Ca2+ channel blockers stimulate ileal and colonic water absorption

GASTROENTEROLOGY

1985;89:858-66

Ca2+ Channel Blockers Stimulate Ileal and Colonic Water Absorption MARK DONOWITZ, SHEILA LEVIN, GEORGE POWERS, GRACE ELTA, PHYLLIS COHEN, and HELEN CHENG Departments of Medicine and Physiology, Tufts University School of Medicine, New England Medical Center, and U.S. Department of Agriculture Human Nutrition Center on Aging at Tufts, Boston, Massachusetts _

The efiects of calcium channel blockers on water transport in the rat ileum and distal colon were studied in vivo using the single-pass perfusion technique. Parenteral but not intraluminal verapamil, and parenteral nifedipine increased ileal water absorption, with effects lasting at least 60 min. In contrast, i-p. verapamil had no e#ect on rat distal colonic water absorption, whereas intraluminal verapamil significantly stimulated colonic water absorption. Similarly, perfusing the rat descending colon with low-Ca2+ Ringer’s_HC03 stimulated coionic water absorption. Verapamil was not antisecretory because the theophylline-induced decrease in ileal water transport was similar in control animals and in animals pretreated with i.p. verapamil. In addition, nifedipine stimulated active Na and Cl absorption in rabbit ileum. These studies demonstrate that the Ca*+ channel blockers verapamii and nifedipine stimulate basal absorption of water in rat ileum and distal colon in vivo, and stimulate active Na and Cl absorption in rabbit ileum in vitro. The verapamil stimulation of colonic water absorption from the luminal surface was duplicated by perfusion with a low-Ca2+ bathing solution. This suggests the presence of apical membrane Ca*+ channels in rat colon, which appear to be involved in regulation of basal water transport, and that these Ca’+ chanReceived November 6, 1984. Accepted April 8, 1985. Address requests for reprints to: Mark Donowitz, M.D., Center for Gastroenterology Research on Absorptive and Secretory Processes (GRASP], GI Unit, Box 217, New England Medical Center, 171 Harrison Avenue, Boston, Massachusetts 02111. This study was supported in part by National Institutes of Health NADDK grant ROI AM26523 and National Institutes of Health NADDK CORE Center for Gastroenterology Research on Absorptive and Secretory Processes, PHS l-P30AM 39428, a World Health Organization Diarrhea1 Disease Program Grant, and by the U.S. Department of Agriculture Nutrition Center on Aging at Tufts. Dr. Donowitz is supported in part by National Institutes of Health RCDA lKOH-00588. 0 1985 by the American Gastroenterological Association 0016-5085/85/$3.30

nels are in a partially open state under basal conditions. Because verapamil stimulates absorption systemically [ileum) as well as intraluminally (colon), Ca2+ channel blockers have properties that might be useful in treatment of diarrhea1 diseases. Although Ca2+ channels were first recognized only in excitable nerve and muscle tissues, gated Ca2+ entry now also appears to occur in tissues previously considered to be nonexcitable, such as fibroblasts and pituitary cells, among others (l-3). This observation is, at the current time, largely due to the responses of these tissues to, and interaction with, Ca2+ channel antagonists [l-3). Based on in vitro studies of rabbit ileum in which verapamil, cadmium, and diltiazem stimulated active Na and Cl we suggested that Ca2+ channels absorption (4-6), exist in the ileal plasma membrane and are in a partially open state under basal conditions. We supported this view by demonstrating that a series of neurohumoral substances normally present in ileal mucosa (serotonin, neurotensin, substance P) act to decrease Na and Cl absorption or increase Cl secretion, or both, by increasing plasma membrane 45Ca2+ entry, whereas other neurohumoral substances, also present in ileal mucosa (catecholamines acting by CQ and dopamine receptors), increase Na and Cl absorption and decrease plasma membrane 45Ca2f entry (4,7--g and Donowitz M, Sharp GWG, unpublished observations). We suggested that this Ca2+ channel was on the ileal basolateral membrane because verapamil [a) stimulated absorption from the serosal but not the mucosal surface and (b) decreased 45Ca2+ influx from the serosal but not the mucosal surface (4-6).

The current studies were undertaken to extend our investigation of effects of Ca2+ channel blockers on Abbreviations monophosphate;

used in this paper: NS, not significant.

CAMP,

cyclic

adenosine

Ca” CHANNEL BLOCKERS AND WATER ABSORPTION

October 1985

intestinal

transport.

whether another stimulated altered

They

specifically

examined

(a) verapamil altered basal transport in species, the rat; (b) Ca2+ channel blockers absorption

colonic

stimulated

in vivo as we]1 as in vitro;

as well

secretion

as ileal

transport;

(c)

(d) altered

as well as basal absorption;

and

(e) an example of another class of Ca2+ channel antagonists, nifedipine, also stimulated intestinal absorption.

Materials and Methods Male Sprague-Dawley rats weighing 250-350 g or New Zealand male albino rabbits weighing 2-2.5 kg were maintained on standard pellet diets with free access to water. Each animal was anesthetized with sodium pentobarbital (65 mg/lOO g body wt). In rats, one ileal or colonic loop was prepared, and in rabbits, distal ileum was removed. Tracheostomies were used only in animals treated with carbachol or carbachol controls in order to clear the increased respiratory secretions.

Intestinal

Transport

In vivo. Intestinal transport was measured in vivo in rat ileum and colon, Ileal loops were -15 cm in length, ending -5 cm proximal to the ileocecal junction. The colonic loops began at the distal end of the transverse colon and extended to the pelvic brim. These were 2-7 cm in length. Influx and efflux catheters were placed into the intestinal loop; intestinal water transport was determined by the single-pass perfusion technique as previously described (10,ll). During this process, body temperature was maintained at 37°C; perfusate was maintained at 37°C on entry into the loop and was perfused at 0.5 mllmin. Unless stated otherwise, the perfusate consisted of Ringer’s_HCO, composed of the following (in mM): NaC1115, NaHCO, 25, K2HP04 2.4, KI-IzPOl 0.4, CaCl, 1.2, MgClz 1.2, mannitol to bring the final osmolarity to 290 mosmol/kg, [‘4C]polyethylene glycol (PEG, molecular weight 4000) and unlabeled PEG 3 g/L as a nonabsorbable water marker; pH 7.4. This fluid was gassed throughout with 95% 02--5% COz. In one series of experiments, Ca2+ was omitted from the solution forming “a low-Ca’+” bathing solution which contained -4-5 PM Ca2+ as determined by atomic absorption spectrometry on the loop effluent. In both ileal and colonic experiments, steady-state periods lasting 60-70 min were followed by up to five 20-min perfusion periods in both the test and control animals studied in parallel experiments in littermates. After perfusion, the intestinal loops were measured in a uniform manner by suspending a 10-g weight for 15 s, and in some cases tissues were prepared immediately for cyclic adenosine monophosphate (CAMP) analyses or histology. Water transport was calculated as previously described (10,ll)and was expressed as microliters per hour per centimeter. Absorption from the lumen was expressed as a positive value, secretion into the lumen as a negative value. Mean values were obtained by averaging the results

859

of the multiple collection periods. [‘4C]Polyethylene glyco1 recovery was determined and periods in which PEG recovery was not 100% k 7% were discarded. In most experiments, Ca2+ channel blockers were studied after determining basal transport in the same intestinal loop, with simultaneously studied time controls performed on littermates. Verapamil was injected intraperitoneally, given intravenously through the jugular vein, or administered intraluminally. Nifedipine was administered intraperitoneally. To determine the effect of verapamil on intestinal secretion, theophylline [CAMP-induced secretion) was perfused intraluminally and the muscarinic cholinergic agonist carbachol (Ca’+-induced secretion) was given intravenously or subcutaneously. In these experiments, it was initially established whether secretagogues had an effect that was constant over the time of study. In describing systemically administered drugs, the drug dose administered is presented as the amount administered per kilogram body weight and also, for comparison, as the concentration that would have resulted if the entire dose were distributed in body water. In vitro. Four pieces of distal rabbit ileal mucosa, with the serosa and muscularis propria removed, were studied simultaneously, after mounting as flat sheets between modified Ussing chambers with a surface area of 1.13 cm2 and bathing with Ringer’s_HCO,. Transmural potential difference (PD) was measured with calomel electrodes attached to tissue by agar bridges containing the corresponding bathing fluid. Except for the PD measurements, direct current was passed through the tissue by means of Ag-AgCl electrodes in sufficient quantity to nullify the spontaneous PD. 22Na and 3”C1 were used to determine the unidirectional mucosal-to-serosal and serosal-to-mucosal ion fluxes on tissue matched to differ in conductance by <25%. Ion flux studies were performed starting 20 min after isotope addition. A negative sign before a net ion flux indicated secretion; a positive sign, absorption. After determining two 20-min basal flux periods 20-60 min after isotope addition, 1 PM nifedipine in a final concentration of 0.05% ethanol and 0.05% PEG (molecular weight 900) or the ethanol plus PEG alone were added to the serosal bath, and one further 20-min flux period was determined 70-90 min after isotope addition. Cyclic

Adenosine

Monophosphate

Assay

Cyclic adenosine monophosphate was determined by a modification of the competitive protein binding assay kit (Amersham of Gilman (11,12)using a commercial Searle Corp., Arlington Heights, Ill.). Mucosal scrapings from the rat ileum perfused in vivo were frozen rapidly in liquid nitrogen, homogenized with a cold ground glass homogenizer, and the CAMP was extracted into acid ethanol (0.2N HCl). After centrifuging, the supernatant was decanted and evaporated to dryness under nitrogen. The residue was redissolved in 0.05 M Tris buffer, pH 7.5, and 4 mM ethylenediaminetetraacetic acid for assay. The protein content of the pellet was determined by the method of Lowry et al. (13). Drugs used in these studies included verapamil, kindly provided by the Knoll Pharmaceutical Company, Whip-

860

DONOWITZ

ET AL.

GASTROENTEROLOGY

pany, N.J.; nifedipine, kindly provided by the Pfizer Chemical Co., Brooklyn, N.Y.; theophylline, from Eastman Kodak Company, Rochester, N.Y.; and carbachol, atropine, and PEG (molecular weight 4000 and 900) from Sigma Chemical Co., St. Louis, MO. Statistical analyses were performed with Student’s ttests for paired and unpaired data and were two-tailed. Kinetic analyses were determined by the method of Woolf-Hanes (14). All results were expressed as mean * SE.

MO)-

Results

IO-Q

Effects of Verapamil on Basal Water Transport in Vivo

’

SALINE TREATEO

60

?I t 3

40

BASAL

Figure

20

40

(0.086mq)

Rat Ileal

Verapamil administered intraperitoneally stimulated ileal water absorption (Figure 1).When 0.86 mg verapamil/250 g body wt was injected intraperitoneally, net water transport in the first 20 min posttreatment was increased compared with basal water transport; the increase in water transport induced by verapamil became maximal in the second 20-min flux period (2040 min after injection) and then remained constant for at least 60 min (Figure 1). In contrast, there was no significant change in basal

60

60

1. Effect of intraperitoneal verapamil on ileal water transport. Net water transport was measured in ileum for three 2&min basal flux periods (basal); then saline or verapamil (0.86 mgI250 g body wt) was injected intraperitoneally and four 20-min flux periods were determined. Net water transport in three basal periods and the periods after verapamil or saline are shown. Positive values represent net absorption and p values refer to comparison of transport during the basal periods and after verapamil or &line during individual flux periods in the same ileal loop (paiied t-test). The times shown refer to the end of the 20-min flux periods after injection; i.e., 20 refers to the periqd q-20 min after saline or verapamil injection. Tisspes from 6 saline-treated and 9 verapamil-treated animais were studied. Solid circles represent verapamil-treated.‘animals; open circles, saline-treated animals wikh injections done at time zero.

Vol. 89, No. 4

3x10-% (026~) I njected

Figure

6x10-% (0.52mg)

IO-% W6mg)

dose of Veropomil

10% (6.6mg)

(mq1250r)

bodyweight)

2. Dose response of i.p. verapamil on ileal water absorption. The maximal increase in net water absorption occurred 20-80 min after the administration of i.p. verapamil (transport after verapamil minus the rate of basal transport before verapamil is shown). The injected dose of verapamil in mg/250 g body wt is shown in parentheses, along with the calculated molar concentrations assuming distribution of the entire dose in body water for the entire time of the study. Verapamil induced a maximal increase in net water transport of 94.0 pi/h. cm, with the concentration kausing a halfmaximal effect of 6 x lo-” M (0.52 mg/250 g body wt). Tissues from 5-10 animals were studied at each dose.

water transport over this time in ileum of control animals intraperitoneally injected with an equal volume of normal saline. The verapamil stimulation of ileal water absorption was dose-dependent (Figure 2). In these studies, the effect at each drug dose shown is the mean increase in water absorption caused by verapamil, 20-80 min after i.p. injection. There was no effect at verapamil doses up to 0.26 mg/250 g body wt; a maximal effect occurred at 0.86 mg/250 g body wt, with the concentration producing 50% of the maximal effect, calculated by the method of Woolf-Hanes (14),being 0.52 mg/250 g body weight. Injection of a supramaximal dose of verapamil (8.6 mg/250 g body wt) caused a significantly smaller effect than 0.86 mg/250 g body wt. A similar inhibition by a supramaximal verapamil concentration was seen when the verapamil effect was studied on rabbit ileal active electrolyte transport in vitro (596). Verapamil (0.86 mg/250 g body wt) injected intravenously caused an increase in ileal water absorption which was maximal in the first 20 min after injection, and the effect was constant for 60 min but then decreased. The maximal effect of i.v. verapamil was lower in magnitude than that of i.p. verapamil (22.8 2 3.3 pi/h. cm compared with a 94.0 2 11.9 pi/h . cm increase in water absorption after i.v. and i.p. injection of 0.86 mg verapamiU250 g body wt, respectively; p < 0.01). In’contrast to the effect of parenteral verapamil,

520 -

Caz+ CHANNEL BLOCKERS AND WATER ABSORPTION

1985

October

Verapamil Treated P”O.025

T

l-

46

F $ 44 II B 40 e z &

IO

’ I

6

Time

different from the increase of net water transport in otherwise untreated control animals treated with the same dose of verapamil (94.0 + 11.9 pi/h - cm). This indicates that the effect of verapamil was not significantly altered by pretreatment with atropine.

Control

Effect of Verapamil on Distal Rat Colonic Basal Water Transport In Vivo

Bsfore After

Figure

861

3. Effect of 10m4 M intraluminal verapamil on rat descending colonic water transport. Net water transport in three 20-min basal periods (labeled before) was determined in animals that were then perfused with Ringer’s_HCO, or verapamil (labeled after). In the time-control animals, Ringer’s_HCO, perfusion was continued and in the verapamil-treated animals, 10m4 M verapamil was perfused for four 20-min periods. Numbers above bars are the numbers of animals studied; p values represent comparison of net water transport in the same loops during periods A and B (paired t-test).

intraluminal perfusion with 10e4 M verapamil for five 20-min periods did not alter basal water transport compared with perfusion with Ringer’s_HCO, [26.2 k 8.0 pllh * cm vs. 32.8 k 10.5 pi/h - cm in control and verapamil-perfused ileal loops, respectively, n = 7, not significant (NS)]. Because of the possibility that parenteral verapamil was stimulating basal ileal water absorption indirectly via altering release of acetylcholine, we determined whether pretreatment with the muscarinic receptor antagonist atropine altered the effect of subsequently added verapamil. After determining the basal rate of ileal water absorption, animals were treated with 1.32 mg atropine1250 g body wt administered &bcutaneously, and the rate of water transport was deterinined for three further 20-min flux periods. Atropine treatment did not alter the rate of water transport [basal water transport, 27.2 2 10.8 pi/h * cm; transport during the first 20-min period after atropine, 18.4 ? 7.7 ~11 he cm; transport during the third 20-min period after atropine (40-60 min after atropine injection), 11.4 + 11.5 pi/h. cm]. Some of these animals were treated with 0.86 mg/250 g body wt of i.p. verapamil 20 min after $tropine injection, and the rate of water transport was determined during one 20-min flux period starting 20 min after verapamil (40-60 min after atropine injection), a time of the maximal effect of verapamil. The rate of water transport after verapamil was 66.5 + 14.1 pi/h - cm. The increase in net water transport caused by verapamil (A verapamil period minus atropine period, 57.8 t 20.6 pi/h. cm) was not significantly

Intraperitoneal verapamil had no effect on rat distal colonic water transport at concentrations as high as 8.6 mg/250 g body wt. Basal transport in distal rat colon was 64.9 + 15.8 pi/h * cm in the three 20-min flux periods before verapamil and 66.0 + 18.6 pllh * cm in the four 20-min flux periods after verapamil (Al.1 * 5.2 pllh . cm, n = 8, NS). There was no significant increase in water absorption in any one individual period after i.p. verapamil. Similarly, in time control, saline-injected animals, which were studied over the same period of time, the rate of net water transport was constant (the change in watet transport over the four 20-min periods after saline injection compared with the three 20-min flux periods before saline was 13.6 * 20.0 pi/h. cm, n = 6, NS). In contrast to the lack of effect of i.p. verapamil, intraluminal verapamil (10M4 M) greatly stimulated net water absorption (Figure 3). The total amount of verapamil perfused intraluminally was approximately equal to that given intraperitoneally. The verapamil-induced increase in colonic absorption was maximal during the first 20 min of perfusion and the effect was constant during at least 80 min of verapamil exposure. The magnitude of the verapamil-induced increase in colonic water absorption (404.1 2 135.8 pi/h . cm, n = 7) was greater than the maximal vetapamil-induced increase in ileal water absorption (94.0 +- 11.9 pi/h. cm, n = 10, p < 0.05). In contrast, there was no significant change in distal rat colonic water transport over the comparable time of study (Figure 3). Effect 6f Verapamil on Cyclic Adenosine Monophosphate-Stimulated Rat Ileal Sbcretion ThB effect of verapamil on stimulated ileal secretion in vivo was determined; specifically, we questioned whether veripamil could prevent or reverse stimulated secretion. The model of CAMPinduced secretion was iiitraluminal perfusion with 10 mM theophylline, which previously had been shown to induce ilkal water and electrolyte secretion at a constant rate for ai least 80 min (10,ll). Although phosphodiesterase inhibitors increase the CAMP and cyclic guanosine monophosphate content in seine tissues, the effects appear restricted to

862

DONOWITZ ET AL.

changes in CAMP content in rat ileum (DeJonge H, private communication]. We initially determined whether verapamil could reverse theophylline-induced secretion. Intraluminal perfusion with theophylline decreased net water transport to a value not significantly different from zero (basal ileal water absorption before theophylline, 42.2 + 11.2 pi/h - cm; net water transport during the 100 min of theophylline perfusion, 9.8 + 16.0 &h - cm; effect of theophylline -32.4 + 7.6 pi/h * cm, p < 0.02). There was no significant difference in the effect of theophylline when one 20-min flux period was compared with any other, indicating that the effect of theophylline was constant for at least 100 min. Verapamil was given intraperitoneally 40 min after theophylline was begun. Verapamil significantly increased absorption 20-60 min after i.p. injection (which was 60-100 min after theophylline perfusion was begun). The increase in water absorption caused by verapamil in theophylline-perfused loops was 54.6 + 14.1 pi/h - cm, n = 7, p < 0.02. In littermate controls treated only with verapamil and studied simultaneously, verapamil increased ileal water absorption by 44.4 k 18.0 pllh * cm, n = 8. There was no significant difference in the effect of verapamil in theophylline-perfused and Ringer’s_HC03-perfused animals. Thus, verapamil stimulated water absorption similarly in untreated control ileal loops and in loops that had decreased net water absorption due to an increased CAMP content in the tissue. Next, we determined whether pretreatment with verapamil prevented the effect of theophylline. An effect on the theophylline-induced decrease in net water transport would suggest a direct antisecretory effect of verapamil. Theophylline perfusion significantly decreased net water transport in verapamiltreated animals by 17.9 + 6.6 pi/h. cm, n = 11, p < 0.05 (transport 20-40 min after i.p. verapamil, 86.4 k 18.0 pi/h . cm; transport O-40 min after theophylline, which was 40-80 min after verapamil, 68.5 + 6.4 pi/h . cm). Theophylline significantly decreased net water transport in simultaneously studied, but otherwise untreated control animals by 27.2 + 7.8 pi/h * cm, n = 7, p < 0.05 (basal transport, 34.2 f 9.0 pi/h . cm; transport o-40 min after theophylline, 7.0 k 6.8 @h . cm). There was no significant difference in the theophylline-induced decrease in net water transport in untreated control animals and in verapamil-treated animals. The failure of verapamil to affect the theophylline-induced secretion was not associated with a significant effect of verapamil on basal ileal CAMP content or the theophyllineinduced increase in CAMP content, as shown in Table 1. Because verapamil specifically inhibited Ca’+-

GASTROENTEROLOGY Vol. 89, No. 4

Table 1. Efiect of Intraperitoneal Verapamil on Basal and Theophylline-Induced Increase in Ileal Cyclic Adenosine Monophosphate Content0 Verapamil (mg/250 g body wt) 0

0

Perfusion

solution

Ringer’s_HCO, Theophylline (10 mM)

n

CAMP content (pmolimg protein]

6 6

5.6 t 1.3b 40.1 r 11.2

6 6

6.1 f l.Zb 27.5 2 6.1'

p < 0.05d 0.86 0.86

Ringer’s_HCO, Theophylline (10 mM) p < 0.05d

a Ileal

cyclic adenosine monophosphate (CAMP) content was determined by the method of Gilman (12) on ileal loops perfused with Ringer’s_HCO, with or without 10 mM theophylline for 100 min. Studies were done in animals treated intraperitoneally with 0.86 mg verapamiU250 g body wt or a similar volume of saline. n refers to the number of animals studied. b,c Comparison of CAMP content in verapamil-treated and untreated control animals (unpaired t-test) was not significant. d Comparison of CAMP content in Ringer’s_HCO,-perfused or theophylline-perfused loops in the same animals (paired t-test).

induced secretory processes, such as those caused by serotonin, substance P, and neurotensin in rabbit ileum studied in vitro (7,15), attempts were made to determine the effect of verapamil on a model of Ca’+-induced ileal secretion in vivo. Attempts to use carbachol subcutaneously or intravenously to cause reproducible ileal secretion were unsuccessful. Carbachol at doses between lop7 M and 3 x lop6 M administered subcutaneously [calculated based on assumed distribution of the carbachol in body water) had no consistent effect on water transport when studied over as short a period as the first 20-min after injection, or studied for as long as 80 min after injection. Rats treated with carbachol did develop bronchorrhea, increased salivation, and increased tearing. The explanation for failure to demonstrate a carbachol-induced effect on ileal water transport in vivo is unclear. Effect of Low Intraluminal Water Absorption

Ca2+ on Colonic

Removing Ca 2f from the luminal perfusing solution of rat descending colon in vivo affected net water transport. After determining the basal rate of water transport, descending colonic loops were perfused with Ringer’s_HCOB solution from which Ca2+ was omitted but which did not contain a Ca2+ chelator [total calcium concentration determined on loop effluent by atomic absorption spectrometry was 4-5 PM). Perfusion with the low-intraluminal-Ca2+ bathing solution caused increased net colonic water

October 1985

Ca*’ CHANNEL BLOCKERS AND WATER ABSORPTION

Ethanol Ccmtrol NS

Nifedip ine Treated PcO.025

u

60

20

Ethanol or Nifedipline

Effect of i.p. nifedipine (0.60 mg/250 g body wt) compared with ethanol control on net ileal water absorption. Three 29-min periods were studied before i.p. injection and three 29-min periods after injection. Numbers above bars are number of animals studied; p values represent comparison of transport before and after i.p. injection in the same loops (paired t-test].

absorption. When studied for five 20-min periods, water absorption significantly increased after the first 20-min period and there was no further significant change over the next 80 min (net water transport in the last 80 min of perfusion with low-Ca2+ bathing solution minus basal, 114.4 -+ 35.6 @l/h * cm, n = 10,p < 0.05), whereas the time control did not change significantly [net water transport in the last 80 min minus basal, 10.4 + 21.5 ,ullh - cm, n = 8, NS). This effect of exposure to a low-Ca” perfusate does not appear to represent damage because when Ca2+ was added back to the luminal perfusate at 1.2 mM after 60 min of perfusion with this low-Cazf solution and net water transport was studied over the next three 20-min periods, the rate of water transport returned to the original level [data not shown). Effect of Nifedipine on Basal Ileal Water Absorption In Vivo and In Vitro To determine whether the verapamil stimulation of ileal water absorption in vivo was unique to verapamil or occurred with other Ca2+ channel blockers, the effect of i.p. nifedipine on basal rat ileal water absorption was determined. In addition, the effect of nifedipine was determined on active ileal Na and Cl transport in rabbit ileum studied in vitro. As shown in Figure 4, nifedipine (0.60 mg/250 g body wt) increased ileal water absorption during the 60 min after i.p. injection. In contrast to the effect of nifedipine, a volume of i.p. ethanol equal to that used as a vehicle for nifedipine (0.35 ml/250 g body wt) did not significantly alter net water transport over the time period studied (Figure 4). The increase in water absorption in the nifedipine-treated animals (68.1 ? lo.5 pi/h . cm) was significantly greater than the change in water transport in the ethanol-treated control animals (14.6 -C-8.9 /-d/h. cm) (p < 0.005).

863

In addition, in studies with the rabbit ileum in vitro, as shown in Figure 5, nifedipine caused a rapid decrease in short-circuit current which was constant for -20 min but returned to baseline by 40 min after addition. The nifedipine effect on active Na and Cl transport was determined during the period of the constant short-circuit current, 10-80 min after the addition of nifedipine. Compared with the time control, nifedipine significantly lowered the PD, increased the conductance, and increased the mucosal-to-serosal and net Na and Cl fluxes, but did not affect the serosal-to-mucosal Na or Cl fluxes (Table 2).

Discussion In these studies, we demonstrated that the Ca2+ channel blockers verapamil and nifedipine stimulate water absorption in vivo in rat ileum, that verapamil stimulates colonic water absorption, and that nifedipine stimulates active Na and Cl absorption in rabbit ileum. These studies extend our previous observations that Ca2+ channel blockers increase rabbit ileal active Na and Cl absorption studied in vitro (4-6) (a) to another species, the rat; (b) to an in vivo model; and (c) to another intestinal segment, the colon. These studies also extend the number of classes of Ca2+ channel blockers shown to stimulate intestinal water or electrolyte absorption, or both, to include nifedipine, such that the major classes of Ca2+ channel blockers now have been shown to increase ileal absorption. That systemic and not intraluminal verapamil increased ileal absorption is consistent with our in vitro studies of the rabbit ileum, in which verapamil increased active Na and Cl absorption when added

30’

<

, -40

-30

-PO

-10

0 TIME

t10

C20

t30

t40

t50

(MINUTES)

Figure 5. Effect of nifedipine [l PM) added to the serosal surface of rabbit ileum in vitro. Ileal short-circuit current (kc) was measured over time in tissue from 9 animals. After stabilization (at the arrow) nifedipine or control ethanol plus polyethylene glycol was added, and the kc was studied for an additional 50 min. The p values refer to comparison of kc in control and nifedipineexposed tissues (paired t-test).

864 DONOWITZET

AL.

GASTROENTEROLOGYVol. 89,No.4

to the serosal but not the mucosal surface, and decreased 45Ca2+ influx from the serosal but not the mucosal surface (4-6).Intraluminal perfusion with verapamil, however, stimulated rat jejunal water and electrolyte absorption when studied in vivo (16). The reason rat ileum and jejunum appear to respond differently to intraluminal verapamil is unclear. The fact that nifedipine, in addition to verapamil, stimulated basal absorption in rabbit ileum in vitro and in rat ileum in vivo and that cadmium and diltiazem, as well as verapamil, increased active Na and Cl absorption in rabbit ileum studied in vitro strongly suggests that these effects are due to the Ca2+ channel blocking effects of these drugs rather than to some of the “non-Ca’+ channel blocking effects” of verapamil (17,18).Our interpretation of the stimulation of basal absorption in rat and rabbit ileum is that Ca2+ entry across the ileal basolateral membrane occurs through Ca’+ channels that are partially open in what is referred to as the basal state. Although a Ca2+ channel that is intrinsically different from classic Ca2+ channels in excitable tissues, which are closed in the basal state, is one possibility, we suggest that, more likely, the partially open state of the Ca‘+ channel in the ileal basolateral membrane is due to effects on Ca2+ entry of neurohumoral substances that are present in the environment of the ileal basolateral membrane in what is called the basal state. Because the ileal mucosa contains a large number of neurohumoral substances, at least several of which appear to increase or decrease Ca2+ entry across the ileal basolateral membrane (4,7-g), local release of these substances may be involved in the physiologic regulation of basal electrolyte transport. The Ca2+ channel involved in the regulation of water absorption in rat colon, in contrast to that in rat ileum, appears to be on the apical membrane rather than on the basolateral membrane. This is suggested because verapamil increased absorption in rat colon from the luminal surface, but not after systemic injection. That this occurred through an alteration of the open status of the Caz+ channel was further suggested as the effect was duplicated by lowering luminal Caz+ in in vivo studies; this is similar to the stimulation of active Na and Cl absorption that occurred in rat descending colon after lowering mucosal Ca 2f (19). This effect of lowering luminal Ca2+ represents the effect of low but not absent luminal Ca2+, and did not appear to result from histologic damage as light microscopic examination was not affected and the transport effects were reversible when Ca2+ was returned to the normally studied level of 1.2mM. What regulates this luminal Ca2+ channel under normal circumstances is not known, Whether neurohumoral substances (such as

October 1685

Table

3.

CaZ+ CHANNEL BLOCKERS AND WATER ABSORPTION

Verapamil E#ect on Basal Water Absorption0

Intestinal segment Ileum Colon

Route of administration Intraluminal

Parenteral

0

T

t

0

a ‘J , increase; 0, no change.

vitamin D, parathyroid hormone, estrogens, or glucocorticoids) or dietary factors (such as phytate or fiber) regulate luminal Ca2+ entry in the rat colon has not been evaluated. Although neurohumoral regulation of apical membrane Ca2+ entry in rat colon appears to be a possibility, this would probably occur by changes in the permeability to calcium of the Ca2+ channel of the apical membrane as it seems less likely that Ca2+ entry is regulated by luminal Ca2+ concentrations. This is because luminal Ca2+ is normally maintained in the millimolar range (20-22); however, the free Ca2+ concentration in the colonic luminal microenvironment at the unstirred layer has not been established. Thus, although the possibility of apical membrane colonic CaZ+ channel regulation by neurohumoral substances exists, it is not known currently whether changes in this Ca2+ entry are regulated physiologically by neurohumoral substances or dietary factors, or if they are affected by disease states. If changes in the apical membrane Ca ‘+ channel permeability do affect colonic water and electrolyte absorption, this could be an unrecognized cause of changes in bowel function. Another explanation, which must be mentioned concerning the effect of the low CaZ+ luminal perfusate and Ca2+ channel blockers on transport, is that the effect might not result from their direct action on plasma membrane Ca’+ channels, but rather, from effects on release of or interactions with the neurohumoral substances present in the intestinal mucosa. We tested one possible way verapamil could be acting via inhibiting release of acetylcholine. In these studies, we showed that pretreatment with atropine did not alter the verapamil effect, indicating that verapamil was not acting via altering acetylcholine release. Furthermore, because lowering mucosal Ca ‘+ but not serosal Ca ‘+ has been shown to alter active colonic electrolyte transport [19), this explanation seems unlikely inasmuch as a selective role for mucosal but not serosal Ca2+ in regulation of release of neurohumoral materials is unlikely; this, however, has not been systematically studied. In the current studies, verapamil was shown not to have an antisecretory effect in a model CAMP secretory system. That is, theophylline caused the same

865

change in water transport in untreated ileum and ileum with increased absorption due to verapamil pretreatment. Although we previously demonstrated that serosal verapamil inhibited the effects of neurohumoral substances, which act by increasing basolateral membrane entry of calcium in rabbit ileum, but did not alter CAMP-induced secretion, we were unable to evaluate whether verapamil had a similar effect in an in vivo rat model of Ca2+-induced ileal secretion. This was because we were unable to develop a reproducible model of Cazf-induced secretion in vivo in rat ileum. In these studies we utilized subcutaneous and intravenous carbachol in concentrations up to 3 x lo-” M; previously we studied the effects of serotonin administration on rats in vivo without establishing an intestinal secretory model (Donowitz M, unpublished observations). The reason for this failure to establish Ca2+induced in vivo models of ileal secretion in the rat is unclear. Because verapamil stimulates ileal and colonic water absorption, it suggests a potential role for this drug in the treatment of diarrhea1 diseases. Some drugs that stimulate intestinal water absorption have been clinically efficacious in the therapy of diarrhea1 diseases, even when they did not have a direct effect on the secretory process (23). Verapamil stimulates absorption systemically (ileum] as well as intraluminally (colon, jejunum) (Table 3). Also, the dose of verapamil that caused a maximal stimulation of ileal water absorption in these studies is equivalent to a dose of 240 mg administered to a patient weighing 70 kg, a standard cardiac therapeutic dose. In addition, after oral ingestion of verapamil, -8% of the material is not absorbed (24,25). Consequently, a verapamil regimen of 240 mg/day would likely result in a colonic verapamil concentration of -8 X 1O-5 M, although this has never been measured directly. Consequently, we suggest that verapamil should be evaluated for its effects in the treatment of acute and chronic diarrhea1 diseases, although we are not aware of published experience with this drug in the treatment of diarrhea.

References Toll L. Calcium antagonists: high-affinity binding and inhibition of calcium transport in a clonal cell line. J Biol Chem 1982;257:13189-92,

Okada Y, Tsuchiya W, Yada T. Calcium channel and calcium pump involved in oscillatory hyperpolarizing responses of L-strain mouse fibroblasts. J Physiol 1982;327:449-61. Hess P, Lansman JB, Tsien RW. Different modes of Ca channel gating behavior favoured by dihydropyridine Ca agonists and antagonists. Nature 1984;311:536-44. Donowitz M. Calcium in the control of active intestinal Na

866

5.

6.

7.

8.

9.

10.

11.

12.

13.

14. 15.

DONOWITZ ET AL.

and Cl transport and involvement in neurohumoral action. Am J Physiol 1983;245:G165-77. Donowitz M, Asarkof N. Calcium dependence of basal electrolyte transport in rabbit ileum. Am J Physiol 1982;243: G28-35. Donowitz M, Cusolito S, Battisti L, Wicks J, Sharp GWG. Role of basolateral membrane Ca+ + entry in regulation of Na and Cl transport in rabbit ileum. In: Heintze K, Skadhauge E, eds. Intestinal absorption and secretion. Lancaster: MTP Press Ltd., 1983;409-16. Donowitz M, Asarkof N, Pike G. Serotinin-induced changes in rabbit ileal active electrolyte transport are calcium dependent and associated with increased calcium uptake. J Clin Invest 1980;66:341-52. Donowitz M, Fogel R, Battisti L, Asarkof N. Calcium content in rabbit ileum is increased by the neurohumoral secretagogues serotonin, carbachol, substance P and neurotensin. Life Sci 1982;31:1929-37. Donowitz M, Cusolito S, Battisti L, Fogel R, Sharp GWG. Dopamine stimulation of active Na and Cl absorption in rabbit ileum: interaction with both cY,-adrenergic and specific dopamine receptors. J Clin Invest 1982;69:1008-16. Charney AN, Donowitz M. Prevention and reversal of cholera enterotoxin-induced intestinal secretion by methylprednisone induction of Na-K-ATPase. J Clin Invest 1976;57:1590-9. Donowitz M, Elta G, Battisti L, Fogel R, Label-Schwartz E. Effects of dopamine and bromocriptine on rat ileal and colonic transport. Gastroenterology 1983;84:516-23. Gilman AGA. A protein binding assay for adenosine 3’;5’cyclic monophosphate. Proc Nat1 Acad Sci USA 1970;67: 305-12. Lowry DH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:263-73. Segel IH. Biochemical calculations. New York: John Wiley & Sons, 1976:236. Kachur J, Miller RJ, Field M, Rivier J. Neurohumoral control

GASTROENTEROLOGY Vol. 89, No. 4

of ileal electrolytic transport. II. Neurotensin and substance P. J Exp Pharm Ther 1981;220:456-63. 16. Fisher SE, Wagner RA. Effect of verapamil on in vivo jejunal water and electrolyte absorption (abstr). Clin Res 1983; 31:286. 17. Bayer R, Henneker R, Kaufmann R, Mannhold R. Inotropic and electrophysiological action of verapamil and D600 in myocardium. III. Effects of the optical isomers on transmembrane action potential. Arch Pharm (Weinheim] 1975; 290:81-97. 18. Katz A, Hager WD, Messineo FC, Pappano AJ. Cellular actions and pharmacology of the calcium channel blocking drugs. Am J Med 1984;77:2-10. 19. Donowitz M, Wicks J, Battisti L, Pike G, DeLellis R. Effect of Senokot on rat intestinal electrolyte transport: evidence of Ca++ dependence. Gastroenterology 1984;87:503-12. 20. Wrong OM, Metcalfe-Gibson A, Morrison RBI, Ing TS, Howard AV. In vivo dialysis of faeces as a method of stool analysis. I. Technique and results in normal subjects. Clin Sci 1965;28:356-75. 21. Wilson DR, Ing TS, Metcalfe-Gibson A, Wrong OM. In vivo dialysis of faeces as a method of stool analysis. III. The effect of intestinal antibiotics. Clin Sci 1968;34:211-21. 22. Wilson DR, Ing TS, Metcalfe-Gibson A, Wrong OM. The chemical composition of faeces in uremia as revealed by in vivo fecal dialysis. Clin Sci 1968;35:197-209. 23. Donowitz M, Wicks J, Cusolito S, Sharp GWG. Pharmacotherapy of diarrhea1 diseases: an approach based on physiologic principles. In: Donowitz M, Sharp GWG, eds. Regulation of intestinal electrolyte transport. New York: Alan R Liss, 1984:329-59. 24. Freedman SB, Richmond DR, Ashley JJ, Kelly DT. Verapamil kinetics in normal subjects and patients with coronary artery spasm. Clin Pharmacol Ther 1981;30:644-52. 25. Schomerus M, Speigelhalder B, Stieren B, Eichelbaum M. Physiological disposition of verapamil in man. Cardiovasc Res 1976;10:605-12.