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Organ culture of fish tissues
Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 3 © 1994 Elsevier Science B.V. All rights reserved.
C H A P T E R 31
Organ culture of fish tissues PETER A . JANSSENS AND J . A . G R I G G Division of Biochemistry
and Molecular Biology, School of Life Sciences, Australian University, Canberra, ACT 0200, Australia
National
I. II.
Introduction The technique of organ culture 1. Principles of the method 2. Apparatus 2.1. Sterile laminar flow cabinet 2.2. Roller tube tissue culture apparatus 2.3. Instruments 2.4. Culture media 3. Culture preparation 4. Stability of tissue in organ culture 5. Effects of hormones on liver tissue in organ culture 6. Analysis III. Conclusion Acknowledgements IV. References
/.
Introduction
The study of metabolic regulation in living animals is difficult because of the complexity and interdependence of the metabolic pathways involved. One need only look at the wall charts prepared by pharmaceutical companies to be aware of this complexity. For study of hepatic metabolism in vertebrates, it became clear very early that an in vitro system in which the metabolism of the liver could be studied in isolation from the rest of the body would greatly facilitate understanding of this complex organ and a number of model systems have been developed since that time. The first to be developed was the liver slice in which liver from mammals such as the rat or mouse was cut into thin slices and incubated in an artificial medium with effectors added to the medium. Liver slices can give some indication of in vivo metabolism but they are generally considered to be a rather unsatisfactory metabolic model for mammalian studies because they have damaged surfaces, have undeterminable anoxia in the centre of the slice, and because the rates of synthesis of a number of products demonstrable in slices is well below those seen in perfused whole livers. For studies on rats and mice, which are predominant in metabolic studies, the iso lated perfused liver became the method of choice for studying hepatic metabolism
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because metabolic rate was high, hormones produced rapid responses , and the effects seen in the perfused liver appeared to be the best in vitro representation of the situation in vivo. There are, however, significant disadvantages with perfused livers among which are the small number of experiments which can be done with a single animal and the difficulty of perfusing livers from large animals. Then, in 2 the late 1960's, techniques for preparing isolated hepatocytes were developed and these have proved extremely useful since there are no problems with diffusion of substrates to, and products from, the cell surface. Furthermore, a large number of parallel studies can be carried out on cells isolated from a single animal providing an internal control and reducing the number of animals which must be used. However, isolated cells have some disadvantages. Damage to the cell surface during perfusion 9 with proteolytic enzymes may change the characteristics of the cellular response , and the part played in metabolic regulation by the tissue architecture is lost from the preparation. Nevertheless, except for a few studies in which radioactive tracers have been used in vivo, almost all studies on regulation of metabolism in mammals in the last 20 years have been conducted using the isolated perfused liver or suspensions of isolated hepatocytes. Studies of metabolic control in nonmammalian vertebrates were rare until the development of interest in comparative endocrinology in the 1960's. Since that time there have been many studies on the effects of injected hormones on parameters such as blood glucose and liver glycogen levels in fish, amphibians and reptiles, but few using perfused livers or isolated hepatocytes until the mid-1970's. One reason for this is that the livers of many fish are diffusely distributed around the coils of the intestine which makes perfusion impossible. One of the first studies with 3 isolated hepatocytes from a teleost fish was that of Birnbaum, Schultz and Fain who determined the effects of hormones on hepatic glycogenolysis in hepatocytes from the goldfish, Carassius auratus. These authors pointed out the difficulties of using liver slices and isolated perfusion for experiments on teleosts and defined a method for the preparation of isolated hepatocytes by shaking minced liver for 2 h in a medium containing 1 mg/ml collagenase. Using this preparation, they demonstrated that both adrenaline and glucagon stimulated glycogenolysis 2+ from hepatocytes in a dose-dependent manner, that cyclicAMP but not C a was integral to this effect, and that the action of adrenaline was blocked by beta-, but not by alpha-, adrenergic antagonists. Since this initial study, there have been many using isolated hepatocytes which are described in detail in Chapter 30 (this volume). 19 2 0 At about the same time, Umminger and his colleagues reported that glucagon and adrenaline, added to liver pieces from the brown bullhead (Ictalurus nebulosus) and the killifish (Fundulus heteroclitus), prevented the decay in glycogen Phosphorylase seen in controls. These experiments were somewhat unsatisfactory because they did not show an activation of the enzyme, just a decrease in the rate 3 of inactivation and appeared to support the criticism of Birnbaum and coworkers that "the use of liver pieces compounds the problems encountered in slices". How ever, also in the mid-1970s, Michael Balls and his colleagues defined systems for long term organ culture of tissue pieces from the amphibian Amphiuma means.
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Liver pieces from A. means maintained relatively constant levels of tissue glycogen, constant activities of several enzymes, and constant rates of urea synthesis and 8 glucose uptake for up to 70 days in culture . Several enzymes retained their normal 8 6 electrophoretic pattern and cultured liver tissue was responsive to hormones . 8 We have therefore used the culture system of Balls and colleagues , initially with liver from the axolotl, Ambystoma mexicanum™, and other amphibians, but later n with liver from the carp, Cyprinus carpio , and the red-fin perch, Perca fluviatilis, (Janssens and Grigg, unpublished experiments) for studies of hormonal regulation of hepatic metabolism, in particular glycogenolysis. Others have used similar sys 718 tems for studies of fish m e t a b o l i s m . These systems have the advantages that they are simple, do not require expensive apparatus or materials, and the findings from them are not complicated by the possibility of damage to cell surfaces by the enzymes used in hepatocyte preparations, and are reproducible and illuminating. They do, however, suffer from the potential disadvantage mentioned by Birnbaum 3 et al. that the level of anoxia inside the pieces cannot be determined, and the rate of diffusion of agonists, substrates and products through the pieces may affect the rate at which metabolic control is brought about and the concentration of effector which is required.
II. The technique of organ culture The roller culture technique is a simple method for organ culture of tissues. The apparatus is simple and the technique can be learned in a short time. We have used it both for research and in undergraduate student classes and have had few problems with contamination. Several hundred cultures can be prepared from the liver of one large fish. 1. Principles of the method The principles are very simple indeed. The tissue is removed from the animal as aseptically as possible and cut into cubes with sides of about 2 mm; the cubes are placed in a small volume of medium in sterile screw-capped incubation tubes. The tubes are then incubated on a roller apparatus which slowly rotates them while they are tilted at an angle of about 4° from horizontal which maximises the surface area of the medium. Under these conditions, we have been able to maintain tissue from 11 goldfish and carp functionally and structurally intact for many days , comparably to 68 the findings of Balls and his collaborators with amphibian t i s s u e . 2.
Apparatus
2.1. Sterile laminar flow cabinet We do all our preparations in a sterile laminar flow cabinet. However, if cultures are not to be kept for more than 3 - 4 days, they may be prepared in less stringent conditions.
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2.2. Roller tube tissue culture apparatus (Fig. lc) Our cultures are incubated in screw-cap glass tubes (150 χ 15 mm) on a roller tube tissue culture apparatus (Matburn, London, UK). This has two circular racks (Fig. lb) each of which holds 210 tubes at an angle of about 4° from horizontal rotating 8 times per hour. We can thus incubate 420 tubes at a time on a piece of apparatus which is about 50 cm wide, 45 cm deep and 55 cm high. We do our incubations in a temperature-controlled cabinet at 20°C, but this may not be necessary in a temperature-controlled building. 2.3. Instruments Surgical scissors and forceps are needed for removing, cubing and manipulating the tissue pieces. All instruments are sterilised by flaming. 2.4. Culture media We use two basic media in our experiments: (a) Carp-MEM (should be suitable for all teleosts) is made by dissolving the ingredients of MEM Eagle (modified) with Earle's salts and glutamine but without sodium bicarbonate (bought as a sachet from Flow Laboratories-Cat # 10-101-26) in 900 ml glass-distilled water. To this is added 3.9 g Na HEPES (making a final concentration of 15 mM), 10,000 IU penicillin, 100 mg streptomycin and 2 mg fungizone. The pH is adjusted to 7.4 with NaOH and the volume brought up to 950 ml with glass-distilled water. The medium is then sterilised by filtering through a 0.22 μπι Millipore filter after which 50 ml sterile fetal bovine serum, previously inactivated at 57°C for 30 min, is added. (b) Glucose-free medium is made by dissolving the salts for MEM together with HEPES in 800 ml glass-distilled water. MEM concentrates of vitamins and amino acids are then added while stirring, together with phenol red (17 mg), 1 mM (final concentration) glutamine, and fungizone, penicillin and streptomycin at the same concentrations as for Carp-MEM. The pH is adjusted to 7.4, the volume made up to 1000 ml with glass-distilled water, and the medium sterilised by filtration through a 0.22 μπι Millipore filter. Other media, for example calcium-free medium, are made in the same way except that specific items, for example calcium chloride, are omitted. All media can be stored frozen at —20°C for several months. We purchase media components from Flow Laboratories Australasia Pty Ltd, but we have used the same technique in other countries using materials from other suppliers with equal success. 3. Culture
preparation
Fish are either killed with a blow to the head or anaesthetised by immersion in a 0.1% solution of MS222 (Metacaine, Sandoz); they are then decapitated, and doubly pithed. The body surface is wiped with a 0.5% (w/v) solution of Hibitane (ICI) in 70% methanol, and the body cavity is opened with sterile instruments and the liver dissected free from associated tissues and placed in a sterile petri dish containing a small volume of Carp-MEM. The liver is cut into slices and then into
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Fig. 1. The preparation of liver pieces. Fig. l a (top) shows a petri dish containing liver pieces in various stages of preparation; Fig. l b (middle) shows the culture tubes containing medium and tissue in the incubation rack; and Fig. l c (bottom) shows the rack on the rotary apparatus.
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cubes while immersed in carp-MEM (Fig. la) and the cubes are placed in sterile glass roller tubes (10-12 cubes weighing about 30 mg in each) containing 2.5 ml carp-MEM (Fig. lb). The tubes are loosely capped to allow gas exchange with air and then placed in the culture apparatus rotating at about 8 times/hour (Fig. lc). If the pieces are kept in culture for more than 3 - 4 days, the medium is removed by aspiration and replaced with fresh on the third and seventh day of incubation, and weekly thereafter. 4. Stability of tissue in organ culture We have routinely kept tissue from goldfish (Carassius auratus), carp (Cypnnus carpio) and red-fin perch {Perca fluviatilis) in organ culture for periods of at least one week and sometimes longer. When liver tissue from carp was cultured in 11 carp-MEM for 7 days , the glycogen content was still nearly 80% of that in fresh liver determined at the time of culture (Fig. 2). During this time there was no fall in the activities of three fundamentally important metabolic enzymes, fructose bisphosphatase which is a potential site of regulation of gluconeogenesis, pyruvate kinase which is rate-limiting in glycolysis, and alanine aminotransferase which is a central link between amino-acid and carbohydrate metabolism (Fig. 2). The tissue also retains its typical histological appearance. Sections of tissue pieces cultured for up to 7 days are indistinguishable from each other and from fresh tissue fixed at the time of preparation of the cultures (Fig. 3). There is no evidence of necrosis in the centre of the tissue pieces and there is no obvious difference in nuclear structure of cells in the centre or on the periphery of the
glycogen pyruvate kinase fructose bisphosphatase alanine aminotransferase
ο ο en ο
% of day 0 value
150
1
2 Days in culture
7
Fig. 2. The concentration of glycogen and the activities of pyruvate kinase, fructose 1,6-bisphosphatase and alanine aminotransferase in carp liver cultured for up to seven days. Values are expressed as percentage of the value in fresh liver and are given as mean ± SEM of four replicates. From Janssens 10 and L o w r e y .
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Fig. 3. Section of liver pieces cut at 4 μηι and stained with haematoxylin and eosin. Figure 3a (top) is of tissue fixed on the day of preparation (day 0); Fig. 3b (middle) is tissue fixed on day 3 and Fig. 3c (bottom) is of tissue fixed on day 7. Magnification χ 140 in each case.
pieces, even after seven days in culture. There is no structural evidence for anoxia within the pieces. These findings suggest that the tissue retains functional and structural integrity and this is supported by the responses of the tissue to agonists and antagonists.
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5. Effects of hormones on liver tissue in organ culture The first studies using organ culture of liver to investigate the actions of hormones on hepatic metabolism were those of Umminger and his colleagues who cut liver into small pieces, incubated these pieces immediately in simple saline media for 30 min with or without the addition of potential agonists, and then determined glycogen Phosphorylase activity. In the brown bullhead, Ictalurus nebulosus, glyco gen Phosphorylase activity decreased to about 40% of the initial activity after 30 min incubation, a decrease which was largely prevented by addition of adrenaline, 19 adrenaline plus 3',5'-cyclicAMP (cAMP), or glucagon plus cAMP to the medium . 2 Similar findings were reported in the killifish, Fundulus heteroclitus ® and in catfish 15 (Ictalurus melas) liver slices . These results do not provide evidence that the treat ments increase glycogen Phosphorylase activity, only that they prevent the decrease in activity seen in control incubations. It may well be that this absence of a positive hormonal effect is attributable to the tissue not having stabilised after the process of preparation; for example glycogen Phosphorylase may already have been activated during the anaesthetisation and killing of the fish. Alternatively, the very simple medium in which the tissue was incubated may have been insufficient to maintain glycogen Phosphorylase activity. 8
The techniques developed by Michael Balls and his group used longer culture periods and more complex media for maintenance of amphibian tissue in culture 10 11 and we adopted these methods in our studies of the axolotl and the carp . In the carp, hormones such as glucagon and the catecholamines, when added to tissue pieces which had previously been held in culture for at least 24 hours, caused substantial increases in the rate of glycogenolysis (Table 1), glycogen Phosphorylase activity (Table 2) and tissue cyclicAMP concentration (Table 3). The effects of the catecholamines could be blocked by β-, but not tv-adrenergic antagonists (Tables 1 and 2) and although the neurohypophysial peptides were powerful glycogenolytic 12 agonists in the axolotl , only isotocin had any effect in the carp and this was rather small (Tables 1, 2 and 3). 18 Sheridan and Muir , using a similar technique but without the overnight pre incubation, showed that catecholamines stimulate glycogen Phosphorylase activity and glucose release from salmon liver in a concentration-dependent manner, and used adrenergic antagonists to show that the hormones produced their effects by 7 binding to ^-adrenergic receptors. Eilertson, O'Connor and Sheridan showed that somatostatin-14 and somatostatin-25 both stimulate hepatic glycogenolysis in liver pieces from rainbow trout incubated in vitro, again in a concentration-dependent 17 way. Sheridan also used liver slices to demonstrate that noradrenaline stimulates hepatic lipid mobilisation in coho salmon (Oncorhynchus kisutch) and that the effect is mediated through β-adrenergic pathways. 6. Analysis At this point it may be instructive to compare results when hormones are injected into living animals, added to the perfusate of isolated liver perfusions, or added to
Organ culture of fish tissues
383 TABLE 1
Glucose release from carp (Cyprinus carpio) liver pieces incubated in the presence of potential agonists and antagonists Agonist
Glucose release (mg/g liver)
Glycogen lost (mg/g liver)
2 h incubation
4 h incubation
Experiment 1 N o n e (control) Arginine vasotocin Arginine vasopressin Lysine vasopressin Lysine vasotocin Mesotocin Oxytocin Isotocin Angiotensin II Glucagon Adrenaline Isoprenaline Noradrenaline Phenylephrine
2.19 1.43 2.22 1.76 2.59 1.39 2.80 3.25 1.42 4.07 9.44 9.26 3.66 1.82
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.29 0.35 0.28 0.46 0.28 0.50 0.35 0.20* 0.18 0.65* 1.73** 1.36** 0.64 0.52
3.55 3.14 3.31 3.27 4.70 3.36 3.86 6.68 2.04 12.78 20.48 22.14 8.09 2.90
± 0.36 ± 0.24 ± 0.57 ± 0.22 ± 0.49 ± 0.26 ± 0.25 ± 0.56* ± 0.23* ±0.93** ± 1.06** ±3.60** ±0.78** ± 0.77
4.8 5.7 3.7 3.9 5.4 5.4 3.6 8.1 3.0 14.2 19.6 23.4 9.1 1.8
Experiment 2 N o n e (control) Adrenaline Adrenaline + propranolol Adrenaline + phentolamine
1.13 4.28 2.03 4.48
±0.07 ±0.55** ± 0.08# ±0.30**
1.64 9.25 20.88 9.73
±0.12 ± 1.24** ± 0.21# ±0.70**
3.0 13.3 7.6 9.7
Values are means ± S.E.M. for 4 replicates. Carp liver pieces were incubated in glucose free medium for four hours with potential agonists added to a final concentration of 1 μ Μ and antagonists to 10 μ Μ . Glucose in the medium was measured at 2 and 4 h. Values significantly different from no addition (*, Ρ < 0.05, **, Ρ < 0.01) and from adrenaline alone ( # , Ρ < 0.01) are indicated. Tissue glycogen content was determined for four cultures before incubation and for all samples after incubation. T h e initial glycogen content in Experiment 1 was 55.3 ± 2.2 mg/g liver and in Experiment 2 was 64.0 ± 2.3 mg/g; the glycogen lost is the difference between this and the mean glycogen content for each group at the end of incubation. These results are typical of at least three experiments with each hormone. 10 Modified from Janssens and L o w r e y . TABLE 2 Glycogen Phosphorylase a activity in carp liver pieces incubated with potential agonists and antagonists Additions
N o addition Adrenaline Adrenaline + propranolol Adrenaline + phentolamine Glucagon Arginine vasotocin Isotocin
Glycogen Phosphorylase a activity (units/g) 2 min
10 min
30 min
0.124 ± 0.034 0.403 ± 0 . 0 3 2 * * 0.156 ± 0.032
0.229 0.683 0.233 0.571
0.123 0.815 0.464 0.743
± 0.026 ±0.119** ± 0.047# ± 0.072**
± 0.041 ±0.093** ± 0.044**# ±0.097**
0.472 ± 0.023** 0.219 ± 0.023 0.348 ± 0.035*
Values are means ± S.E.M. for 6 replicates. Carp liver pieces were incubated in glucose-free medium for the times indicated. Hormones were at 100 nM and antagonists at 1 μ Μ . Values significantly different from no addition (**, Ρ < 0.01; *, Ρ < 0.05) and from adrenaline alone ( # , Ρ < 0.01) are indicated. 10 These results are typical of three similar experiments performed. Modified from Janssens and L o w r e y .
384
P.A. Janssens and J.A. Grigg TABLE 3 Cyclic A M P content of carp liver incubated with various potential agonists for different times
Agonist
N o addition Arginine vasotocin Arginine vasopressin Isotocin Glucagon Adrenaline Isoprenaline Phenylephrine
Tissue content of cyclic A M P (pmol/g) 0 min
2 min
5 min
10 min
248 ± 9
233 ± 27
220 ± 17 247 ± 26 260 ± 20
264 ± 26 547 ± 4 0 * * 465 ± 28**
304 737 630 257
199 260 238 276 310 703 650 179
± 10** ±61** ±39** ± 46
± 21 ± 29 ± 55 ± 20* ±29* ± 153** ±57** ±4
Values are means ± S.E.M. from five replicates. Carp liver pieces were incubated in glucose-free medium for the times indicated with agonists added at a concentration of 1 μ Μ . Incubation was terminated by addition of 50 μΐ 27V HCl after which the tissue was dispersed in the medium with a tissue disperser and then incubated in a boiling water bath for 10 min. The dispersate was stored at - 2 0 ° C until assayed for its cyclic A M P content. Values significantly different from no addition at the same time (*, Ρ < 0.05; **, Ρ < 0.01) are indicated. These values are typical of three similar 10 experiments with each hormone. From Janssens and L o w r e y .
the incubation media of liver slice or piece incubations or of isolated hepatocytes. Such a comparison can be made for the catfish from studies over the past few years by Luigi Brighenti, Celestina Ottolenghi and their group. When adrenaline was injected into catfish, there was a rapid and substantial increase in blood glucose concentration and small and statistically insignificant falls in the concentration of 14 glycogen in liver and muscle . The interpretation of these findings is difficult because of the large variability between animals and because of the large number of responses which may follow injection of adrenaline. Among these responses, 14 as outlined in discussion by Ottolenghi and colleagues , are changes in rates of glycogenolysis and/or gluconeogenesis in several tissues, and changes, either increases or decreases, in the secretion of other hormones, including insulin and glucagon. Further, the concentration of adrenaline in the blood and tissue of the fish was not known. When adrenaline was added to the medium of an isolated perfused catfish liver, 15 the glycogen content fell and glucose release increased but the mechanism for this was not clear since glycogen Phosphorylase activity was not determined and, unfortunately, the concentration of adrenaline in the perfusion medium could not be determined. In liver slices, glycogen Phosphorylase activity fell in both control and adrenaline-treated incubations although the fall was smaller in the presence 1516 of a d r e n a l i n e ; this indicates, albeit not wholly convincingly, that adrenaline activates glycogen Phosphorylase, thereby causing glycogenolysis. 45 The position becomes clearer when isolated hepatocytes are e m p l o y e d . Addi tion of adrenaline to the incubation medium was followed by an increase in glycogen Phosphorylase activity, increased glucose release and a fall in tissue glycogen con centration. Nevertheless, Phosphorylase activity increased in control incubations 4 in the absence of adrenaline and this increase was not seen in the presence of
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tissues
385
propranolol, which suggests that it was attributable to activation via β-adrenergic receptors. These findings are difficult to interpret but it seems possible that, despite 14 thorough washing of the hepatocytes , some catecholamine released during hepatocyte preparation may have remained bound to the receptor, only being displaced in the presence of propranolol which was added at the high concentration of 100 4 μ Μ . Unfortunately, the effect of adrenaline has not been determined in catfish 11 liver cultured using the technique which we used with carp liver . However, our 18 results and those of Sheridan indicate that glycogen Phosphorylase activity is reasonably stable in liver pieces cultured in a complex medium and that changes in glucose release and glycogen content can be readily determined. It is also unfortunate that a direct comparison between organ culture and isolated perfusion has not been made in a teleost. However, in the axolotl, results from the isolated, perfused liver confirm completely those gained from organ culture, the only substantial difference being an increase of about an order of magnitude of the 12 EC50 for hormone, in this case arginine vasotocin, in the culture . There seems no good reason why a similar finding would not be true in a teleost.
Ill
Conclusion
It seems to us that all the techniques mentioned in this chapter have their uses. Clearly, the effects of a hormone in vivo are critically important even though they are difficult to interpret, and the effects in the isolated perfused liver are likely to be a better representation of the response of the organ than are isolated liver pieces or hepatocytes. However, only one treatment can be given to an intact animal and the number of experiments which can be performed on an isolated perfused liver preparation is limited. Organ culture or isolated hepatocytes are less demanding of time and animals and the findings from both can be extremely useful. 13 Mommsen and Storey have argued that hepatocytes have an advantage over cultured liver pieces because they are experimentally uniform. This is probably correct and hepatocytes are undoubtedly very useful for many experimental anal yses. However, they have been treated with enzymes to disperse them which may 9 affect their responses to agonists and antagonists , and they have completely lost the cellular interrelationships which they have in the intact tissue. Findings from experiments using isolated organ culture have been extremely productive and this simple, easy technique deserves full consideration when experiments on metabolic activities of nonmammalian vertebrate tissues are planned. Acknowledgements. This work was supported by the Australian Research Council and the Faculties Research Fund, Australian National University. We are grateful to Luke Wensing for preparation of the photographs.
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IV References 2+
1. Altin, J.G. and FL. Bygrave. Second messenger and the regulation of C a - m o b i l i z i n g agonists in rat liver. Biol. Rev. 63: 5 5 1 - 6 1 1 , 1988. 2. Berry, M.N. and D.S. Friend. High-yield preparation of isolated rat liver parenchymal cells. A biochemical and structural study./. Cell Biol. 43: 506-520, 1969. 3. Birnbaum, M.J., J. Schultz and J.N. Fain. Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes. Amer. J. Physiol. 231: 191-197, 1976. 4. Brighenti, L., A. C. Puviani, Μ. E. Gavioli and C. Ottolenghi. Mechanisms involved in cate cholamine effect on glycogenolysis in catfish isolated hepatocytes. Gen. Comp. Endocrinol. 66: 3 0 6 313, 1987. 5. Brighenti, L., A. C. Puviani, Μ. E. Gavioli and C. Ottolenghi. Interaction of salmon glucagon, glucagon-like peptide, and epinephrine in the stimulation of Phosphorylase a activity in fish isolated hepatocytes. Gen. Comp. Endocrinol. 82: 131-139, 1991. 6. Brown, D., N. Fleming and M. Balls. Hormonal control of glucose production by Amphiuma means liver in organ culture. Gen. Comp Endocrinol. 27: 380-388, 1975. 7. Eilertson, C.D., P.K. O'Connor and M.D. Sheridan. Somatostatin-14 and somatostatin-25 stimu late glycogenolysis in rainbow trout, Oncorhynchus mykiss, incubated in vitro: a systemic role for somatostatins. Gen. Comp. Endocrinol. 82: 192-196, 1991. 8. Fleming, N., D . Brown and M. Balls. Hepatocyte function in long-term organ culture of Amphiuma means liver./. Cell Science 18: 533-544, 1975. 9. Ichihara, Α., Τ. Nakamura and K. Tanaka. Use of hepatocytes in primary culture for biochemical studies on liver functions. Mol. Cell. Biochem. 43: 145-160, 1982. 10. Janssens, P.A., A.G. Caine and J. Dixon. Hormonal control of glycogenolysis and the mechanism of action of adrenaline in amphibian liver in vitro. Gen. Comp. Endocrinol. 47: 477-484, 1983. 11. Janssens, P.A. and P.E. Lowrey. Hormonal regulation of hepatic glycogenolysis in the carp, Cyprinus carpio. Amer. J. Physiol. 252: R 6 5 3 - R 6 6 0 , 1987. 2+ 12. Janssens, P.A., J. Kleineke and A.G. Caine. C a - i n d e p e n d e n t stimulation of glycogenolysis by arginine vasotocin and catecholamines in liver of axolotl (Ambystoma mexicanum) in vitro. J. Endocrinol. 109: 7 5 - 8 4 , 1986. 13. Mommsen, T.P. and K.B. Storey. Hormonal effects on glycogen metabolism in isolated hepatocytes of a freeze-tolerant frog. Gen. Comp. Endocrinol. 87: 4 4 - 5 3 , 1992. 14. Ottolenghi, C , A. C. Puviani, A. Baruffaldi and L. Brighenti. Effect of insulin on glycogen metabolism in isolated catfish hepatocytes. Comp. Biochem. Physiol. 78A: 705-710, 1984. 15. Ottolenghi, C , A. C. Puviani, Μ. E. Gavioli and L. Brighenti. Epinephrine effect on carbohydrate metabolism in isolated and perfused catfish liver. Gen. Comp. Endocrinol. 59: 219-229, 1985. 16. Ottolenghi, C , A. C. Puviani, Μ. E. Gavioli and L. Brighenti. Epinephrine effect on glycogen Phosphorylase activity in catfish liver and muscles. Gen. Comp. Endocrinol. 61: 4 6 9 - 4 7 5 , 1986. 17. Sheridan, M.A. Effects of epinephrine and norepinephrine on lipid mobilization from coho salmon liver incubated in vitro. Endocrinology 120: 2234-2239, 1987. 18. Sheridan, M.A. and N.A. Muir. Effects of epinephrine and norepinephrine on glucose release from chinook salmon (Oncorhynchus tshawytscha) liver incubated in vitro. J. Exp. Zool. 248: 155-159, 1988. 19. Umminger, B.L. and D . Benziger. In vitro stimulation of hepatic glycogen Phosphorylase activity by epinephrine and glucagon in the brown bullhead, Ictalurus nebulosus. Gen. Comp. Endocrinol. 25: 9 6 - 1 0 4 , 1975. 20. Umminger, B.L., D . Benziger and S. Levy. In vitro stimulation of hepatic glycogen Phosphorylase activity by epinephrine and glucagon in the killifish, Fundulus heteroclitus. Comp. Biochem. Physiol. 51C: 111-115, 1975.
C H A P T E R 31
Organ culture of fish tissues PETER A . JANSSENS AND J . A . G R I G G Division of Biochemistry
and Molecular Biology, School of Life Sciences, Australian University, Canberra, ACT 0200, Australia
National
I. II.
Introduction The technique of organ culture 1. Principles of the method 2. Apparatus 2.1. Sterile laminar flow cabinet 2.2. Roller tube tissue culture apparatus 2.3. Instruments 2.4. Culture media 3. Culture preparation 4. Stability of tissue in organ culture 5. Effects of hormones on liver tissue in organ culture 6. Analysis III. Conclusion Acknowledgements IV. References
/.
Introduction
The study of metabolic regulation in living animals is difficult because of the complexity and interdependence of the metabolic pathways involved. One need only look at the wall charts prepared by pharmaceutical companies to be aware of this complexity. For study of hepatic metabolism in vertebrates, it became clear very early that an in vitro system in which the metabolism of the liver could be studied in isolation from the rest of the body would greatly facilitate understanding of this complex organ and a number of model systems have been developed since that time. The first to be developed was the liver slice in which liver from mammals such as the rat or mouse was cut into thin slices and incubated in an artificial medium with effectors added to the medium. Liver slices can give some indication of in vivo metabolism but they are generally considered to be a rather unsatisfactory metabolic model for mammalian studies because they have damaged surfaces, have undeterminable anoxia in the centre of the slice, and because the rates of synthesis of a number of products demonstrable in slices is well below those seen in perfused whole livers. For studies on rats and mice, which are predominant in metabolic studies, the iso lated perfused liver became the method of choice for studying hepatic metabolism
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because metabolic rate was high, hormones produced rapid responses , and the effects seen in the perfused liver appeared to be the best in vitro representation of the situation in vivo. There are, however, significant disadvantages with perfused livers among which are the small number of experiments which can be done with a single animal and the difficulty of perfusing livers from large animals. Then, in 2 the late 1960's, techniques for preparing isolated hepatocytes were developed and these have proved extremely useful since there are no problems with diffusion of substrates to, and products from, the cell surface. Furthermore, a large number of parallel studies can be carried out on cells isolated from a single animal providing an internal control and reducing the number of animals which must be used. However, isolated cells have some disadvantages. Damage to the cell surface during perfusion 9 with proteolytic enzymes may change the characteristics of the cellular response , and the part played in metabolic regulation by the tissue architecture is lost from the preparation. Nevertheless, except for a few studies in which radioactive tracers have been used in vivo, almost all studies on regulation of metabolism in mammals in the last 20 years have been conducted using the isolated perfused liver or suspensions of isolated hepatocytes. Studies of metabolic control in nonmammalian vertebrates were rare until the development of interest in comparative endocrinology in the 1960's. Since that time there have been many studies on the effects of injected hormones on parameters such as blood glucose and liver glycogen levels in fish, amphibians and reptiles, but few using perfused livers or isolated hepatocytes until the mid-1970's. One reason for this is that the livers of many fish are diffusely distributed around the coils of the intestine which makes perfusion impossible. One of the first studies with 3 isolated hepatocytes from a teleost fish was that of Birnbaum, Schultz and Fain who determined the effects of hormones on hepatic glycogenolysis in hepatocytes from the goldfish, Carassius auratus. These authors pointed out the difficulties of using liver slices and isolated perfusion for experiments on teleosts and defined a method for the preparation of isolated hepatocytes by shaking minced liver for 2 h in a medium containing 1 mg/ml collagenase. Using this preparation, they demonstrated that both adrenaline and glucagon stimulated glycogenolysis 2+ from hepatocytes in a dose-dependent manner, that cyclicAMP but not C a was integral to this effect, and that the action of adrenaline was blocked by beta-, but not by alpha-, adrenergic antagonists. Since this initial study, there have been many using isolated hepatocytes which are described in detail in Chapter 30 (this volume). 19 2 0 At about the same time, Umminger and his colleagues reported that glucagon and adrenaline, added to liver pieces from the brown bullhead (Ictalurus nebulosus) and the killifish (Fundulus heteroclitus), prevented the decay in glycogen Phosphorylase seen in controls. These experiments were somewhat unsatisfactory because they did not show an activation of the enzyme, just a decrease in the rate 3 of inactivation and appeared to support the criticism of Birnbaum and coworkers that "the use of liver pieces compounds the problems encountered in slices". How ever, also in the mid-1970s, Michael Balls and his colleagues defined systems for long term organ culture of tissue pieces from the amphibian Amphiuma means.
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Liver pieces from A. means maintained relatively constant levels of tissue glycogen, constant activities of several enzymes, and constant rates of urea synthesis and 8 glucose uptake for up to 70 days in culture . Several enzymes retained their normal 8 6 electrophoretic pattern and cultured liver tissue was responsive to hormones . 8 We have therefore used the culture system of Balls and colleagues , initially with liver from the axolotl, Ambystoma mexicanum™, and other amphibians, but later n with liver from the carp, Cyprinus carpio , and the red-fin perch, Perca fluviatilis, (Janssens and Grigg, unpublished experiments) for studies of hormonal regulation of hepatic metabolism, in particular glycogenolysis. Others have used similar sys 718 tems for studies of fish m e t a b o l i s m . These systems have the advantages that they are simple, do not require expensive apparatus or materials, and the findings from them are not complicated by the possibility of damage to cell surfaces by the enzymes used in hepatocyte preparations, and are reproducible and illuminating. They do, however, suffer from the potential disadvantage mentioned by Birnbaum 3 et al. that the level of anoxia inside the pieces cannot be determined, and the rate of diffusion of agonists, substrates and products through the pieces may affect the rate at which metabolic control is brought about and the concentration of effector which is required.
II. The technique of organ culture The roller culture technique is a simple method for organ culture of tissues. The apparatus is simple and the technique can be learned in a short time. We have used it both for research and in undergraduate student classes and have had few problems with contamination. Several hundred cultures can be prepared from the liver of one large fish. 1. Principles of the method The principles are very simple indeed. The tissue is removed from the animal as aseptically as possible and cut into cubes with sides of about 2 mm; the cubes are placed in a small volume of medium in sterile screw-capped incubation tubes. The tubes are then incubated on a roller apparatus which slowly rotates them while they are tilted at an angle of about 4° from horizontal which maximises the surface area of the medium. Under these conditions, we have been able to maintain tissue from 11 goldfish and carp functionally and structurally intact for many days , comparably to 68 the findings of Balls and his collaborators with amphibian t i s s u e . 2.
Apparatus
2.1. Sterile laminar flow cabinet We do all our preparations in a sterile laminar flow cabinet. However, if cultures are not to be kept for more than 3 - 4 days, they may be prepared in less stringent conditions.
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2.2. Roller tube tissue culture apparatus (Fig. lc) Our cultures are incubated in screw-cap glass tubes (150 χ 15 mm) on a roller tube tissue culture apparatus (Matburn, London, UK). This has two circular racks (Fig. lb) each of which holds 210 tubes at an angle of about 4° from horizontal rotating 8 times per hour. We can thus incubate 420 tubes at a time on a piece of apparatus which is about 50 cm wide, 45 cm deep and 55 cm high. We do our incubations in a temperature-controlled cabinet at 20°C, but this may not be necessary in a temperature-controlled building. 2.3. Instruments Surgical scissors and forceps are needed for removing, cubing and manipulating the tissue pieces. All instruments are sterilised by flaming. 2.4. Culture media We use two basic media in our experiments: (a) Carp-MEM (should be suitable for all teleosts) is made by dissolving the ingredients of MEM Eagle (modified) with Earle's salts and glutamine but without sodium bicarbonate (bought as a sachet from Flow Laboratories-Cat # 10-101-26) in 900 ml glass-distilled water. To this is added 3.9 g Na HEPES (making a final concentration of 15 mM), 10,000 IU penicillin, 100 mg streptomycin and 2 mg fungizone. The pH is adjusted to 7.4 with NaOH and the volume brought up to 950 ml with glass-distilled water. The medium is then sterilised by filtering through a 0.22 μπι Millipore filter after which 50 ml sterile fetal bovine serum, previously inactivated at 57°C for 30 min, is added. (b) Glucose-free medium is made by dissolving the salts for MEM together with HEPES in 800 ml glass-distilled water. MEM concentrates of vitamins and amino acids are then added while stirring, together with phenol red (17 mg), 1 mM (final concentration) glutamine, and fungizone, penicillin and streptomycin at the same concentrations as for Carp-MEM. The pH is adjusted to 7.4, the volume made up to 1000 ml with glass-distilled water, and the medium sterilised by filtration through a 0.22 μπι Millipore filter. Other media, for example calcium-free medium, are made in the same way except that specific items, for example calcium chloride, are omitted. All media can be stored frozen at —20°C for several months. We purchase media components from Flow Laboratories Australasia Pty Ltd, but we have used the same technique in other countries using materials from other suppliers with equal success. 3. Culture
preparation
Fish are either killed with a blow to the head or anaesthetised by immersion in a 0.1% solution of MS222 (Metacaine, Sandoz); they are then decapitated, and doubly pithed. The body surface is wiped with a 0.5% (w/v) solution of Hibitane (ICI) in 70% methanol, and the body cavity is opened with sterile instruments and the liver dissected free from associated tissues and placed in a sterile petri dish containing a small volume of Carp-MEM. The liver is cut into slices and then into
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379
Fig. 1. The preparation of liver pieces. Fig. l a (top) shows a petri dish containing liver pieces in various stages of preparation; Fig. l b (middle) shows the culture tubes containing medium and tissue in the incubation rack; and Fig. l c (bottom) shows the rack on the rotary apparatus.
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cubes while immersed in carp-MEM (Fig. la) and the cubes are placed in sterile glass roller tubes (10-12 cubes weighing about 30 mg in each) containing 2.5 ml carp-MEM (Fig. lb). The tubes are loosely capped to allow gas exchange with air and then placed in the culture apparatus rotating at about 8 times/hour (Fig. lc). If the pieces are kept in culture for more than 3 - 4 days, the medium is removed by aspiration and replaced with fresh on the third and seventh day of incubation, and weekly thereafter. 4. Stability of tissue in organ culture We have routinely kept tissue from goldfish (Carassius auratus), carp (Cypnnus carpio) and red-fin perch {Perca fluviatilis) in organ culture for periods of at least one week and sometimes longer. When liver tissue from carp was cultured in 11 carp-MEM for 7 days , the glycogen content was still nearly 80% of that in fresh liver determined at the time of culture (Fig. 2). During this time there was no fall in the activities of three fundamentally important metabolic enzymes, fructose bisphosphatase which is a potential site of regulation of gluconeogenesis, pyruvate kinase which is rate-limiting in glycolysis, and alanine aminotransferase which is a central link between amino-acid and carbohydrate metabolism (Fig. 2). The tissue also retains its typical histological appearance. Sections of tissue pieces cultured for up to 7 days are indistinguishable from each other and from fresh tissue fixed at the time of preparation of the cultures (Fig. 3). There is no evidence of necrosis in the centre of the tissue pieces and there is no obvious difference in nuclear structure of cells in the centre or on the periphery of the
glycogen pyruvate kinase fructose bisphosphatase alanine aminotransferase
ο ο en ο
% of day 0 value
150
1
2 Days in culture
7
Fig. 2. The concentration of glycogen and the activities of pyruvate kinase, fructose 1,6-bisphosphatase and alanine aminotransferase in carp liver cultured for up to seven days. Values are expressed as percentage of the value in fresh liver and are given as mean ± SEM of four replicates. From Janssens 10 and L o w r e y .
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Fig. 3. Section of liver pieces cut at 4 μηι and stained with haematoxylin and eosin. Figure 3a (top) is of tissue fixed on the day of preparation (day 0); Fig. 3b (middle) is tissue fixed on day 3 and Fig. 3c (bottom) is of tissue fixed on day 7. Magnification χ 140 in each case.
pieces, even after seven days in culture. There is no structural evidence for anoxia within the pieces. These findings suggest that the tissue retains functional and structural integrity and this is supported by the responses of the tissue to agonists and antagonists.
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5. Effects of hormones on liver tissue in organ culture The first studies using organ culture of liver to investigate the actions of hormones on hepatic metabolism were those of Umminger and his colleagues who cut liver into small pieces, incubated these pieces immediately in simple saline media for 30 min with or without the addition of potential agonists, and then determined glycogen Phosphorylase activity. In the brown bullhead, Ictalurus nebulosus, glyco gen Phosphorylase activity decreased to about 40% of the initial activity after 30 min incubation, a decrease which was largely prevented by addition of adrenaline, 19 adrenaline plus 3',5'-cyclicAMP (cAMP), or glucagon plus cAMP to the medium . 2 Similar findings were reported in the killifish, Fundulus heteroclitus ® and in catfish 15 (Ictalurus melas) liver slices . These results do not provide evidence that the treat ments increase glycogen Phosphorylase activity, only that they prevent the decrease in activity seen in control incubations. It may well be that this absence of a positive hormonal effect is attributable to the tissue not having stabilised after the process of preparation; for example glycogen Phosphorylase may already have been activated during the anaesthetisation and killing of the fish. Alternatively, the very simple medium in which the tissue was incubated may have been insufficient to maintain glycogen Phosphorylase activity. 8
The techniques developed by Michael Balls and his group used longer culture periods and more complex media for maintenance of amphibian tissue in culture 10 11 and we adopted these methods in our studies of the axolotl and the carp . In the carp, hormones such as glucagon and the catecholamines, when added to tissue pieces which had previously been held in culture for at least 24 hours, caused substantial increases in the rate of glycogenolysis (Table 1), glycogen Phosphorylase activity (Table 2) and tissue cyclicAMP concentration (Table 3). The effects of the catecholamines could be blocked by β-, but not tv-adrenergic antagonists (Tables 1 and 2) and although the neurohypophysial peptides were powerful glycogenolytic 12 agonists in the axolotl , only isotocin had any effect in the carp and this was rather small (Tables 1, 2 and 3). 18 Sheridan and Muir , using a similar technique but without the overnight pre incubation, showed that catecholamines stimulate glycogen Phosphorylase activity and glucose release from salmon liver in a concentration-dependent manner, and used adrenergic antagonists to show that the hormones produced their effects by 7 binding to ^-adrenergic receptors. Eilertson, O'Connor and Sheridan showed that somatostatin-14 and somatostatin-25 both stimulate hepatic glycogenolysis in liver pieces from rainbow trout incubated in vitro, again in a concentration-dependent 17 way. Sheridan also used liver slices to demonstrate that noradrenaline stimulates hepatic lipid mobilisation in coho salmon (Oncorhynchus kisutch) and that the effect is mediated through β-adrenergic pathways. 6. Analysis At this point it may be instructive to compare results when hormones are injected into living animals, added to the perfusate of isolated liver perfusions, or added to
Organ culture of fish tissues
383 TABLE 1
Glucose release from carp (Cyprinus carpio) liver pieces incubated in the presence of potential agonists and antagonists Agonist
Glucose release (mg/g liver)
Glycogen lost (mg/g liver)
2 h incubation
4 h incubation
Experiment 1 N o n e (control) Arginine vasotocin Arginine vasopressin Lysine vasopressin Lysine vasotocin Mesotocin Oxytocin Isotocin Angiotensin II Glucagon Adrenaline Isoprenaline Noradrenaline Phenylephrine
2.19 1.43 2.22 1.76 2.59 1.39 2.80 3.25 1.42 4.07 9.44 9.26 3.66 1.82
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.29 0.35 0.28 0.46 0.28 0.50 0.35 0.20* 0.18 0.65* 1.73** 1.36** 0.64 0.52
3.55 3.14 3.31 3.27 4.70 3.36 3.86 6.68 2.04 12.78 20.48 22.14 8.09 2.90
± 0.36 ± 0.24 ± 0.57 ± 0.22 ± 0.49 ± 0.26 ± 0.25 ± 0.56* ± 0.23* ±0.93** ± 1.06** ±3.60** ±0.78** ± 0.77
4.8 5.7 3.7 3.9 5.4 5.4 3.6 8.1 3.0 14.2 19.6 23.4 9.1 1.8
Experiment 2 N o n e (control) Adrenaline Adrenaline + propranolol Adrenaline + phentolamine
1.13 4.28 2.03 4.48
±0.07 ±0.55** ± 0.08# ±0.30**
1.64 9.25 20.88 9.73
±0.12 ± 1.24** ± 0.21# ±0.70**
3.0 13.3 7.6 9.7
Values are means ± S.E.M. for 4 replicates. Carp liver pieces were incubated in glucose free medium for four hours with potential agonists added to a final concentration of 1 μ Μ and antagonists to 10 μ Μ . Glucose in the medium was measured at 2 and 4 h. Values significantly different from no addition (*, Ρ < 0.05, **, Ρ < 0.01) and from adrenaline alone ( # , Ρ < 0.01) are indicated. Tissue glycogen content was determined for four cultures before incubation and for all samples after incubation. T h e initial glycogen content in Experiment 1 was 55.3 ± 2.2 mg/g liver and in Experiment 2 was 64.0 ± 2.3 mg/g; the glycogen lost is the difference between this and the mean glycogen content for each group at the end of incubation. These results are typical of at least three experiments with each hormone. 10 Modified from Janssens and L o w r e y . TABLE 2 Glycogen Phosphorylase a activity in carp liver pieces incubated with potential agonists and antagonists Additions
N o addition Adrenaline Adrenaline + propranolol Adrenaline + phentolamine Glucagon Arginine vasotocin Isotocin
Glycogen Phosphorylase a activity (units/g) 2 min
10 min
30 min
0.124 ± 0.034 0.403 ± 0 . 0 3 2 * * 0.156 ± 0.032
0.229 0.683 0.233 0.571
0.123 0.815 0.464 0.743
± 0.026 ±0.119** ± 0.047# ± 0.072**
± 0.041 ±0.093** ± 0.044**# ±0.097**
0.472 ± 0.023** 0.219 ± 0.023 0.348 ± 0.035*
Values are means ± S.E.M. for 6 replicates. Carp liver pieces were incubated in glucose-free medium for the times indicated. Hormones were at 100 nM and antagonists at 1 μ Μ . Values significantly different from no addition (**, Ρ < 0.01; *, Ρ < 0.05) and from adrenaline alone ( # , Ρ < 0.01) are indicated. 10 These results are typical of three similar experiments performed. Modified from Janssens and L o w r e y .
384
P.A. Janssens and J.A. Grigg TABLE 3 Cyclic A M P content of carp liver incubated with various potential agonists for different times
Agonist
N o addition Arginine vasotocin Arginine vasopressin Isotocin Glucagon Adrenaline Isoprenaline Phenylephrine
Tissue content of cyclic A M P (pmol/g) 0 min
2 min
5 min
10 min
248 ± 9
233 ± 27
220 ± 17 247 ± 26 260 ± 20
264 ± 26 547 ± 4 0 * * 465 ± 28**
304 737 630 257
199 260 238 276 310 703 650 179
± 10** ±61** ±39** ± 46
± 21 ± 29 ± 55 ± 20* ±29* ± 153** ±57** ±4
Values are means ± S.E.M. from five replicates. Carp liver pieces were incubated in glucose-free medium for the times indicated with agonists added at a concentration of 1 μ Μ . Incubation was terminated by addition of 50 μΐ 27V HCl after which the tissue was dispersed in the medium with a tissue disperser and then incubated in a boiling water bath for 10 min. The dispersate was stored at - 2 0 ° C until assayed for its cyclic A M P content. Values significantly different from no addition at the same time (*, Ρ < 0.05; **, Ρ < 0.01) are indicated. These values are typical of three similar 10 experiments with each hormone. From Janssens and L o w r e y .
the incubation media of liver slice or piece incubations or of isolated hepatocytes. Such a comparison can be made for the catfish from studies over the past few years by Luigi Brighenti, Celestina Ottolenghi and their group. When adrenaline was injected into catfish, there was a rapid and substantial increase in blood glucose concentration and small and statistically insignificant falls in the concentration of 14 glycogen in liver and muscle . The interpretation of these findings is difficult because of the large variability between animals and because of the large number of responses which may follow injection of adrenaline. Among these responses, 14 as outlined in discussion by Ottolenghi and colleagues , are changes in rates of glycogenolysis and/or gluconeogenesis in several tissues, and changes, either increases or decreases, in the secretion of other hormones, including insulin and glucagon. Further, the concentration of adrenaline in the blood and tissue of the fish was not known. When adrenaline was added to the medium of an isolated perfused catfish liver, 15 the glycogen content fell and glucose release increased but the mechanism for this was not clear since glycogen Phosphorylase activity was not determined and, unfortunately, the concentration of adrenaline in the perfusion medium could not be determined. In liver slices, glycogen Phosphorylase activity fell in both control and adrenaline-treated incubations although the fall was smaller in the presence 1516 of a d r e n a l i n e ; this indicates, albeit not wholly convincingly, that adrenaline activates glycogen Phosphorylase, thereby causing glycogenolysis. 45 The position becomes clearer when isolated hepatocytes are e m p l o y e d . Addi tion of adrenaline to the incubation medium was followed by an increase in glycogen Phosphorylase activity, increased glucose release and a fall in tissue glycogen con centration. Nevertheless, Phosphorylase activity increased in control incubations 4 in the absence of adrenaline and this increase was not seen in the presence of
Organ culture offish
tissues
385
propranolol, which suggests that it was attributable to activation via β-adrenergic receptors. These findings are difficult to interpret but it seems possible that, despite 14 thorough washing of the hepatocytes , some catecholamine released during hepatocyte preparation may have remained bound to the receptor, only being displaced in the presence of propranolol which was added at the high concentration of 100 4 μ Μ . Unfortunately, the effect of adrenaline has not been determined in catfish 11 liver cultured using the technique which we used with carp liver . However, our 18 results and those of Sheridan indicate that glycogen Phosphorylase activity is reasonably stable in liver pieces cultured in a complex medium and that changes in glucose release and glycogen content can be readily determined. It is also unfortunate that a direct comparison between organ culture and isolated perfusion has not been made in a teleost. However, in the axolotl, results from the isolated, perfused liver confirm completely those gained from organ culture, the only substantial difference being an increase of about an order of magnitude of the 12 EC50 for hormone, in this case arginine vasotocin, in the culture . There seems no good reason why a similar finding would not be true in a teleost.
Ill
Conclusion
It seems to us that all the techniques mentioned in this chapter have their uses. Clearly, the effects of a hormone in vivo are critically important even though they are difficult to interpret, and the effects in the isolated perfused liver are likely to be a better representation of the response of the organ than are isolated liver pieces or hepatocytes. However, only one treatment can be given to an intact animal and the number of experiments which can be performed on an isolated perfused liver preparation is limited. Organ culture or isolated hepatocytes are less demanding of time and animals and the findings from both can be extremely useful. 13 Mommsen and Storey have argued that hepatocytes have an advantage over cultured liver pieces because they are experimentally uniform. This is probably correct and hepatocytes are undoubtedly very useful for many experimental anal yses. However, they have been treated with enzymes to disperse them which may 9 affect their responses to agonists and antagonists , and they have completely lost the cellular interrelationships which they have in the intact tissue. Findings from experiments using isolated organ culture have been extremely productive and this simple, easy technique deserves full consideration when experiments on metabolic activities of nonmammalian vertebrate tissues are planned. Acknowledgements. This work was supported by the Australian Research Council and the Faculties Research Fund, Australian National University. We are grateful to Luke Wensing for preparation of the photographs.
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IV References 2+
1. Altin, J.G. and FL. Bygrave. Second messenger and the regulation of C a - m o b i l i z i n g agonists in rat liver. Biol. Rev. 63: 5 5 1 - 6 1 1 , 1988. 2. Berry, M.N. and D.S. Friend. High-yield preparation of isolated rat liver parenchymal cells. A biochemical and structural study./. Cell Biol. 43: 506-520, 1969. 3. Birnbaum, M.J., J. Schultz and J.N. Fain. Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes. Amer. J. Physiol. 231: 191-197, 1976. 4. Brighenti, L., A. C. Puviani, Μ. E. Gavioli and C. Ottolenghi. Mechanisms involved in cate cholamine effect on glycogenolysis in catfish isolated hepatocytes. Gen. Comp. Endocrinol. 66: 3 0 6 313, 1987. 5. Brighenti, L., A. C. Puviani, Μ. E. Gavioli and C. Ottolenghi. Interaction of salmon glucagon, glucagon-like peptide, and epinephrine in the stimulation of Phosphorylase a activity in fish isolated hepatocytes. Gen. Comp. Endocrinol. 82: 131-139, 1991. 6. Brown, D., N. Fleming and M. Balls. Hormonal control of glucose production by Amphiuma means liver in organ culture. Gen. Comp Endocrinol. 27: 380-388, 1975. 7. Eilertson, C.D., P.K. O'Connor and M.D. Sheridan. Somatostatin-14 and somatostatin-25 stimu late glycogenolysis in rainbow trout, Oncorhynchus mykiss, incubated in vitro: a systemic role for somatostatins. Gen. Comp. Endocrinol. 82: 192-196, 1991. 8. Fleming, N., D . Brown and M. Balls. Hepatocyte function in long-term organ culture of Amphiuma means liver./. Cell Science 18: 533-544, 1975. 9. Ichihara, Α., Τ. Nakamura and K. Tanaka. Use of hepatocytes in primary culture for biochemical studies on liver functions. Mol. Cell. Biochem. 43: 145-160, 1982. 10. Janssens, P.A., A.G. Caine and J. Dixon. Hormonal control of glycogenolysis and the mechanism of action of adrenaline in amphibian liver in vitro. Gen. Comp. Endocrinol. 47: 477-484, 1983. 11. Janssens, P.A. and P.E. Lowrey. Hormonal regulation of hepatic glycogenolysis in the carp, Cyprinus carpio. Amer. J. Physiol. 252: R 6 5 3 - R 6 6 0 , 1987. 2+ 12. Janssens, P.A., J. Kleineke and A.G. Caine. C a - i n d e p e n d e n t stimulation of glycogenolysis by arginine vasotocin and catecholamines in liver of axolotl (Ambystoma mexicanum) in vitro. J. Endocrinol. 109: 7 5 - 8 4 , 1986. 13. Mommsen, T.P. and K.B. Storey. Hormonal effects on glycogen metabolism in isolated hepatocytes of a freeze-tolerant frog. Gen. Comp. Endocrinol. 87: 4 4 - 5 3 , 1992. 14. Ottolenghi, C , A. C. Puviani, A. Baruffaldi and L. Brighenti. Effect of insulin on glycogen metabolism in isolated catfish hepatocytes. Comp. Biochem. Physiol. 78A: 705-710, 1984. 15. Ottolenghi, C , A. C. Puviani, Μ. E. Gavioli and L. Brighenti. Epinephrine effect on carbohydrate metabolism in isolated and perfused catfish liver. Gen. Comp. Endocrinol. 59: 219-229, 1985. 16. Ottolenghi, C , A. C. Puviani, Μ. E. Gavioli and L. Brighenti. Epinephrine effect on glycogen Phosphorylase activity in catfish liver and muscles. Gen. Comp. Endocrinol. 61: 4 6 9 - 4 7 5 , 1986. 17. Sheridan, M.A. Effects of epinephrine and norepinephrine on lipid mobilization from coho salmon liver incubated in vitro. Endocrinology 120: 2234-2239, 1987. 18. Sheridan, M.A. and N.A. Muir. Effects of epinephrine and norepinephrine on glucose release from chinook salmon (Oncorhynchus tshawytscha) liver incubated in vitro. J. Exp. Zool. 248: 155-159, 1988. 19. Umminger, B.L. and D . Benziger. In vitro stimulation of hepatic glycogen Phosphorylase activity by epinephrine and glucagon in the brown bullhead, Ictalurus nebulosus. Gen. Comp. Endocrinol. 25: 9 6 - 1 0 4 , 1975. 20. Umminger, B.L., D . Benziger and S. Levy. In vitro stimulation of hepatic glycogen Phosphorylase activity by epinephrine and glucagon in the killifish, Fundulus heteroclitus. Comp. Biochem. Physiol. 51C: 111-115, 1975.