Zinc sulphate attenuates chloride secretion in Human colonic mucosae in vitro

European Journal of Pharmacology 696 (2012) 166–171

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Pulmonary, gastrointestinal and urogenital pharmacology

Zinc sulphate attenuates chloride secretion in Human colonic mucosae in vitro Mekki Medani a,1, Victoria A Bzik 1,b, Ailin Rogers a,b, Danielle Collins a, Rory Kennelly a, Des C Winter a, David J Brayden b, Alan W Baird 1,b,n a b

Institute for Clinical Outcomes Research and Education (ICORE), Department of Surgery, St Vincent’s University Hospital, Dublin 4, Ireland UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 July 2012 Received in revised form 4 September 2012 Accepted 17 September 2012 Available online 26 September 2012

Zinc’s usefulness in the treatment of diarrhoea is well established as an addition to oral rehydration. Mechanisms of action of zinc have been explored in intestinal epithelia from rodents and in cell lines. The aim was to examine how zinc alters ion transport and signal transduction in human colon in vitro. Voltage clamped colonic sheets obtained at the time of surgical resection were used to quantify ion transport responses to established secretagogues. Nystatin permeabilisation was used to study basolaterally-sited ion channels. Direct actions of zinc were determined using preparations of colonic crypts isolated from human mucosal sheets. Electrophysiological measurements revealed zinc to be an inhibitor of electrogenic ion transport stimulated by forskolin, PGE2, histamine and carbachol in isolated human colonic epithelium. Basolateral addition of zinc sulphate had no direct effect on the epithelium. To further outline the mechanism of action, levels of secondary intracellular messengers (3’, 5’-cyclic adenosine monophosphate; cAMP) were determined in isolated colonic crypts, and were found to be reduced by zinc sulphate. Finally, indirect evidence from nystatin-permeabilised mucosae further suggested that zinc inhibits basolateral K þ channels, which are critical for transepithelial Cl  secretion linked to water flux. Anti-secretory, and therefore anti-diarrhoeal, actions of exogenous zinc are due, at least in part, to direct basolateral epithelial K þ channel inhibition. & 2012 Elsevier B.V. All rights reserved.

Keywords: Zinc sulphate Colon Potassium channel Secretion Anti-diarrhoeal

1. Introduction Even mild zinc deficiency is associated with diarrhoea (Hambidge, 2000) and zinc supplementation is a cheap and effective anti-diarrhoeal agent (Robberstad et al., 2004). There is substantial evidence of the clinical efficacy of zinc as an antidiarrhoeal agent (Baqui et al., 2002; Bhatnagar et al., 2004; Dutta et al., 2000; Lukacik et al., 2008) and the physiological principles underlying the effects of zinc have been explored in animal models and human cell lines (Berni Canani et al., 2010; Bzik et al., 2012; Carlson et al., 2006; Hoque et al., 2005). However zinc actions on human intestinal tissues remain to be elucidated. We and others have previously shown that the anti-secretory effects of zinc on rat intestinal epithelia are due, at least in part, to direct blockade of basolateral channels on colonic epithelial cells, which is achieved through permeation from the apical side (Bzik et al., 2012; Hoque et al., 2009). Here, we have extended and developed

Abbreviations: cAMP, Cyclic adenosine monophosphate; Isc, Short-circuit current n Corresponding author. Tel.: þ353 1716 6220. E-mail address: [email protected] (A. Baird). 1 Authors contributed equally 0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.09.017

this approach to explore actions of zinc in a human model of colonic electrogenic ion transport. Electrophysiological measurements revealed zinc in the form of zinc sulphate to be an inhibitor of electrogenic chloride secretion in isolated human colonic epithelium. Basolateral addition of zinc sulphate had no direct effects on the epithelial short-circuit current (Isc). To further outline the mechanism of action, levels of the secondary intracellular messenger (3’, 5’-cyclic adenosine monophosphate; cAMP) were determined in isolated human colonic crypts, and were unaffected by zinc sulphate. Finally, evidence from nystatinpermeabilised mucosae was obtained to further suggest that zinc inhibits basolateral K þ channels, which are essential for epithelial Cl  secretion (Bachmann et al., 2011; Murek et al., 2011).

2. Materials and methods 2.1. Preparation of epithelia and ion transport studies Ethical approval and informed patient consent were obtained for the purposes of this study. Fresh specimens of human colon were collected from macroscopically-normal proximal resection margins of colorectal carcinoma specimens at St Vincent’s University

M. Medani et al. / European Journal of Pharmacology 696 (2012) 166–171

Hospital, Dublin, Ireland. Tissues were obtained from both male and female patients, age median 65 years, range 35–88. The specimens were transported to the laboratory in pre-oxygenated Krebs– Henseleit (KH) solution that contained (mM) NaCl 118, KCl 4.7, CaCl 2.5, MgSO4 1.1, KH2PO4 1.2, NaHCO3 25, and D-Glucose 11.1; pH 7.4. The basolateral sero-muscular layer was micro-dissected from the mucosa, which was then mounted in an Ussing chamber (0.63 cm2 aperture); World Precision Instruments (WPI, Stevenage, UK), and bathed bilaterally with KH solution (5 ml). The buffer was continually perfused with a mixture of 95% O2 and 5% CO2 gas lift, and maintained at 37 1C. Tissues were studied under voltage-clamp conditions (EVC-4000 amplifier, WPI). Transepithelial potential difference (PD, mV) and short-circuit current (Isc, mA cm  2) were monitored using a Pro-4 timing device (WPI) via Ag–AgCl agar–salt bridge electrodes (3% agar in 3 M KCl). Data was recorded using a MacLabs data acquisition system (AD Instruments, Hastings, UK). Transepithelial electrical resistance (TEER, O cm2 ) was calculated from Isc and PD using Ohm’s law. Zinc was added to the basolateral bathing solution of treatment groups in the form of zinc sulphate. After a 30 min equilibration period, secretagogues (forskolin, prostaglandin E2, histamine, or carbachol) were added to the basolateral compartment and subsequent inward Isc measured. The viability of individual tissues was assessed at the end of each experiment by measuring secretory capacity; structural integrity was confirmed histologically using haematoxylin and eosin (H&E) staining.

2.2. Tight junction modulation using cytochalasin D To ascertain the specificity of the action of zinc on the basolateral membrane, we incubated colonic mucosa with apical zinc sulphate (1 mM) and the tight junction opener, cytochalasin D (8 mM), for 60 min and then stimulated the tissues with basolaterally-added forskolin (10 mM).

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2.5. Reagents Zinc sulphate, carbachol, forskolin, histamine, prostaglandin E2 (PGE2), and nystatin were purchased from Sigma-Aldrich Ltd. (Dublin, Ireland). Stock solutions were made up in de-ionised water (zinc sulphate, carbachol, histamine), DMSO (carbachol, nystatin), or ethanol (forskolin, PGE2) and diluted to working concentrations in physiological solution. The concentration of ethanol and dimethyl sulphoxide never exceeded 0.1% in the final solution. 2.6. Intracellular cAMP measurement Following specimen collection, preparation, and isolation of crypts as mentioned above, crypt pellets were incubated with zinc sulphate or vehicle for 20 min before addition of the adenylyl cyclase activator, forskolin. After 10 min, crypts were lysed by incubating them with 0.1 M HCl for 30 min and then centrifuged at 450g for 10 min. The supernatant was extracted and snap frozen in liquid nitrogen, and run in the assay in accordance with the manufacturer’s guidelines. 2.7. Light microscopy assessment of mucosal damage Following addition of basolateral or apical zinc sulphate (100mM–1mM) in the presence or absence of cD (8 mM) for 60 min, specimens were fixed in 10% buffered formalin for at least 48 h before being embedded in paraffin wax. 5 mm sections were obtained from a mictrotome (Leitz 1512; GMI, USA), mounted on adhesive coated slides, and then stained with haematoxylin and eosin (H&E) or Alcian blue 8GX (AB). A light microscope (Labophot—2A; Nikon, Japan) was used to acquire the images, which were captured using a high-resolution camera (Micropublisher 3.3 RTV; Qimaging, Canada) and Image-Pro Pluss software version 6.3 (Media Cybernetics Inc, USA).

2.3. Permeabilization of the apical membrane

2.8. Statistical analysis

To investigate basolateral potassium ion (K þ ) transport, the apical membrane was bathed in a high K þ solution (120 mM) to mimic intracellular concentrations and then permeabilised with nystatin. The basolateral membrane remained bathed in low K þ (5 mM) solution. Under these conditions, the transepithelial current is generated by the K þ conductance of the basolateral membrane. Permeabilization experiments were performed using an apical solution with the following ionic composition (mM): NaCl, 17; CaCl2, 0.3; MgSO4, 3.0; KH2PO4, 1.2; K2HPO4, 2.9; glucose, 11; K gluconate, 123 mM; and HEPES, 5 mM. The corresponding basolateral solution comprised (mM) NaCl, 20; Na gluconate, 100; K gluconate, 5; NaHCO3, 25; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; and glucose, 11.

Results are expressed as mean 7standard error of mean (S.E.M.). Changes in Isc are reported as DIsc (mA cm  2) or DIK in nystatin-permeabilised mucosa. Statistical analyses were performed using Wilcoxon’s test and ANOVA as used to compare mean values. Values of IC50 were generated from concentration– response curves analysed by non-linear fitting of parameters in the Hill equation; p o0.05 was considered statistically significant.

2.4. Isolation of colonic crypts Segments of colonic mucosa were weighed and incubated in hyperosmolar EDTA solution (composition in mM: NaCl 96, KCl 1.5, HEPES/Tris 10, NaEDTA 27, Sorbitol 45, Sucrose 28, pH 7.4) for 30 min and agitated gently every 10–15 min. The hyperosmotic nature of the solution combined with the chelating effects of EDTA separated the underlying lamina propria and submucosa, leaving intact colonic crypts. A pellet of isolated crypts was obtained by centrifugation at 100 rpm. for 1 min. The supernatant was removed and crypts re-suspended in KH solution and kept on ice until use. The viability of crypts was assessed by trypan blue exclusion. Trypan blue viability was 91.8% 72.4% (n ¼5).

3. Results 3.1. Effects of zinc on baseline bioelectrical parameters of human colonic mucosae The baseline values for human colonic mucosa were Isc: 104.3712.2 mA cm  2, PD: 8.871.0 mV, and TEER: 10278 O cm2 (n¼36). These values were stable for up to 60 min and were not altered by application of zinc sulphate at concentrations up to 1 mM. 3.2. Zinc inhibits forskolin- and PGE2-stimulated electrogenic ion secretion in human colonic mucosae Following pre-incubation with 100 mM Zn þ the basolateral sidetissues were exposed to forskolin (10 mM). Addition of forskolin to the basolateral side caused a rapid onset, sustained increase in Isc, Fig. 1(A). Pre-incubation with basolateral zinc sulphate attenuated the forskolin-induced DIsc in a concentration-dependent manner with

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80

200

ΔISC (μA.cm-2)

Isc (μA.cm-2)

FSK 10μM

150 100

60 40 20

50

0

0 0

5

10

15

20

Control

25

Time (min)

Treated

Fig. 3. Ion transport responses to PGE2 (1 mM) were attenuated by pre-incubation with basolateral zinc sulphate (100 mM; * po 0.05; n¼ 4).

200 cAMP (fmol/mg)

ΔIsc (μA.cm2)

100

50

0 Control

100μM

1mM

Fig. 1. (A) Forskolin applied to the basolateral bathing solution caused a rapid onset, sustained increase in Isc (n ¼16). (B) Basolateral zinc sulphate inhibited responses to forskolin (10 mM) in a concentration-dependent manner with an IC50 of 4.5 mM (95% CI ¼1.2  10  6–1.8  10  5 M; n¼8 for each; * p o 0.05, ** p o 0.01).

150 100 50 0 FSK

Zn + FSK

Fig. 4. Pre-treatment of colonic crypts with zinc sulphate (100 mM) attenuated the forskolin-induced rise in intracellular cAMP (* p o 0.05, n¼ 5).

ΔIsc (μA.cm2)

200

These results indicate that sensitivity to apical (luminal) zinc sulphate can be conferred by enhancing paracellular permeability with cytochalasin D.

150 100

3.4. Zinc attenuates secondary intracellular messenger production 50 0 Control

cD

cD + Zn (A)

Fig. 2. Apical zinc sulphate (1 mM ) was without effect upon ion transport responses to forskolin. However, in the presence of cytochalasin D (8 mM), apical zinc sulphate (1 mM) inhibited the forskolin-induced ISC in a manner similar to that of basolateral zinc sulphate in the absence of cytochalasin D (** p o0.01, n¼ 4).

To determine whether zinc inhibits cAMP-mediated secretion by inhibiting the production of this secondary intracellular signalling molecule, we measured its levels in isolated colonic crypts following stimulation by forskolin (10 mM). Pre-incubation with zinc attenuated the forskolin-induced rise in intracellular cAMP: control 133760 fmol mg  1, treated 28 78 fmol mg  1; po0.05, n ¼5, Fig. 4). 3.5. Zinc inhibits calcium-mediated chloride secretion

an IC50 of 4.5 mM (95% CI¼1.7–21; n¼8 for each. Fig. 1(B). Apical treatment with zinc sulphate had no significant effect on the forskolin-induced rise in Isc control; 124.2711.5 mA cm  2 vs treated; 108.7712.1 mA cm  2; n¼6). The addition of PGE2 (1 mM) to the basolateral bathing solution also induced a rapid sustained increase in Isc that was inhibited by pre-incubation with zinc sulphate (100 mM; n¼4, po0.05; Fig. 3). 3.3. Tight junction modulation enables apically applied zinc to inhibit forskolin-stimulated Isc Treatment of tissues with apical addition of the tight junction opener, cytochalasin D, caused a drop in TEER compared to control (Fig. 2). However, in the presence of cytochalasin D, apical zinc sulphate attenuated the forskolin-induced Isc similar to basolateral zinc sulphate in the absence of cytochalasin D (n ¼3, p o0.01; Fig. 2). Importantly, apical zinc sulphate had no effect on the forskolin-induced rise in ISC in the absence of cytochalasin D.

Basolateral addition of histamine (100 mM) or carbachol (10 mM) evoked a rapid, transient increase in Isc. In each case, pre-incubation with zinc sulphate (100 mM) attenuated the responses (Fig. 5A and B). 3.6. Effect of zinc on nystatin-permeabilised mucosa Addition of nystatin to the apical membrane unveils a transepithelial current that reflects channel-mediated K þ flow across the basolateral membrane, the trans-basolateral K þ current (IK). Nystatin added apically (100 mg ml  1) caused a sustained increase in IK. A decrease in the nystatin-induced IK was apparent in response to basolaterally-applied zinc (Fig. 6). Carbachol (10 mM), but not forskolin) induces a rise in IK in nystatinpermeabilised mucosae by activating basolateral potassium channels, an effect that was also attenuated by pre-incubation with basolateral zinc (Fig. 7).

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zinc sulphate (1 mM), tissues were fixed for histology. Tissue architecture was normal and treated tissues could not be distinguished from their respective vehicle controls (data not shown).

ΔISC (μA.cm-2)

200 150

4. Discussion

100 50 0 Control

Treated

ΔISC (μA.cm-2)

200 150 100 50 0 Control

Treated

Fig. 5. (A) Responses to carbachol (10 mM) were inhibited by basolateral pretreatment with zinc sulphate (** p o 0.01; n¼ 4). (B) Responses to histamine (100 mM) were also attenuated by pre-incubation with basolateral zinc sulphate (* p o0.05; n¼ 4).

80

ΔIK (μA.cm-2)

60

40

20

0 Control

Treated

Fig. 6. Pre-incubation with basolateral zinc sulphate inhibited the nystatininduced IK across the basolateral membrane (* p o 0.05; n ¼4).

100 ΔIK (μA.cm-2)

169

50

0 Control

Zn 10μM

Zn 100μM

Fig. 7. Carbachol (10 mM)-induced IK was also attenuated by basolateral zinc sulphate pre-treatment (p o 0.05; n¼ 4)

3.7. Zinc þ exerts its effects without disrupting colonic mucosal architecture At the end of experiments during which voltage-clamped human colonic mucosae were incubated with apical or basolateral

A growing body of literature supports zinc as an inexpensive and useful intervention in treatment of diarrhoea (Frohna, 2011; Gitanjali and Weerasuriya, 2011; Kulkarni et al., 2012). Mechanisms of zinc’s actions have been studied in vitro using human cell lines (Canani et al., 2005; Canani, Ruotolo, 2006; Berni Canani et al., 2011) and also isolated tissues from rodent or pig (Carlson et al., 2006). The resting parameters and secretagogue-stimulated changes we report here are consistent with previous reports using voltage clamped rat colon in vitro (Collins et al., 2009). We examined chloride secretion stimulated pharmacologically by two separate transduction pathways (Kunzelmann, 2002). Forskolin and PGE2 elevate intracellular cAMP to cause Cl  secretion, and carbachol and histamine elevate intracellular Ca2 þ to stimulated epithelial Cl  secretion (Keely et al., 1995; Chough et al., 1993). The presence of zinc in the basolateral (but not apical) compartment significantly attenuated the stimulated transepithelial Cl  secretion induced by both cAMP- and Ca2 þ mediated secretagogues. Such sidedness was a feature of our previous study in rats (Bzik et al., 2012). This inhibitory effect may be an important component in the effectiveness of zinc as an anti-diarrhoeal agent. Zinc alone was without effect on basal electrophysiological parameters, in contrast to other reports of potassium channel blocking agents reducing resting short circuit current and enhancing electrical resistance and epithelial barrier function (Wang et al., in press). Colonic chloride secretion and consequent fluid secretion are becoming well understood (Bachmann et al., 2011; Barrett and Keely, 2000). Enterocyte cAMP, which is increased by forskolin and PGE2, is an important signalling molecule during secretory responses to cholera toxin and other heat-labile bacterial endotoxins (Field, 2003). cAMP activates protein kinase A, which directly phosphorylates apical Cl  channels (Cohn et al., 1992; Berger et al., 1993). We examined whether zinc influenced intracellular levels of cAMP in isolated crypts of human colonic mucosa following stimulation with forskolin. Forskolin caused a dramatic increase in levels of intracellular cAMP that was attenuated in the presence of zinc, indicating that zinc acts directly to inhibit cAMP-stimulated Cl  secretion via a mechanism that is independent of cAMP production, findings in keeping with those in colonic cell lines and animal models (Canani et al., 2005; Feng et al., 2006; Hoque et al., 2005). Further evidence to support the anti-secretory effects of zinc was acquired by stimulating Cl  secretion in isolated human colonic mucosae using carbachol and histamine agents that induce transepithelial Cl  secretion by increasing intracellular levels of Ca2 þ (Barrett and Keely, 2000). Zinc attenuated the Ca2 þ -induced secretory response when added to the basolateral membrane. These findings are supported by the observations that zinc treatment of Caco-2 epithelial cells and porcine small bowel attenuates ion transport (Berni Canani et al., 2010; Carlson et al., 2006). Zinc has also been reported as an inhibitor of gastric acid secretion (Kirchhoff et al., 2011). Contraction of intestinal smooth muscle, which was not examined in this study, has been reported to be attenuated by zinc which acts as a spasmolytic in vitro (Cho and Teh, 1991; Schnieden and Small, 1971). Since zinc appeared to act as a non-specific inhibitor of Cl  transport we asked whether its actions may be due to an effect on basolateral ion transporters involved in colonic anion secretion (Bachmann et al., 2011). We found that a likely target of exogenous

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zinc is upon basolateral K þ channels, upon which Cl  secretion depends (Kunzelmann, 2002). We utilised nystatin to permeabilise the apical membrane and enable the study of basolateral K þ transport (Lewis et al., 1977). Under these conditions, evidence was obtained to suggest that zinc inhibits basolateral K þ transport in permeabilised isolated segments of colonic human mucosae, consistent with findings in colonic cell lines. The effect of zinc on sodium absorption was not examined in the present studies. However, (Hoque et al., 2005) showed, using rodent ileum, that zinc did not affect Na–K–2Cl co-transport, which was estimated by the bumetanide-sensitive component of 86Rb uptake which (as a surrogate of K) was determined in the presence of ouabain and barium. Zinc is known to influence voltage- and ligand-gated channels in a wide range of tissues (Harrison and Gibbons, 1994; Jiang et al., 2012; Smart et al., 1994) and may act through zinc-dependent Ca2 þ signalling via the putative ZnR pathway (Hershfinkel et al., 2007). We found that zinc inhibits both cAMP- and Ca2 þ -dependent epithelial ion transport processes in human colonic mucosae. Such non-specific inhibition is similar to that of other K þ channel-blocking agents, including loperamide (Taylor and Baird, 1995), berberine (Taylor et al., 1999), clotrimazole (Rufo et al.,1997), and levamisole (Mun et al., 1998). These drugs inhibit Cl  secretion regardless of the stimulus, possibly broadening their potential therapeutic utilizations. Fully understanding the mechanism of action via which zinc exerts its effects will move us closer to incorporating it into everyday treatment of diarrhoea in adult and infant populations (Kulkarni et al., 2012; Buccigrossi et al., 2010; Patel et al. 2011; Irlam et al., 2010). In conclusion, zinc-inhibited chloride transport in voltage clamped isolated human colonic epithelia. Zinc’s inhibitory action was promiscuous in that responses to a range of secretagogues were prevented. The anti-secretory action of zinc required access to the basolateral side of the mucosal sheet and was mediated by K channel blockade. Apical-side sensitivity to zinc was conferred by using cytochalasin D. The mechanism of action appears to involve inhibition of basolateral potassium conductance, since zinc also inhibited ion transport changes in permeabilised tissues. The specifc target(s) or sites of zinc’s anti-secretory action remain to be identified. These data establish a mechanism which accounts for clinical benefits of zinc supplementation which, in combination with oral rehydration therapy, has shown to significantly reduce the duration and severity of acute and persistent childhood diarrhoea.

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