The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components

174 ~ o c h i m i c a e t B i ~ c a . , 4 c t a . !115(1991) 174-179 i~ 1~1 Ehev/er Sc/cnce PublLshcrs B.V. All r/ehls reser~-cd 0.~4-4165/91/.fd)3.50

BBAGEN Z3622

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components

L y n d a A . S e l l e r s ~, A d r i a n A l l e n ~, E d w i n R . M o r r i s ~- a n d S i m o n B. R o s s - M u r p h y 3

I Deparm'~mt o f P~,siological Sc/er.ces, The Med&al Scho~ Unii-ersity o f Newcastle upon Tyne, New,cable ~q~on Tyne (U.KJ, 2 Cranfiwld ltastzt~:e o f T e c h n o ~ ; Silsoe Cagege, Silso¢. Bedford (U.K) and 3 D e p a r t ~ o f PtO, sics , C a L ' ~ LabaraloO;

Camb~ge ¢ U.K_)

tRecehred 1 Malrch 1991) { Revised man~scl/p~ rccci,,-ed ~ August 1991)

Kt.~- v.-,nd,,: Mucus; Sma|l intestine; Cobn , Mechan/can s l ~ c t ~ Visotmlasli¢ gel; Mucin g~c~co~cha

Mechanical spectn~seopy has been used to study the structure and properties of ~ small inlesfiaal and coleaic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but tha t from the small intestine was substantially weaker in qualily. Small intestinal mucus gel was disrupted by acid (pH I), detergents (bile) and protein denaturants while tha t from the colon remained stable following these treatments. Co~eatra_lioa of colonic mucin produced a gel with the same rheelogical properties as the native secretion. ~ .small i n ~ mucin when concentrated lmalneed a stronger gel than the native secretion and, in oeatlrast to the latter, one which was not disrulRed by acid or denaturants . The instability of native small intestinal mucus was shown not to be a functio~ of the mucin components (which alone could account for the gel-forming preperfies), lint to arise from the presence of insoluble material largely from sloughed mucesal cells. These studies show (!) tha t mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution tha t should be exercised when interpreting the rlteolegiral properties of mucus preparations, particularly with respect to their content of mucosal cellular materiaL

Introduction

The gastrointestinal mucus barrier is a layer of gel adherent it, the mucosal surface. The median thickness of this mucus layer is 180 t tm in human stomach and 80 t~m in rat stomach or duodenum respectively, and 150 t tm in rat colon; in these tissues the layer is continuous [1-3]. Rheological studies show adherent mucus preparations gently scraped from the surface of human or pig stomach and pig duodenum are viscoelastic gels [4.5]. Similar viscoelastic gel properties have been shown for respiratory mucus secretions al- though, in relation to mucociliary dearance, they have more tendency to flow (deform) than the more 'rigid' gastrointestinal mucus gels [6-8]. Gastroduodenal mu-

Correspondence: A. Allen. Department of Physiological Sciences. The Medical Scherzi, Framlington Place. Newcastle upon Tyae. NE2 4HH, U.K.

cus gel structure is not changed by exposure to acid, detergents, e.g. bile, and protein denaturants, e.g. 6 M guanidinium chloride or 8 M urea [4]. When the purified mucins from gastroduodenal mucus, free from detectable protein, nucleic acid and fipid, are reconstituted at their in vivo concentrations, they form a gel with the same rheological properties as the native secretion [4,5]. One model which has been proposed for gastrointestinal mucus structure is where gel-forming interactions arise between the polymeric mucin molecules by interdigitation of their carbobydrate side chains [4,9]. Other models of gel-formation in mucus include those involving more specific "lectin type" interactions [10].

The mucin pul~meric structure of subunits joined by interchain disulphide bridges is essential for the molecule to participate in gel formatinn [9,11]. How- ever mucins which differ widely in the composition and length of their carbohydrate side chains can all form gels with the characteristic viscoelastic gel structure [9,12].

This paper describes studies which demonstrate that pig small intestinal mucus, in comparison with other gastrointestinal mucus secretions including that from the colon, has a weaker gel structure which collapses ~vhen exposed to denaturants. This instability of small intestinal mucus was shown not to be a function of :.he mucin components which possess full gel-forming properties, but to arise from the vresence in the mucus of insoluble material, largely from sloughed mucosal cells.

l~Laterials and Methods

Pig small intestinal and colonic mucus gel was obtained by gently scraping the surface of the washed mucosa with a small plastic scoop avoiding, as far as possible, the removal of epithelial cells. Mucus from either the intestine or the colon was pooled and either investigated immediately or stored at - 2 0 ° C until required. The glycoprotein content of the mucus secretions was assayed by the modified periodic acid-Schiff (PAS) method [13] after the sample of gel had first been exhaustively digested with papain (EC 3.4.22.2) and diah,~sed against 0.2 M NaCI-0.02% (w/v) NaN~ to remo~:e hydrolysed protein.

The mucus gel samples were solub" ~dL~ed individually by brief homogenisation (1 rain) in 0.2 M NaCI-0.02% (w/v) NaN 3 (4 vols.). Samples were centrifuged (1000 x g, 10 mitt) to remove cell debris, the supernatant from each s~mple was loaded on to a Sepharose CL-2B column (1.5 × 140 cm) and eluted with 0.2 M NAC1- 0.02% (w/v) NaN 3. Fractions (3 ml) were collected and analysed for glycoprotein and protein [13,14]. The percentage of total material recovered was 84-93%. The amount of excluded relative to included glycoprotein (fractions 19-31 and fractions 32-70, respectively) was calculated. The smafl PAS peak in the total volume (fractions 71-85) was ignored, since this was totally accounted for by protein interference in the PAS assay, and mucin purified by equih'brium centrifugation in CsCi did not contain this peak.

Mucin glycoprotein was isolated from small intestinal or colonic mucus, purified and reconstituted into a gel by concentration to 28-33 mg nd -~ and 19-20 mg ml- l , respectively. Pooled mucus scrapings in 4 times their own vol. of ice-cold 0.2 M NaCI-0.02% (w/v) NaN 3 were homogenised in a Waring blender at full speed for 1 rain at 4 ° C. After centrifugation (6000 × g, 1 h), the supernatant was strained through glass wool to remove insoluble lipid-rich material. The glycoprotein was separated from protein and nucleic acid by equih'brium centrifugation in a CsC! density gradient [15]. Mucus glycoprotein fractions from the first gradient were further purified by subsequent centrifugation in a CsCI gradient. After centrifugation, the glycoprotein fractions were pooled, dialysed against ¢.}.2 M NaCI-0.02% (w/v) NAN3, concentrated by vacuum

dialysis and redialysed against 0.2 M NaCI-0.02% (w/v) NaN 3. The is¢lated glycoprotein was concentrated to 28-33 mg mi- i and 19-22 mg ml- i for small intestinal and colonic mucus, respectively, to give reconstituted gels with glycoprotein concentrations comparable to those of the native secretions. Glycoprotein samples used to prepare each batch of gel (400-500 p.g of glycoprotein) were run on 7.5% polyacrylamide disc gels (7 cm long) in the presence of sodium dodecyl sulphate [16] and stained for protein with Coomassie blue. All mucin samples purified by fr,,~*.ionat;~on in CsCI were shown, by this method, to be free of detectable protein. In some experiments muein glycoprotein purified in a CsCI density gradient was mixed with either protein fractions from the same gradient or the pellet following centrifugation of the homogenised mucus. This mucin mixed with protein or pellet was concentrated by vacuum dialysis at 4°C to give equiva- lent mucin con~ntrations to that in the unfractionated native gel secretion.

Dynamic oscillatory measurements on native gel samples or gels reconstituted from purified mucins were performed on a Rheometrics Mechanical Spec- trometer (Rheometrics, Piscataway, U.S.A.) using a cone and plate system (cone diameter 25 ram, cone angle 0.1 radian). Environmental control of the sample chamber was by forced air convection, the air being supplied from a thermostatted water bath and humidi- fied at the working temperature of 25°C. Sample temperature was regulated to _+ 0.3 °C and measured by use of a platinum resistance thermometer. The storage modulus G ' (elastic component) and the loss modulus G" (viscous coraponent) were expressed as functions of frequency (frequency range 10-2-102 rad s - t ) at a constant strain of 20%.

After loading, all samples were allowed to equili- brate (15 min) to the measuring temperature and to relax from any major stresses that may have been induced by the loading procedure. The mechanical properties of the mucus samples were also unchanged over four successive frequcncy scans (2.5 h), showing that dehydration of the gel or other mechanical dam- age was not a factor in the measurements. Strain scans (!0 tad s - l) on all mucus gels showed that both moduli were independent of strain in the range 5-95%.

Native mucus or gels reconstituted from isolated mucin were dialysed against 0.1 M HCI glycine buffer pH 1, 6 M gnanidininm chloride or 8 M urea. Gels were also incubated with undiluted pig bile. The native mucus and reconstituted mucin gels were reduced by dialysis against 0.2 M mercaptoethanol in 0.2 M sodium phosphate buffer at pH 8.0 (0.02% (w/v) NaN 3) at 4 °C. For all treatments, control samples of gel were incubated or dialysed over the same time-course against control buffers, after which their mechanical spectra was shown not to be significantly changed by the maxi-

176

mum 3% change in the absolute value o f any parame- t e l

Results and Discussion

Colonic mucus was present as a thick covering o f t ranslucent gel firmly adheren t to the mucosai surface. This gel, removed from the mucosa by gentle scraping, had a mechanical spectrum indistinguishable from that shown previously for gastric and duodenal mucus and characterist ic o f a weak ,~scoelastic gel [4,5,17]. Throughout the frequency range s tudied ( 1 0 - ' - 1 0 2 rad s - t ) the s torage (elastic) modulus {G' , characteristic o f solid proper t ies) was substantially grea ter than the loss (viscous) modulus (G" , characterist ic o f liquid proper t ies) and both showed little frequency dependence (Fig. 1). Dialysis o f fresh colonic mucus gel for 24 h at 4°C against HCI p H I or pH 2, phosphate buffer pH 7.5 or t rea tment with bile (direct from pig gall b ladder) did no~ significantly change it~ mechanical profile. The mechanical proper t ies of the gel under different condit ions are expressed (Fig. 2) in terms o f log tan 6, the ratio o f the viscous to elastic modulus ( G " / G ' ) , where a small value of log tan 6 ( < I) indicates a predominant ly elastic response and where a larger tan 6 ( > 1) indicates a predominant ly ~Jscous response.

Purified colonic mucin was p repa red by homogenisation and centrifugation (10000 x g - 6 0 min) and two fract ionations o f the resulting soluble mucus in a caesium chloride gradient . This colonic mucin preparation, shown by SDS gel e lectrophoresis to be free o f

Fig. 1. Viscoelasticity G', storage modulus (filled symbo~ continuous lines) and the loss modulus G" (open symbots, broken lines) for:. fresh colonic mucus (ll E3~ fresh pig small intestinal mucus (o o~ small intestinal mucus reconstituted from purified glycoprotein ( • ~ ); and small intestinal mucus after exposure to HC1, pH ! for 24 h 4°C (0 <>). The scan was over a frequency range of 10-2-10 2 tad s- t at 20% strain, with three measurements being recorded per

frequency decade.

2 ~\~\%%-%.\\%%.\xx%,%-%~

N\~"%'~X%\%~N\~%%~~~%~X%N~\\\\\\\\\\\\\\\\\N\\\N\\\\N\N/\N ~

.ca .o~ ,o~ o -az -el -c-~ -~e -~

F~g. ~ Comparison of the ratio of loss ~ t t s to s/~rage modulus, tan 6 (at I tad s- a 20% su'-,.m) fc~ pig c o ~ c mucus.- ( | ) fresh mucus; (2) mucus exposed to HCL pH t, 24 h, 4 ~ (3) mucus exposed to bile 24 h, 4~C: (4) mucus ~ to 6 M ~ u m chl~'kk, 24 h, 4°C; (5) mucus exposed to 0.2 ° M mercaptoellmm~ 24 h, 4"C; (6) mucus digested by papa/n, 24 h, 37~C; (7) gel rec~asfituted

from colonic toucan -= 2~ ~g m] -~ .

non-covalently bound protein, was concent ra ted to form gels with a concentrat ion o f 19-20 mg n d - s (mean 20, n = 15), which was within the range of mucin concentrat ion found in the native secretion. The reconst i tuted colonic mucin gel bad a , ~ o c l a s t i c response indistinguishable from the native secretion. This toge ther with the resistance o f the colonic mucus gel to acid or bile t rea tment provides fur ther evidence that colonic mucus has the same type o f gel s tructure as that f rom the s tomach and duodenum.

The colonic mucin preparat ion, purif ied in a CsC! density gradient , was shown to be free o f detectable non-mncin protein and, from o the r studies, f ree f rom detectable nucleic acid and lipid [18,19]. This and similar s tudies with purified gastroduodenal mucins [4,5,9] demonst ra te that the purif ied mucin can repro- duce the gebforming proper t ies o f the native secretion. Brief homogenisat ion (1 rain) used in these studies to solubilise the native mucus gel does not therefore impair the gel-forming ability o f the result ing purif ied mucins. The purified colonic mucin was extracted in 0.2 M NaCI, without added proteinasc inhi'bitors, and by analogy with o the r mucin preparat ions it will almost certainly have undergone limited proteobjtic nicking during isolation [20-22]. However such mucin preparations, which are sensitive themselves to incubatiort with added proteinase, clearly retain the key structural ele- ments necessary for gel formation and provide a good model for studying the molecular interactions forming the gel matrix. This is particularly relevant since pig gastric mucin extracted with proteolytic inhibitors and guanidinium chloride, condition~ which rigorously ex- clude proteolysis [20,21], precipitates at gel-forming concentrat ions [22]. A reasonable explanation o f this is that the tertiary structure o f the mucin, essential for its gel-forming propert ies, is lost on exposure to guanidinium chloride. The absence of effect o f guanidinium chloride on the native colonic mucus presumably re-

flects the stability, in this denaturant, of the non-cova- lent gel-forming interactions, which maintain the tertiary structure of the mucus.

Small intestinal mucus, present in copious amounts and very easily removed from the washed mucosal surface, was opaque and heterogeneous in appearance. Histological staining of this mucus preparation showed it to be very heavily contaminated with mucosal cells despite its gentle removal from the mucosa. The mechanical profile for fresh small intestinal mucus was of a noticeably weaker, poorer quality gel than that from the colon, duodenum and stomach. Thus the storage (elastic) modulus (G ' ) and the loss (viscous) modulus (G °) were much closer together and both showed a strong dependence on frequency (Fig. 1). The gel structure of fresh small intestinal mucus was completely collapsed following dialysis for 24 h at 4°C against HCI pH 1 ~3r against pig gall bladder bile, whilst gel quality was substantially reduced in 6 M guanidininm chloride. The frequency profile for fresh small intestinal mucus following exposure to acid pH 1 (Fig. 1) was characteristic of a viscous liquid with the loss modulus (G °) dominant over the storage modulus (G ' ) throughout the frequency range. Following exposure to bile (pig gall bladder), small intestinal mucus gave a liquid emulsion which was too weak for rheological measure° merits to be made. This instability of fresh small intestinal mucus was again in contrast to the other gastrointestinal mucus secretiop~s.

Small intestinal mucin was pwified by fractionation in a double CsC! gradient and concentrated to form gels of 28-33 mg ml - I (mean 30 mg ml -! , n = 8) within the range found for the fresh mucus secretion. These reconstituted mucin gels were distinctly stronger than the fresh small intestinal mucus secretion and had characteristic gel-like spe~ra comparable to those of colonic and gas-:ruduodenal mucus. Thus the storage modulus of the reconstituted small intestinal muein gel was essentially independent af frequency and substantially greater than the loss modulus throughout the frequency range assessed (Fig. 1). The reconstituted small intestinal mucin gel was stable foll6~ing exposure to HCI (pH 1) for 24 h at 4°C with an unchanged mechanical profde (Fig. 3), again in sharp contrast to the native mucus secretion.

Studies on gastrointestinal secretions have demonstrated that gel formation is dependent on the mucin polymeric stpacture which is destroyed by reduction of disulphide bridges or proteolysis [5,11]. Pig small intestinal and colonic mucus have both been shown to have a polymeric structure [23,24]. The relative amounts of polymeric mncin (excluded on Sepharose CL-2B) to lower sized degraded mucin (included on Seph.,rose CL-2B) can be quantitated by size-exclusion chro- matography [9,13]. For fresh and reconstituted small intestinal and colonic mucus as well as in the same

177

1 I ~ \ \ \ \ \ \ \ \ \ \ \ \ ~ .

~ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ' . x ~

s ~ N N N N N N ~ ' ~

I

, i [ i i i i ' t I i

*02 0 -O.a -O.4 -0.6 -Oe Log "ra, 6

Fig. 3. A comparison of the ratio of loss modulus to storage modulus, tan fi (at I red s -I, 20% strain) for pig small intestinal mucus: (1) fresh mucus: (2) mucus exposed to acid HCI, pH 1, 24 h, 4°C; (3) mucus exposed to 6 M guanidinium chloride 24 h, 4°C; (4) reconstituted mucin gel ~-30 mg ml-t; (5) reconstituted mucin exposed to HCI, pH 1, 24 h, 4°C; (6) reconstituted mucin exposed to 6 M guanidinium chloride, 24 h, 4°C; (7) reconstituted mucin, 30 mg ml- I

mucin, with resospended pellet (! : 1 by volume).

preparations following treatment with HCI, pH 1 or protein denaturants (Table 1) and including the collapsed fresh small intestinal mucus preparations, the amount of polymeric mucin was greater than 70% of the total mucin present. This showed that deficiencies in mucin polymeric structure are not the explanation for the weakened gel structure of fresh small intestinal mucus or for its collapse in acid and protein denaturants.

Exposure to 0.2 M mercaptoethanol for 24 h or proteolysis caused .q complete collapse of gel structure in both fresh small intestinal and colonic mucus secretions as well as in the corresponding reconstituted mucin gels. The profiles of the reduced gels showed liquid-like mechanical spectra with the loss modulus dominant over the storage modulus and both showing a

TABLE l

Gel filtration of natice small intestinal mucin

Gel fdtration was by Sepharose CL-2B and a similar result was obtained for three different mucus preparations lot each treatment (average value given)

Treatment Time Excluded Storage (h) glycoprotein modulus G'

(% of total (Pa at eluted) 1 tad s- t)

Fresh mucus 76 3.83 pH 7.O 18 75 4.22 pH 1.0 18 75 0.5 6 M urea 18 75 0.7 8 M guanidinium

chloride 18 74 0.9 0.2 M mercaptoethanol 3 65 0.03 0.2 M mercaptoethanol 12 47 0.01 0.2 M mercaptoethanol 24 27 0.01

Reconstituted mucus 0.2 M NaCI - 75 3.82

178

"/:,7

oo~ '¢

, ' Y / l " ~ ...... %2; .........

Fig. 4. Tan 6 (at I tad s t lrt+m the frequent.3 profiles) of fresh I1) and reconstituted ( ~ } pig cobntc mucus gels: fresh (o} and reconstituted (0) pig small intestinal mucus, gels. All samples were incubated with 0.2 M mercaptoethamfl for various time intervals at 4 or 37=C. The percentage of pot',merit mucin in the total mu¢in gl~,coprotein

was cuk:ulated by the amount excluded by Scpharos¢ CL-2B.

strong frequency dependence. The dependence of gel structure on mucin polymcrisation was demonstrated by plotting the percentage polymeric mucin in the sample (Table 1) as a function of mechanical properties (measured as tan fi) over the time-course of reduction where tan fi increases as the sample progressively he- comes more liquid-like. Log tan fi increases linearly with decreasing content of mucin that is excluded by Sepharose CL-2B (polymer) during the progressive reduction of reconstituted small intestinal mucin, fresh colonic mucin or reconstituted colonic mucin (Fig. 4). This linear relationship, previously observed for pig gastroduodenal mucus secretions, shows the direct dependence of gel quality and strength on mucin polymer;c structure [9,12]. it also provides further evidence that colonic mucus and reconstituted colonic or small intestinal mucin gels have essentially the same viscoelastic structure as those from stomach and duodenum.

The structure of fresh small intestinal mucus (as judged by mechanical spectroscopy) collapsed substantially quicker than that for the corresponding reconstituted mucin gel at the same concentration. For exam- ple, after 3 h reduction at 4°C in 0.2 M mercaptoethanol, fresh intestinal mucus had completely collapsed with a log tan ~ of > 1 while the reconstituted intestinal mucin gel still retained its gel-forming char- acteristics after 12 h under the same conditions, with a log tan fi of < 1. Also a plot of log tan fi ver0us the percentage of residual polymeric mucin at various times

of reduction of small intestimd mucus was not linear, in contrast to those of the reconstituted small intestinal mucin gel and other mucus geLs (Fig. 4), but showed a sharp decrease in gel quality for a relatively small decrease in the percentage of mucin in the polymeric form. This~ together with the stable reconstituted mucin gel having a similar content of polymeric mucin, indicates other factors (than cleavage of the pol~aeric structure) contribute to the rap/d collapse of small intestinal mucus on reduction in mercaptoethanol. These results again emphasise the instability of the fresh small intestinal mucus secretion, in this case to disulphide bond-breaking agents, in contrast to the reconstituted mucin gel and other gastrointestinal mucus secretions.

The above studies pointed to non-mucin components removed during mucin isolation that were re- sponsible for the poor mechanical properties and instability of the fresh small intestinal mucus secretion. Therefore studies were designed to investigate the effect on the theological properties of adding back to the reconstituted small intestinal mucin gel the soluble protein and insoluble cell debris removed during pu- rification. After brief homogenisation to dissoVqe the native mucus gel, the insoluble pellet obtained following centrifugation was always substantially greater from small intestinal mucus (approx. 80-90% of the volume) compared to that from colonic mucus (approx. 30% of the volume) and this reflected a much larger cellular content of the former (observed from comparison of intestinal and colonic mucns mi~oseop/cally). Analysis of the soluble small intestinal mucus gave means of glycoprotein 28%, protein 68% and nucleic acid 4% by weight (p = 15 preparations) while analysis of soluble colonic mucus gave means of glycoprotein 47%, protein 51% and nucleic acid 2% by weight (n = 25 preparations).

Purified small intestinal mucin was mixed with samples of the protein rich fractions from the first caesium chloride fractionation of the water soluble mucus in the ratio of glycoprotein to protein of 4:1, 3 :2 or 2:3 by wt., at a glycoprotein concentration of 30 mg ml- t. All mixtures of protein and mucin gave mechanical spectra more characteristic of the reconstituted purified small intestinal mucin gel than the original unfractionated secretion. This showed that the protein, com- prising about 70% by weight of the water soluble mucus, does not noticeably influence the gel forming properties of the constituent mucin. In contrast a mixture of one volume of pelleted cellular material (separated from the homogenised mucus by centrifugation) to one volume of purified mucin (at 30 mg ml - t final concentration) gave a mechanical spectrum very similar to that of the unstable weaker gel characteristic of the native mucus secretion (Fig. 3). A sample containing twice the volume of pellet to glycoprotcin was ex-

t remely he t e rogeneous with mechanica l propet ies ap- proximat ing to those o f a liquid (loss modu lus > s torage m o d u l u s at all frequ~.ncies). T h e s e resul ts a re shown in Fig. 3. showing increases in the value o f log tan 6 on going f rom the reconst i tu ted pure small intest inal muc in gel to the native m u c u s and the fur ther in- c reases for a reconst i tu ted mixture o f pu re mucin and excess o f insoluble pellet. Th i s s tudy shows tha t the anom a lous proper t ies o f small intest inal m u c u s gel a re due to the excessive a m o u n t s o f mucosa l cells, which precipi ta te as the insoluble pellet following homogeni - sat ion and centr i fugat ion. T h e insoluble pellet was essential ly res is tant to papa in digest ion bu t was solu- bilised by bile, in keeping with a p redominan t ly m e m - b rane lipid content , i t is not c lear w h e t h e r this gross cel lular con tamina t ion is a fea ture o f the intest inal m u c u s secret ion in vivo o r if it reflects removal o f mucosa l t i ssue dur ing the collection o f the m u c u s gel by scraping f rom the mucosa l surface. Native gastric, duodena l and colonic m u c u s scrapings all have a substantially lower prote in con ten t (17.4 m g m l - J , 13.3 m g m l - t and 22 m g ml - t, respectively) compared to small intest inal m u c u s (72.8 m g ml -~) indicative o f a m u c h lower cellular con ten t in the fo rmer secret ions.

i t has been sugges ted that non-muc in pro te ins and lipids e n h a n c e the viscous and gel-forming proper t ies o f m u c u s gels [25-27]. O u r resul ts do not suppor t this hypothesis ; as poin ted out above, gels with the same rbeological proper t ies as the native m u c u s secret ions can be ob ta ined by concent ra t ion o f the purif ied mucins . Fur ther , in the case o f small intestinal mucus , insoluble (p redominant ly lipid) cel lular mater ia l causes a substant ia l weaken ing ra ther than a s t r eng then ing o f gel s t ructure . Also adding back the soluble prote in to the recons t i tu ted purif ied muc in gel had no effect on its theological propert ies . O n e c o m p o n e n t tha t can substant ial ly e n h a n c e viscosity o f m u c u s secret ions is D N A [18]. However the a m o u n t o f nucleic ac/d p resen t in the m u c u s prepara t ions was small ( 2 - 4 % by wt.) and was removed by densi ty grad ien t centr i fugat ion from the purif ied muc in used to reconst i tu te the gels.

W h a t is clear f rom this s tudy is tha t the presence o f an excessive a m o u n t o f cel lular mater ia l in an o ther - wise typical m u c u s secret ion can cause ser ious instability with respect to a variety o f agents . Fu r the r experi- men ta t ion is requi red to establish the mechan i sm o f this effect, bu t one plausible explanat ion is tha t the cell surfaces by b inding the mucin disrupt the gel matr ix and thus cause its instability. Whi le the genera l appli- cability o f this p h e n o m e n o n canno t be assumed , it is impor tan t in rheological s tudies on o the r m u c u s preparations, particularly those f rom pathological samples , tha t the effects o f cellular mater ia l a re assessed. Th i s might explain why pig colonic m u c u s and, f rom previ- ons s tudies , gastr ic and duodena l mucus [4] (relatively

179

low in cell conten t ) are res is tant to acid dena tu ran t s and bile while o ther mucus prepara t ions are similar to native pig intestinal mucus (high cell content ) and are d is rupted by such agents [6,28].

Admowledgement

The au thors thank Mr. R.M, Coan for technical suppor t .

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