the proximal incision, and the luminal contents were flushed through the distal incision with warm 0.9% NaCl. The colon was handled in a similar manner: the cecal-colonic junction was ligated and the luminal contents were flushed out the rectum by a cannula placed through an incision just distal to the proximal occluding ligature; residual saline was emptied by gentle manual expression. Three intestinal loops of -15-cm length were created with ligatures in the jejunum (beginning 2 cm distal to the ligament of Treitz), in the ileum (beginning 2 cm proximal to the ileocecal valve), and in the colon (beginning 2 cm below the cecal-colonic junction and extending distally to the peritoneal reflection]. In
565
Studies
In vitro studies were performed on ileum that was directly proximal to the ileal loop. These tissues were remdved immediately after the animal was killed, cut open along the mesenteric border, and incubated with a solution of the following ionic composition (mM): Na, 143; K, 5: Mg, 1.1; Ca, 1.25; Cl, 124; HC03, 25; HPO,, 1.65; HzP04, 0.3; and fructose, 40. The pH was 7.4 when gassed with 5% CO, in 95% O2 at 37°C. Ileal segments were placed serosal-side-facing-up on a plexiglass dissecting board, and were stripped of their serosa and underlying longitudinal muscle layer using a flat-edged dissecting forceps. Mucosal strips were then mounted in Ussing chambers, and transmural electric potential difference, resistance, and short-circuit current (I,,) were determined as previously described (6). Response to tyramine. Norepinephrine stores in ileal mucosa were estimated by I,, response to the serosal addition of tyramine (10 PM). Tyramine effectively releases 80% of all stored norepinephrine (7). Theophylline (1 mM) was added to the serosal reservoir 15 min before the addition of tyramine to stimulate the tissues to secrete, thus maximizing the I,, response to tyramine. Pargyline (50 ELM),a monoamine oxidase inhibitor, was added to the serosal reservoir 10 min before tyramine to prevent tyramine metabolism (7). After the response to tyramine, o-glucose was added to both reservoirs, and the resulting I,, was determined as a measure of tissue viability. In no instance was this ~20 PA/cm”. Epinephrine dose-response relationship. Transmural short-circuit current responses to accumulative additions of I-epinephrine were determined in ileal mucosa of clonidine-treated and untreated chronic diabetic and control rats. The concentration of epinephrine producing a response that was 50% of maximal was estimated in each set of tissues in which I,, responses to accumulative additions were determined. Results were then subjected to Student’s t-test analysis for unpaired variables.
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Results Animal
Groups
Two groups of chronically diabetic rats were studied: one group received no treatment and the other was treated with clonidine. Littermates of each group served as controls. All chronically diabetic rats had persistent hyperglycemia, failure to gain weight (see Table l), polyuria, and persistent watery diarrhea. Although stool and urine could not be separated for independent quantitations, the diarrhea in the clonidine-treated chronically diabetic rat group appeared to improve, as assessed by the stool consistency and degree of fecal soilage in each cage. Intestinal
Fluid Absorption
In Vivo
As shown in Figure 1, control rats had a net fluid absorption in jejunum, ileum, and colon. Untreated chronically diabetic rats, however, exhibited net fluid secretion in ileum and colon, but not in jejunum. This was the case whether results were expressed as microliters per gram dry weight (data not shown) or as microliters per length (in centimeters) of bowel (Figure 1).These results are in agreement with our previous observations (l), with the exception that net secretion had not previously been noted in the colon of chronically diabetic rats. Treatment of chronically diabetic rats with clonidine reversed the net fluid secretion in ileum and colon to net fluid absorption (Figure 1). This dose of clonidine did not, however, completely correct fluid malabsorption in chronically diabetic rats, and clonidine at this dose did not alter basal fluid absorptive rates in control rats or increase fluid absorption in the jejunum of chronically diabetic rats. Ileal Transmural Measurements
Short-Circuit
Current
; Figure
1.
rcolO”{
1. Effect of clonidine treatment (closed bars) on in vivo intestinal fluid absorption per centimeter in control and diabetic animals. Location of jejunum, ileum, and colon is as described in Materials and Methods. Values are mean f 1 SEM for 5 rats.
follow I,, alone rather than attempt to measure changes in net fluxes of individual ions. Transmural short-circuit current changes to catecholamines reflect an inhibition of electrogenic anion secretion (8). In the presence of theophylline, decreases in I,, stimulated by these agents are caused by an inhibition of active chloride secretion (8). Transmural short-circuit current responses to theophylline (1 mM) were the same in chronically diabetic rats and littermate, nondiabetic controls (59 + 5 vs. 69 + 7 pAlcm2). The I,, response to tyramine in chronically diabetic rats was 29 2 6 pAlcm2, significantly less than the 48 + 6 PA/cm2 response in controls (n = 5, p < 0.05). The response relationship between the concentration of exogenously added I-epinephrine and I,, is shown in Figure 2. There was no apparent difference in the EDso for I-epinephrine, i.e., the concentration stimulating 50% of the maximal effect, in the ileum of control and treated and untreated chronically diabetic rats. The EDso values for each group were as follows (in nanomoles per liter): control, 12.6 + 2.6 (n = 6); control (clonidine treated), 26.8 t 9.6 (n = 8); chronically diabetic rats, 10.4 k 1.2 (n = 10); and chronically diabetic rats
As Na, Cl, and HC03 transport responses to theophylline and I-epinephrine have been well characterized in vitro and correlate well with oppositely directed I,, reponses to these agents, we elected to
Table
{}
8o/
100 ??
Dlab&c/untreated
A Control/untreated
0 Dmbetlc treated ~Control treated
Animal Group Profile
Group Chronic diabetes Clonidine-treated Untreated Control Clonidine-treated Untreated
Body weight
Blood glucose
(g)
(mg/dl)
5 5
255 2 16 235 " 5
483 2 17 417 t 36
5 5
440 f 14 445 k 7
79 +- 10 85 2 8
n
n, number of rats. Values are mean T SEM.
0
’6*T?“’
9
1
6
5
Epmephrlne HogM)
Figure
2. Effect of varying doses of I-epinephrine on in vitro intestinal short-circuit current (1.J determinations. Diabetic and control untreated groups (closed circles, closed triangles, respectively) are compared with the diabetic and control clonidine-treated groups (open circles, open triangles, respectively). Values are for n 2 6 animals for all groups.
September 1986
(clonidine treated), 11.3 _+ 2.5 (n = 12).There was, however, a significant increase (p < 0.05) in the maximal I,, response to lop5 M I-epinephrine in untreated chronically diabetic rats compared with controls (68 -+ 8 vs. 45 + 10 PA/cm’, respectively). As we had previously noted (l), the I,, response to I-epinephrine was inhibited by the az-antagonist yohimbine (10U5 M) but not by the P-receptor antagonist propanalol (10m5 M) or the al-antagonist prazosin (lob5 M). Treatment of chronically diabetic rats with clonidine decreased the maximal I,, response so that it was no different from controls. Clonidine did not alter basal I,, in untreated or treated chronically diabetic rats (6.9 5 1.2 vs. 11 + 2.1 pAlcm2, respectively) or in untreated or treated control rats (13.8 +- 2.9 vs. 19.2 2 4.7 PA/cm’, respectively). Although not shown in Figure 2, clonidine treatment had no effect on the I,, responses to exogenously added I-epinephrine in nondiabetic controls.
Discussion Adrenergic innervation has a major role in the regulation of fluid and electrolyte transport in the intestinal tract. In vitro stimulation of postsynaptic cu,-adrenergic receptors on ileal enterocytes causes an active absorption of Na and Cl and inhibits active secretion of Cl and HCO, (8). These effects have been shown by Tapper et al. (7) to also occur when norepinephrine is released from endogenous stores in rabbit ileal mucosa by tyramine. Wu and Gaginella (9) demonstrated the presence in rat colonic mucosa of a functional noradrenergic neural network associated with the epithelium. Newsome et al. (10) have shown electron-micrographic evidence of actual synapses between putative noradrenergic fibers and basal granulated epithelial (crypt) cells. Autonomic neuropathy and, in particular, adrenergic neuropathy, is a frequent complication of chronic diabetes mellitus. Hensley and Soergel (11) reported lesions in the dendritic processes of diabetic prevertebral and paravertebral sympathetic ganglia. Schmidt et al. (2) have demonstrated, in rats made chronically diabetic with streptozocin, histologic evidence of visceral nerve destruction and biochemical evidence of cholinergic as well as adrenergic denervation. More recently, Lincoln et al. (3) suggested that there might be a selective destruction of adrenergic nerves of the myenteric plexus of streptozocin-treated diabetic rats. Impaired adrenergic regulation of mucosal fluid and electrolyte transport can, therefore, alter the homeostatic balance exerted by neuroendocrine elements to favor net secretion. This increased secretory tone could also be due to marked changes in the
CLONIDINEIN DIABETICDIARRHEA 567
pattern of innervation by nerve fibers to the intestinal mucosa. For example, cholinergic and vasoactive intestinal polypeptide-like immunoreaction innervation to the submucosa and myenteric plexus appear to be increased in the ileum and proximal colon of streptozocin-treated, chronically diabetic rats (3,12). Basal secretion would be increased because of the release of these neurosecretagogues, in the absence of adrenergic tone, to the intestinal mucosa. In support of this contention, recent observations in chronically diabetic rats showed impaired absorption of fluid by the ileum and colon (1). These chronically diabetic rats also showed depletion of mucosal norepinephrine stores as estimated in vitro by the I,, response to tyramine. After sympathetic denervation with 6-hydroxydopamine, the pattern and magnitude of impaired fluid absorption in nondiabetic rats was the same as that observed in chronically diabetic rats. It was concluded that impaired intestinal mucosal absorption of fluid and electrolytes develops slowly in rats made diabetic with streptozocin, and that this absorptive impairment is due to a loss of normally present noradrenergic innervation of enterocytes. The present studies confirm our previous study, except that frank net secretion was observed in both the ileum and colon of chronically diabetic rats. A possible explanation for this difference is that the chronically diabetic rats in this study were anesthetized with ketamine, a dissociative anesthetic, rather than ether. As a consequence, rats (particularly chronically diabetic rats) were better able to tolerate abdominal surgery and were hemodynamically more stable. In our previous study we noted a significant increase in the I,, response to exogenously added I-epinephrine in jejunum of chronically diabetic rats, suggesting the possibility of postsynaptic denervation hypersensitivity. This was not noted in ileum, where the I,, responses were more variable. To explore this further, we reexamined the effects of exogenously added I-epinephrine on I,, in the ileum. The dose-response relationship shown in Figure 2 suggests that postsynaptic hypersensitivity is also present in ileum of chronically diabetic rats. There was no change in the EDs0 to I-epinephrine, suggesting that receptor affinity was not altered, but a significant increase in the I,, response to maximal concentrations of I-epinephrine was noted. It is unlikely that this increase was due to mucosal hypertrophy, as I,, responses to theophylline were no different in chronically diabetic rats than in controls. Basal I,, was unaffected by clonidine treatment. Therefore, it is very likely that any residual clonidine bound to receptors was washed out during the preparation and preincubation periods. This
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CHANG ET AL.
assumption is supported by the finding that yohimbine had no effect on basal I,, in tissues from treated or untreated chronically diabetic rats. These results suggest that postsynaptic denervation hypersensitivity may be a consequence of increased numbers of cr2-adrenergic receptors on enterocytes. Similar findings have been reported in other tissues. Denervation produces a moderate increase in postsynaptic cu2-adrenergic receptor density in membranes prepared from whole rat submandibular glands (IS), without a significant increase in binding affinity. Denervation with intraventricular injection of 6-hydroxydopamine also increased postsynaptic a2-receptor density, but not binding affinity, in rat cerebral cortex (l&15). The denervation hypersensitivity of the ileum of chronically diabetic rats can be corrected by treatment with the a2-adrenergic agonist clonidine. The implications of these findings are that postsynaptic denervation hypersensitivity is a reversible adaptation to diminished adrenergic tone. Clonidine may therefore be potentially useful in the treatment of diabetic diarrhea. In normal subjects, clonidine appears to enhance intestinal fluid and electrolyte absorption by an enteropooling effect and by a direct action on mucosal function (5,16). We previously reported 3 cases in which patients with severe diabetic diarrhea responded to oral therapy with clonidine (5). Goff (17) has reported a similar experience using the a2-adrenergic agonist lidamidine. In chronically diabetic rats, clonidine, at a dose nearly equivalent in milligrams per kilogram body weight to that used in patients with diabetic diarrhea, reversed net fluid secretion in ileum and colon to net absorption. Clonidine treatment was continued for 3 days in order to reach a steady-state serum concentration. This dose of clonidine did not, however, alter basal absorptive rates in control rats. In rats, this dose appears to be well below the EDS0 (0.06-0.12 mg/kg) for clonidine to have an effect on stimulated intestinal secretion (17-19). However, with the techniques employed in this study to measure intestinal fluid absorption, small changes of
cal sympathectomy with 6-hydroxydopamine (1). Furthermore, the decrease in I,, response to the in vitro addition of I-epinephrine was smaller in the jejunum than in the colon and ileum. Diarrhea in streptozocin-induced, chronically diabetic rats appears to be a good model for the study of diabetic diarrhea in humans. Although these animals uniformly develop diarrhea, wherereas most patients have intermittent diarrhea, we think the difference may be attributable to the fact that chronically diabetic rats have diabetes for prolonged periods without the benefit of treatment with insulin. This circumstance is rarely encountered in a patient population. Intractable diarrhea in these rats most likely represents a predominantly adrenergic rather than cholinergic neuropathy. Hence, the impairment of adrenergic innervation of the intestinal mucosa shifts neuroendocrine regulation of ion transport in favor of net secretion. The present studies demonstrate that this impairment of adrenergic innervation of the intestinal epithelium causes a denervation hypersensitivity of postsynaptic a2-adrenergic receptors. This alteration in receptor responses appears to be a result of increased receptor numbers rather than of any change in receptor-binding affinity for catecholamines. CQAdrenergic agonists in this model also appear to have a role in reversing intestinal secretion and the observed a2-receptor denervation hypersensitivity, suggesting potential clinical utility of these agents for the treatment of diabetic diarrhea.
References 1.
Chang EB, Bergenstal RM, Field M. Diarrhea in streptozocintreated rats. J Clin Invest 1985;75:1666-70. 2. Schmidt RE, Nelson JS, Johnson EM. Experimental diabetic autonomic neuropathy. Am J Path01 1981;103:210-25. 3. Lincoln J, Bokor JT, Crowe R, Griffith SG, Haven AJ, Burnstock G. Myenteric plexus in streptozocin-treated rats. Neurochemical and histochemical evidence for diabetic neuropathy in the gut. Gastroenterology 1984;86:654-61. 4. Durbin T, Rosenthal L, McArthur K, Anderson D, Dharmsathaphorn K. Clonidine and lidamidine stimulate sodium and chloride absorption in the rabbit intestine. Gastroenterology 1982;82:1352-8. 5. Fedorak RN, Field M, Chang EB. Treatment of diabetic diarrhea with clonidine. Ann Intern Med 1985;102:197-9. 6. Field M, Fromm D, McCall I. Ion transport in rabbit ileal mucosa. I. Na and Cl fluxes and short-circuit current. Am J Physiol 1971;220:1388-94. 7. Tapper EJ. Bloom JS, Lewand DL. Endogenous norepinephrine release induced by tyramine modulates intestinal ion transport. Am J Physiol 1981;229:86-92. a. Field M, McCall I. Ion transport in rabbit ileal mucosa. III. Effects of catecholamines. Am J Physiol 1973;225:852-7. 9. Wu ZC, Gaginella TS. Functional properties of noradrenergic nervous system in rat colonic mucosa: uptake of [3H] norepinephrine. Am J Physiol 1981;241:G13742. 10. Newsome B, Ahlman H, Dahlstrom A, Das Gupta TK, Nyhus
September
11. 12.
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14.
1986
LM. On the innervation of the ileal mucosa in the rat - a synapse. Acta Physiol Stand 1979;105:387-9. Hensley GT, Soergel KH. Neuropathological findings in diabetic diarrhea. Arch Path01 1968;85:587-97. Belai A, Lincoln J, Mulner P, Crow R, Loesch A, Burnstock G. Enteric nerves in diabetic rats: increase in vasoactive intestinal polypeptide but not substance P. Gastroenterology 1985;89:967-76. Arnett CD, Davis JN. Denervation-induced changes in alpha and beta adrenergic receptors of the rat submandibular gland. J Pharmacol Exp Ther 1979;211:394-400. U’Prichard CD, Bechtel WD, Ronuot BM, Snyder SH. Multiple apparent alpha-noradrenergic receptor binding sites in rat brain: effect of 6-hydroxydopamine. Mol Pharmacol 1979; 16:47-60.
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15. Dausse JP, LeQuan-bui KH, Meyer P. Alpha-l and alpha-2 adrenoreceptors in rat cerebral cortex: effects of neonatal treatment with 6-hydroxydopamine. Eur J Pharmacol 1982; 78:15-20. 16. Schiller LR, Santa Ana CA, Morawski SG, Fordtran JS. Effect of clonidine on experimental diarrhea in humans (abstr]. Gastroenterology 1984;86:1235. 17. Goff JS. Diabetic diarrhea and lidamidine. Ann Intern Med 1984;101:874-5. 18. Nakaki T, Chang PC, Tokunaga Y, Kato R. Alpha,adrenoceptors modulating diarrhoea in morphine-dependent rats. J Pharm Pharmacol 1981;33:397-9. 19. Spraggs, CF, Bunce KT. Alpha,-adrenoceptors and the delay of castor oil-induced diarrhoea by clonidine in rats. J Pharm Pharmacol 1983;35:321-2.