the time-course of appearance and net accumulation ... - Science Direct

Camp. Biochem. Physiol. Vol. 88A. No. 3. pp. 507-510. Printed in Great Britain

0300-9629187 $3.00 + 0.00 Q 1987 Pergamon Journals Ltd

1987

THE TIME-COURSE OF APPEARANCE AND NET ACCUMULATION OF HORSERADISH PEROXIDASE (HRP) PRESENTED ORALLY TO RAINBOW TROUT SALMO GAIRDNERI (RICHARDSON) School

of Biomedical

Sciences,

E. MCLEAN and R. ASH* University of Bradford, Bradford, Telephone: (0274) 733466 (Receiced

West

Yorkshire

BD7

IDP,

UK.

I3 March 1987)

Abstract-l. A sensitive enzyme-linked immunosorbent assay (ELISA) technique was used in order to determine horseradish peroxidase (HRP) uptake from the rainbow trout gut. 2. HRP was detected in blood plasma and various tissues within 15 min of oral intubation. 3. The time-course of net accumulation (uptake-degradation) over a 75 min period was recorded. 4. The presence of HRP reached a maximum in the body tissues approximately 45 min after intubation

and on a rig/g wet weight basis the order of accumulation within the tissues was liver > spleen = kidney > plasma > heart. 5. The total organ accumulation (net) was in the order liver > plasma > kidney > spleen > heart.

INTRODUCTION It is

now well established (for review see Ash, 1985) that absorptive enterocytes located within specific regions of the teleost gut possess the ability to internalize intact protein molecules of large molecular weight (>40,000 daltons). Furthermore, such luminally presented proteins have also been shown to gain access to the intercellular spaces between absorptive cells (Noaillac-Depeyre and Gas, 1976) and have been located within the lamina propria (Iida and Yamomoto, 1984) and certain cell types associated with the intestine, e.g. leucocytes (Rombout et al., 1985). and plasma cells presumed (Noaillac-Depeyre and Gas, 1979). More recently, unequivocal evidence for the detection of such intact proteins (or at least significantly large polypeptide components thereof), within the blood and body tissues of fish has been presented (McLean and Ash, 1986). A variety of hypotheses have been proposed in an attempt to explain the physiological significance of these observations. Thus, Ezeasor and Stokoe (1981) and Georgopoulou et al. (1986) have suggested that the non-selective absorption of intact proteins may represent an extension of the normal digestive capacity of the fish gut. Alternatively, Stroband and van der Veen (1981) consider that this mechanism provides a safeguard against periods of digestive enzyme insufficiency (i.e. during larval development, immediate post-aestivation and other periods of temporary food deprivation) when protein digestion might be compromised. On the other hand, Hofer and Schiemer (198 1) and Hofer (1982) have proposed that this phenomenon may be linked to the provision of an enteropancreatic circulation of digestive enzymes (for review see Xu, 1985), while Noaillac-Depeyre and Gas (1979) and Davina et al. (1982) have sug--__ ___*Author

to whom

correspondence

should

be addressed.

gested that the absorption of intact proteins may be linked to antigen sampling and the subsequent mounting of an immune response in a similar manner to that described for the mammalian M cell (for reviews see Wolf and Bye, 1984; Egberts et al., 1985). However, given the paucity of quantitative data currently available concerning the extent of uptake of intact, dietary-derived proteins and their subsequent fate within the body of the fish, the physiological significance of this phenomenon must remain largely a matter for conjecture. Experiments described in this report were therefore designed to investigate the time-course of appearance (tissue net presence) of a specific protein (horseradish peroxidase) presented orally to the rainbow trout (Salmo gairdneri R.). Information so obtained would, in combination with previously published data (McLean and Ash, 1986) enable comparisons to be made between aspect of intact protein absorption in representative species of economically important “gastric” and “agastric” fish. MATERIALS AND METHODS

Rainbow trout (S&no guirdneri) of approx. 200 g weight were obtained from a local fish farm (Ripponden Fish Farm, Ripponden, W. Yorks. UK) and maintained in a commercially available holding facility as previously described (Ash, 1980). Subsequent to a 48 hr period of food withdrawal. horseradish peroxidase (HRP type I, Sigma Chemical Co.. Poole, Dorset, UK) was administered to the fish by oral intubation (20 mg HRP/ml 0.9% NaCI). Control animals were intubated with amylase (BDH Chemicals, Poole, Dorset, UK) rather than HRP. At selected post intubation time intervals ((r75 min), fish were removed from their holding facility, anaesthetized in a solution of MS 222 (95 mg/l; Thompson & Joseph Ltd, Norwich, Norfolk, UK), and blood collected into 2ml heparinized vials (LIP Ltd, Shipley, W. Yorks, UK) via puncture of the dorsal aorta. Immediately after blood withdrawal, fish were killed by a blow to the head and 507

E. MCLEAN and R. ASH

508

Table I. Horseradish peroxidase presence in various tissues of rainbow trout at stxcific times followine oral intubation of a 20 me load in I ml of 0.9% sahne Time

HRP presence (“g/g* wet weight ~~~~ _~~ ~~ ~~

_~

(min) I5 30 45 60 75

Plasma (5)

Kidney (5)

Spleen (5)

3.4 18.5 9.6 3.6 2.0

4.0 15.2 21.4 17.5 10.3

8.5 20.0 20.0 21.9 14.1

k 0.7 i 2.9 _+ I.1 k I .4 + 0.7

f I.3 f 2.8 i_ 4.0 + 3.6 + I.5

Number in parentheses = number *per ml plasma. Values are means f SEM.

+ f + i +

I.4 4.9 6.1 2.2 1.8

of tissue)

Liver (5) 6.3 + 3.0 14.3 5 I.3 22.6 k 4.5 21.6 f 3.3 11.4+5.1

Heart (5) 5.6 12.6 9.9 6.8 3.0

HRP ng g-’ tissue+

20

10

analysis. The marker protein (HRP) was detected by the enzymelinked immunosorbent assay described by Ambler and Peters (1984) and samples read spectrophotometrically at 490 nm, against an assay blank. using an MR-590 ELISA Minireader (Dynatech Europe, Guernsey,UK).

RESULTS

The presence of HRP within various tissues of the rainbow trout at specific time intervals after oral intubation is presented in Table 1 and Fig. 1. The results demonstrate that HRP was detected within the plasma (arterial), liver, kidney, spleen and heart at the earliest time after oral loading (15 min) that practical measurements were made. No detectable levels (results not shown) of HRP (-like) activity were observed in control fish and thus the data presented is considered to represent true. transmucosal passage of the orally introduced tracer protein. Peak values for HRP presence within the heart and plasma were recorded at 30min whilst for kidney, spleen, and liver, maximum values occurred between 45 and 60min (see Fig. 1). The rank order of maximum HRP presence expressed in terms of rig/g wet weight of tissue (or /ml plasma) was: However, liver > spleen = kidney > plasma > heart. when ranked in terms of maximum content of HRP was: liver > order the organ whole per plasma > kidney > spleen > heart (see Table 2).

Tissue Total plasma volume1 Kidney Spleen Liver Heart

2.7 0.2 2.3 2. I 0.9

of fish

the spleen, heart, liver and kidney removed. Tissues were homogenized in phosphate buffered saline (pH 7.4) containing 0.5% Tween-20 (Sigma Chemical Co., Poole. Dorset, UK). All homogenates were subjected to sonication (6 x 5 set) in a Pulsatron 125 sonicator (Kerry Ultrasonics Ltd, Hitchin, Herts, UK) prior to centrifugation at 12.000 g in a type 320 microcentrifuge (Burkard Scientific Sales, Rickmansworth, Herts, UK). The resultant supernatants were utilized, with appropriate dilution, for all subsequent

Table 2. Maximum

+ + * + +

40

80

t [minsl Fig. 1. The time-course of appearance of horseradish peroxidase in arterial plasma (dorsal aorta) and various body tissues of the rainbow trout, Sulmo gairdneri, following a single oral intubation of 20mg in 1 ml of 0.9% saline. +ng HRP/ml plasma.

DISCUSSION

Sufficient morphological (Kuperman and Kuz’mina. 1984; Kuperman et al., 1985; Elba1 and Agulleiro, 1986). histochemical (Iida and Yamamoto, 1984), and biochemical (McLean and Ash, 1985, 1986) evidence now exists to indicate that, in fish, irrespective of their state of maturity or morphology, intact protein absorption appears to be a characteristic feature of the gut, or certain sections thereof. Although the exact mechanism(s) by which such proteins are sequestered remains to be clarified, considerable evidence in support of transcellular routes of absorption have recently been reported (Rombout et al., 1985; Georgopoulou et al., 1984, 1985; Iida and Yamomoto, 1985; Iida et al., 1986). However, it is also possible that intercellular pathways (see Volkheimer and Schultz. 1968; Volkheimer, 1972, 1975)

intact HRP content per tissue in rainbow trout subsequent 20 mg HRP in I ml of 0.9% saline

to a” oral intubation

of

% Body weight

Total mea” wet weight

Peak HRP presence? (“g/g* wet weight)

Maximum intact HRP content per organ

3 2.98 0.644 5.74 0.624

5.982 I.490 0.322 2.870 0.312

18.5 21.4 21.9 22.6 12.6

52.03 31.9 14.1 129.5 7.8

*per ml plasma. t30 minutes plasma, 45 minutes kidney, liver, spleen. fTotal plasma volume as derived from the relationship (Thornson, 1961; cited by Conte ef al., 1963). Packed cell volume = 47.19 f 2.33%.

blood

volume

(trout) = 3% body

weight

Uptake of horseradish peroxidase from rainbow trout gut

may provide quantitatively significant routes for absorption. Regardless of the exact route(s) of uptake, however, it is evident from the present study that HRP (mol. wt 40,OOOdaltons) was detectable within arterial plasma and body tissues of the 48 hr fasted rainbow trout within 15 min of oral intubation. It should be stressed that, because the analytical procedure (Ambler and Peters, 1984) utilized to detect HRP presence depends upon the continued potency of both antigenic determinants and the active site of the molecule, it is therefore assumed that detection subsequent to intubation must reflect the continued presence of either the intact parent molecule or a large polypeptide derivative thereof. Localization of HRP within the spleen and kidney as observed in this study has previously been described for a variety of other antigenic substances (e.g. Aeromonas hydrophila, Salmonella pullorum), introduced either via immersion (Nelson et al., 1985); intramuscular injection (Maas and Bootsma, 1982); intraperitoneal inoculation (Secombes and Manning, 1980); intravenous injection (Ferguson, 1984); oral intubation (McLean and Ash, 1986); or the anal route (Rombout et al., 1985; Johnson and Amend, 1983). Localization of antigenic material within these organs (kidney and spleen) is consistent with the status of both organs within the fish reticuloendothelial system and their high apparent phagocytotic potential (Rijkers, 1980; Maas and Bootsma, 1982; Lamers. 1986). From the results of the present study it would also appear that the liver and, to a much lesser extent, the heart, sequester HRP. Such results are consistent with those of both Dannevig et al. (198 1) and Smedsrud et al. (1984) who previously reported participation of the liver in the clearance and accumulation of various intravenously administered proteins (see also MacArthur et al., 1983). In contrast to the results of these workers the present study would appear to imply that, relative to the other tissues studied, the importance of the liver is seemingly greater than that previously suggested. Such a result may simply arise as a consequence of the fact that an oral route of antigen delivery was employed in the present study and the liver, as first passage organ of hepatic portal vein blood, would be the organ of initial receipt of absorbed HRP (assuming uptake by the lymphatic system can be largely ignored). Alternatively, but less likely, it is conceivable that HRP may be “handled” in a different manner to the antigenic materials employed in previous studies. However, it must be appreciated that the tissue HRP levels tabulated in the present report provide no indication of total HRP uptake by any individual tissue since the rates of HRP degradation within these tissues remain unknown. Thus, the presence of HRP at any given time represents a net value (uptake-degradation) and hence the relative order of importance of the individual tissues in clearing antigenie material cannot be assessed from the data presented. Nevertheless, the results of the present study are of significance since they demonstrate unequivocally that orally administered antigenic substances, in this case HRP, can be absorbed intact and be subsequently localized within various tissues of the rambow

trout.

509

It is of considerable interest to note that a direct comparison of the results obtained in the present study for rainbow trout with those previously published (McLean and Ash, 1986) for common carp (Cyprinus carpio) reveals that, in all cases, the tissue presence of orally administered HRP is significantly (lOOO-fold) greater in the “agastric” carp than in the “gastric” trout. Such differences may reflect fundamental differences between the gastrointestinal physiology of the two species. In particular, the importance of the stomach in the initial predigestion (acid/pepsin) of ingested protein must be considered. In addition, Stroband et al. (1979) have shown that the absorptive enterocytes of the mid-intestine, which represents some 25% of the total gut length, of the grass carp (Ctenopharyngodon idella), an agastric species, are capable of pinocytotic uptake of intact proteins. In contrast, the results of Noaillac-Depeyre and Gas (1983) indicate that only 3% of the gut of the catfish (Ameiurius nebulosus), a gastric species, contains such enterocytes. From such data, and the fact that the total length of the catfish gut is considerably shorter than that of the grass carp, it is possible to infer that, in agastric species, greater quantities of intact or partially digested proteins may gain access to the distal gut and hence be available for absorption. The results of the studies presented in this and the previous paper (McLean and Ash, 1986) are therefore consistent with such differences in gastrointestinal physiology between trout and carp. Agastric species of fish may therefore have to consistently contend with relatively high loads of dietaryderived antigenic material within the distal sections of the intestine. The facility to adequately sample such material might therefore be of high value in maintaining the immuno-competence of such species.

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