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Induction of liver glycogenolysis by phenylephrine: Comparison between superfusion and perfusion techniques
Conzp. Biochem. Ph.wiol. Vol. SOC, No. 2, pp. 371-374. 1985 Printed in Great Britain
0306.4492!85 $3.00 + 0.00 8~“ 1985 Pergamon Press Ltd
INDUCTION OF LIVER GLYCOGENOLYSIS BY PHENYLEPHRINE: COMPARISON BETWEEN SUPERFUSION AND PERFUSION TECHNIQUES V. LESKOVAC, S. TRIVX? and M. KAL& Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Faculty of Technology, Vlahovika 2, 21000 Novi Sad, Yugoslavia. Telephone: (021) 5.5-622
V.
(Received I June 1984) Abstract-l. Superfusion of liver tissue slices was performed by a technique specially developed for that purpose, and compared to the whole organ perfusion technique of Jakob and Diem in order to assess the merits of the former techniaue. 2. Activation ot’ liver glycogenolysis of phenylephrine has been examined by both techniques. 3. Both techniques showed important similarities and some dissimilarities. It was concluded that the application of superfusion techniques requires careful quantitative assessment of each experimental detail for each physiological event examined.
INTRODUCTION
Various techniques are currently in use for the investigation of pharmacological events in the liver, such as: recirculating and non-recirculating whole organ perfusion, metabolite determination using micro-light guides, NMR spectroscopy and others. The older technique of superfusion of tissue slices is inferior to the above techniques, since they resemble more closely itz aim physiological conditions. Nevertheless, so far only two techniques, whole organ perfusion and the superfusion of tissue slices, have found a general application, due to their technical simplicity. Only liver and a few other organs are accessible to organ perfusion techniques; most organs and tissues are not. Therefore, superfusion techniques may become of interest for tissues not amenable to perfusion techniques. For this reason, we have compared perfusion of the whole liver with the superfusion of liver tissue slices, in order to assess the merits of the latter technique. A standard organ perfusion technique, well characterized in the literature, has been chosen as a reference and compared to the tissue slice superfusion technique developed by ourselves for this purpose. In both systems, induction of liver glycogenolysis by phenylephrine has been examined under comparable experimental conditions. Comparison between two techniques indicates numerous similarities and some dift’erences between them. Most important, experimental evidence presented in this communication indicates that the tissue slice system has its merits, but requires a careful assessment of each detail of each particular physiological event examined, in order to reach a proper interpretation of the same. In addition, this communication features a novel. simple superfusion apparatus, almost entirely made from commercially available composite parts; experimental evidence indicates that a wide variety of experimental protocols may be realized in our device.
MATERIALS
Materials
Phenvlephrine, tolazoline, substrates and enzymes for enzyma&hetermination of glucose (Bergmeyer, 1971) were nurchased from the Sigma Chemie GmbH, Miinchen, Tauf&hen, Germany, N.M.R.1. white mice have been obtained from Dr M. Petrovii: of the Pasteur Institute, Novi Sad, Yugoslavia. Twice distilled water from an ail-glass assembly was used throughout this work.
A block diagram of the whole superfusion apparatus is presented in Fig. 1. The superfusion medium was continously pumped from its reservoir, with the aid of a peristaltic pump, into the superfusion cell; the open reservoir with the medium was constantly aerated with the aid of a small air-compressor: such open aeration precludes the application of volatile buffers, such as bicarbonate. Effluent from the superfusion cell was directly collected into the fraction collector. A wide variety of commercial types are satisfactory for the air-compressor, peristaltic pump and the fraction collector.
The superfusion cell was constructed by a simple modi~cation ofa Clarc-type oxygen electrode. commercially
0 C
I I
b-n SC
--
R
F‘;n . + 1. Block diagram of the superfusion apparatus. Compressor for aeration (C), reservoir for the superfusion medium (M), peristaltic pump (P), superfusion cell (SC), fraction collector (FC) and recorder to the oxygen electrode (R). Full lines represent the Bow of superfusion medium.
371 c8P so?C K
AND METHODS
V.
312 IN
LESKOVAC et
al.
Ceil content was continously mixed at 37”C, and the effluent collected into the fraction collector; concentration of glucose in the effluent was estimated enzymatically (Bergmeyer, 1971) at appropriate time intervals. The superfusion medium was externally aerated by a small air-compressor, and oxygen tension in the cell continuously monitored on an oxygraph recorder. The slices consume oxygen in the cell at a rate of approx. 0.2/rmoles O,/min/g liver wt, at 37°C. Therefore, a flow rate of 5 ml/min was sufficient to keep 0, concentration at 0.2mM, simply by aeration with an air-compressor. Each experiment presented in Fig. 4 was repeated several times, but only the results of a single representative experiment are presented each time.
OUT
RESULTS
I
Perfusion of the whole liver through the vascular bed has been compared with the superfusion of liver tissue slices, in order to assess the merits of the latter technique. A standard non-recirculating organ perfusion technique, well characterized in the literature Williamson et al., 1971; Jakob and Diem, 1974, 1975), has been chosen as a reference, and compared with the tissue slice superfusion technique developed by ourselves for this purpose. In both systems, induction of glycogenolysis by an a -adrenergic agonist, phenylephrine, has been examined under comparable conditions (Table 1). Induction of liver glycogenolysis by phenyleph~ne has been chosen as a physiological event easily reproduced in both systems. Figures 3 and 4 show the induction of glycogenolysis by phenylephrine in both systems. In the perfusion system, the rate of glucose release into the effluent decreased gradually, but non-linearly, with the progress of perfusion. Continuous infusion of phenylephrine increased immediately the rate of glucose release approx. two-fold (Fig. 3). In the superfusion system, the rate of glucose release into the ellluent decreased linearly with time (Fig. 4). In the absence of effecters, 3.2-3.8 g of glucose per 100 g of liver tissue were liberated into the medium, within the first 2 hr of superfusion (Fig. 4A); large amounts of liberated glucose indicates that its origin is in liver glycogen, set free by glycogenolysis. Three different modes of phenylephrine induction of glycogenolysis have been examined: (1) continuous infusion of phenylephrine from the beginning of the experiment (Fig. 4A), (2) co~ltinuous infusion of phenylephrine some time after the beginning of experiment (Fig. 4B) and (3) brief exposure of slices to phenylephrine prior to the experiment, followed by a superfusion without effecters (Fig. 4C). Figure 4A shows that the infusion of phenylephrine from the beginning of experiment increased the rate of glucose release (the rate of glycogenolysis)
I
M
Fig. 2. Schematic drawing of the superfusion cell. Stirring motor (M), polarographic electrode (PE), magnetic follower, flea (F), water-jacket (WJ), tissue slices (S), plunger closing the cell (PL), inlet (IN) and outlet stainless tube (OUT).
manufactured by Hansatech Ltd., King’s Lynn, Norfolk, UK (Delieu and Walker, 1972). Nothing has been changed in the originaf design of the commercial oxygen electrode, with one exception. The original plunger of the electrode chamber has been replaced by a plastic plunger of the same size, shown in Fig. 2. Two stainless tubes were driven through the plunger, one the inlet, with both ends open, and the other the outlet, with one end closed; the outlet tube was closed at the cell side, in order to prevent escape of small tissue particles. Free Aow from the outlet tube was made possible by the perforations on the side walls of the tube. A small perforated plastic holder was attached to the lower part of the cell side of the inlet tube, mounting the tissue slices (Fig. 2). A very small clearance between the plastic holder and the cell wall and the rapid upward movement of the superfusion medium prevented the loss of tissue particles. Internal volume of the cell is 2 ml. Supei$isior2 protocol
Livers were removed from well fed young N.M.R.I. white mice, both sexes, weighing 14-l7g. No anaesthetic was used; the animals were taken from stables early in the morning, immediately decapitated, liver removed and several tissue slices cut by a razor blade across one of the lobes, quickly weighed and mounted into the perfusion chamber. Slice thickness was approx. 1 mm and total wet wt 185270mg. Immediately after mounting the slices, a Krebs-Ringer solution (with 0.5mM Ca2+) was continuously pumped through the superfusion cell at 5 ml/mm.
Table
f.
Comparison between conditions for the su~r~usion and the perfusion technique Flow rate (ml/min/g
Animals -___
Technique Perfusion (Jakob and Diem, Superfusion (this paper)
1975)
Adult rats Young mice
Medium ___..__~.
Concentration of phenylephrine (PM) (nmoles/min/g of tissue) .~___. _
of tissue)
Temp.
Bicarbonate*
2.5
37°C
0.5
0.2
Krebs-Ringer? (with Ca’ ‘)
18.5-27
37°C
20.0
0.74-I ~08
-...
“In mM: bicarbonate 25, NaCl I IS, KC1 4.7, KHJ’O, 1.2, MgSO, 1.2 and CaCI, 1.3. tin mM: Na,HPO, 15, NaCl 1.50, KC1 5.8, K&PO, 1.5, MgSO, 1.5 and CaCl, 0.5.
Induction of liver glycogenolysis by pbenylephrine
373
140ymoles glucose/hi+/100 g animal wt in the pertechnique (Fig. 3) and fusion 185 pmoles glu~ose/br~lO~g animal wt in the superfusion technique (Fig. 4A). In the superfusion technique, the rate of glucose release decreases in a strictfy linear manner over a prolonged period of time (Fig. 4). For the perfusion technique, the rates of glucose release are
qO20 MINUTES
Fig. 3. Effect of phenylephrine on the rate of glucose release in the perfusion system. Redrawn from Jakob and Diem (1975), Fig. 5; the arrow shows the addition of phenylephrine (OS PM) to the perfusion medium.
by approx. 80%. This is comparable to the similar increase in the rate of giycogenoIysis effected by phenyiephrine in the perfusion system (Fig. 3). Figure 4B shows that the continuous infusion of phenylephrine, 75 min after the beginning of the experiment, increased the rate of glucose release to the same extent (approx. SOY,). A similar effect has been achieved by starting the infusion of phenylephrine any time within the time span of the experiment (2 hr) (data not shown). This indicates that the liver slices were viable and responsive to hormone throughout the whole experiment. Continuous infusion of 1 mM tolazoline, an cr-adrenergic antagonist, was without effect on the rate of glycogenolysis; continuous infusion of the a-antagonist prior to the application of pllenyiephrine, completely abolished the effect of the a-agonist (data not shown). This clearly indicates that the induction of glycogenolysis by phenyleph~ne is mediated via a-adrenergic receptors. Figure 4C shows that the brief exposure of tissue slices to phenylephrine (30sec), prior to the experiment, followed by a continuous superfusion without effecters, greatly decreased the rate of glycogenolysis. This strong inhibition of glycogenolysis by phenylephrine has not been further examined, although such inhibitory effects have not been reported in the literature on perfusion systems. So far, the above observation serves only to illustrate the potentialities of the superfusion system. DISCUSSION
The above experimental evidence clearly indicates the important similarities between the perfusion and the superfusion techniques. Despite the protocol and experimental differences, the rate of glucose release in the absence of effecters, 20 min after the beginning of (su)perfusion, is comparable for both techniques:
1
0
20
10
60
80
100
120
MINUTES
Fig. 4. Effects of phenylephrine (20 p M) on the rate of glucose release in the superfusion system. (A) Continuous infusion of phenylephrine from the beginning of experiment (0). control: infusion of medium without phenyiephrine (A); animal wt 14.1 g, weight of tissue slices 222.4 mg. (B) Continuous infusion of pheny~ephrine 75min after the beginning of experiment (a), control: infusion of phenylephrine from the beginning of experiment (0); animal 15.1 g, tissue slices 266.7 mg. (C) Brief exposure of slices (30 set) to phenylephrine prior to the experiment, followed by a superfusion without effecters (A), control: infusion of phenylephrine from the beginning of experiment (0); animal 15.4g, tissue slices 185.7 mg.
374
V.
hSKOVAC
reported only for limited periods of time (Fig. 3); nevertheless, the reported literature data indicate that the rate of glucose release probably also decreases in the perfusion technique as well (Jakob and Diem, 1974, 1975). The principal difference between the two techniques should be expected on the grounds of the difference in rates of diffusion of glucose and phenylephrine through the liver tissue. In the perfusion technique, the whole vascular bed is perfused evenly, providing balanced conditions for the diffusion of phenylephrine into the cells, and the diffusion of glucose from the cells into the vascular bed. In the superfusion technique, phenylephrine must reach the deeper cell layers by diffusion from the surface of slices, and glucose must constantly diffuse from the deeper cell layers to the surface of slices. The differences between both techniques are shown in Figs 3 and 4. In the perfusion technique, infusion of phenylephrine rapidly raised the rate of glucose release from the lower to the higher level (Fig. 3). In the superfusion technique, the response to phenylephrine is different. Here, the rate of glucose release is constantly and linearly decreasing; infusion of phenylephrine increased this rate to a new and constant level (Fig. 4B). The two different responses of glucose release to phenylephrine must obviously be attributed to different diffusion routes by which phenylephrine and glucose must reach the cells, in the two different techniques. The perfusion technique is superior to the superfusion technique, since it resembles more closely in
et al. vivo physiological
conditions. However, only liver and few other organs are routinely accessible to perfusion techniques, and most organs and tissues are not. Therefore, in most cases, the potential advantages of superfusion techniques may gain in importance. This communication is restricted to liver tissue, but it is obvious that the successful application of superfusion techniques to other tissues as well requires careful quantitative assessment of the technique itself for each particular tissue and each physiological event in turn. Experimental data presented here indicate that the application of superfusion techniques requires quantitative assessment of each detail in each physiological event, in order to avoid artefacts and reach a proper interpretation of the same. REFERENCES
Bergmeyer H. U. (1971) Methoden der enzymatischen Analyse. Chemie-Verlag, Weinheim. Deheu T. and Walker D. A. (1972) Measurement of photosynthetic oxygen evolution. New Phytol. 71, 201-220. Jakob A. and Diem S. (1974) Activation of glycogenolysis in perfused rat liver by glucagon and metabolic inhibitors. Biochim. biophys. Acta 362, 469-479. Jakob A. and Diem S. (1975) Metabolic response of perfused rat livers to alpha- and beta-adrenergic agonists, glucagon and cyclic AMP. Biochim. biophys. Acta 404, 57-66. Williamson J. R., Browning E. T. and Scholz R. (1969) Control mechanisms of gluconeogenesis and ketogenesis. J. biol. Chem. 244, 46074627.
0306.4492!85 $3.00 + 0.00 8~“ 1985 Pergamon Press Ltd
INDUCTION OF LIVER GLYCOGENOLYSIS BY PHENYLEPHRINE: COMPARISON BETWEEN SUPERFUSION AND PERFUSION TECHNIQUES V. LESKOVAC, S. TRIVX? and M. KAL& Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Faculty of Technology, Vlahovika 2, 21000 Novi Sad, Yugoslavia. Telephone: (021) 5.5-622
V.
(Received I June 1984) Abstract-l. Superfusion of liver tissue slices was performed by a technique specially developed for that purpose, and compared to the whole organ perfusion technique of Jakob and Diem in order to assess the merits of the former techniaue. 2. Activation ot’ liver glycogenolysis of phenylephrine has been examined by both techniques. 3. Both techniques showed important similarities and some dissimilarities. It was concluded that the application of superfusion techniques requires careful quantitative assessment of each experimental detail for each physiological event examined.
INTRODUCTION
Various techniques are currently in use for the investigation of pharmacological events in the liver, such as: recirculating and non-recirculating whole organ perfusion, metabolite determination using micro-light guides, NMR spectroscopy and others. The older technique of superfusion of tissue slices is inferior to the above techniques, since they resemble more closely itz aim physiological conditions. Nevertheless, so far only two techniques, whole organ perfusion and the superfusion of tissue slices, have found a general application, due to their technical simplicity. Only liver and a few other organs are accessible to organ perfusion techniques; most organs and tissues are not. Therefore, superfusion techniques may become of interest for tissues not amenable to perfusion techniques. For this reason, we have compared perfusion of the whole liver with the superfusion of liver tissue slices, in order to assess the merits of the latter technique. A standard organ perfusion technique, well characterized in the literature, has been chosen as a reference and compared to the tissue slice superfusion technique developed by ourselves for this purpose. In both systems, induction of liver glycogenolysis by phenylephrine has been examined under comparable experimental conditions. Comparison between two techniques indicates numerous similarities and some dift’erences between them. Most important, experimental evidence presented in this communication indicates that the tissue slice system has its merits, but requires a careful assessment of each detail of each particular physiological event examined, in order to reach a proper interpretation of the same. In addition, this communication features a novel. simple superfusion apparatus, almost entirely made from commercially available composite parts; experimental evidence indicates that a wide variety of experimental protocols may be realized in our device.
MATERIALS
Materials
Phenvlephrine, tolazoline, substrates and enzymes for enzyma&hetermination of glucose (Bergmeyer, 1971) were nurchased from the Sigma Chemie GmbH, Miinchen, Tauf&hen, Germany, N.M.R.1. white mice have been obtained from Dr M. Petrovii: of the Pasteur Institute, Novi Sad, Yugoslavia. Twice distilled water from an ail-glass assembly was used throughout this work.
A block diagram of the whole superfusion apparatus is presented in Fig. 1. The superfusion medium was continously pumped from its reservoir, with the aid of a peristaltic pump, into the superfusion cell; the open reservoir with the medium was constantly aerated with the aid of a small air-compressor: such open aeration precludes the application of volatile buffers, such as bicarbonate. Effluent from the superfusion cell was directly collected into the fraction collector. A wide variety of commercial types are satisfactory for the air-compressor, peristaltic pump and the fraction collector.
The superfusion cell was constructed by a simple modi~cation ofa Clarc-type oxygen electrode. commercially
0 C
I I
b-n SC
--
R
F‘;n . + 1. Block diagram of the superfusion apparatus. Compressor for aeration (C), reservoir for the superfusion medium (M), peristaltic pump (P), superfusion cell (SC), fraction collector (FC) and recorder to the oxygen electrode (R). Full lines represent the Bow of superfusion medium.
371 c8P so?C K
AND METHODS
V.
312 IN
LESKOVAC et
al.
Ceil content was continously mixed at 37”C, and the effluent collected into the fraction collector; concentration of glucose in the effluent was estimated enzymatically (Bergmeyer, 1971) at appropriate time intervals. The superfusion medium was externally aerated by a small air-compressor, and oxygen tension in the cell continuously monitored on an oxygraph recorder. The slices consume oxygen in the cell at a rate of approx. 0.2/rmoles O,/min/g liver wt, at 37°C. Therefore, a flow rate of 5 ml/min was sufficient to keep 0, concentration at 0.2mM, simply by aeration with an air-compressor. Each experiment presented in Fig. 4 was repeated several times, but only the results of a single representative experiment are presented each time.
OUT
RESULTS
I
Perfusion of the whole liver through the vascular bed has been compared with the superfusion of liver tissue slices, in order to assess the merits of the latter technique. A standard non-recirculating organ perfusion technique, well characterized in the literature Williamson et al., 1971; Jakob and Diem, 1974, 1975), has been chosen as a reference, and compared with the tissue slice superfusion technique developed by ourselves for this purpose. In both systems, induction of glycogenolysis by an a -adrenergic agonist, phenylephrine, has been examined under comparable conditions (Table 1). Induction of liver glycogenolysis by phenyleph~ne has been chosen as a physiological event easily reproduced in both systems. Figures 3 and 4 show the induction of glycogenolysis by phenylephrine in both systems. In the perfusion system, the rate of glucose release into the effluent decreased gradually, but non-linearly, with the progress of perfusion. Continuous infusion of phenylephrine increased immediately the rate of glucose release approx. two-fold (Fig. 3). In the superfusion system, the rate of glucose release into the ellluent decreased linearly with time (Fig. 4). In the absence of effecters, 3.2-3.8 g of glucose per 100 g of liver tissue were liberated into the medium, within the first 2 hr of superfusion (Fig. 4A); large amounts of liberated glucose indicates that its origin is in liver glycogen, set free by glycogenolysis. Three different modes of phenylephrine induction of glycogenolysis have been examined: (1) continuous infusion of phenylephrine from the beginning of the experiment (Fig. 4A), (2) co~ltinuous infusion of phenylephrine some time after the beginning of experiment (Fig. 4B) and (3) brief exposure of slices to phenylephrine prior to the experiment, followed by a superfusion without effecters (Fig. 4C). Figure 4A shows that the infusion of phenylephrine from the beginning of experiment increased the rate of glucose release (the rate of glycogenolysis)
I
M
Fig. 2. Schematic drawing of the superfusion cell. Stirring motor (M), polarographic electrode (PE), magnetic follower, flea (F), water-jacket (WJ), tissue slices (S), plunger closing the cell (PL), inlet (IN) and outlet stainless tube (OUT).
manufactured by Hansatech Ltd., King’s Lynn, Norfolk, UK (Delieu and Walker, 1972). Nothing has been changed in the originaf design of the commercial oxygen electrode, with one exception. The original plunger of the electrode chamber has been replaced by a plastic plunger of the same size, shown in Fig. 2. Two stainless tubes were driven through the plunger, one the inlet, with both ends open, and the other the outlet, with one end closed; the outlet tube was closed at the cell side, in order to prevent escape of small tissue particles. Free Aow from the outlet tube was made possible by the perforations on the side walls of the tube. A small perforated plastic holder was attached to the lower part of the cell side of the inlet tube, mounting the tissue slices (Fig. 2). A very small clearance between the plastic holder and the cell wall and the rapid upward movement of the superfusion medium prevented the loss of tissue particles. Internal volume of the cell is 2 ml. Supei$isior2 protocol
Livers were removed from well fed young N.M.R.I. white mice, both sexes, weighing 14-l7g. No anaesthetic was used; the animals were taken from stables early in the morning, immediately decapitated, liver removed and several tissue slices cut by a razor blade across one of the lobes, quickly weighed and mounted into the perfusion chamber. Slice thickness was approx. 1 mm and total wet wt 185270mg. Immediately after mounting the slices, a Krebs-Ringer solution (with 0.5mM Ca2+) was continuously pumped through the superfusion cell at 5 ml/mm.
Table
f.
Comparison between conditions for the su~r~usion and the perfusion technique Flow rate (ml/min/g
Animals -___
Technique Perfusion (Jakob and Diem, Superfusion (this paper)
1975)
Adult rats Young mice
Medium ___..__~.
Concentration of phenylephrine (PM) (nmoles/min/g of tissue) .~___. _
of tissue)
Temp.
Bicarbonate*
2.5
37°C
0.5
0.2
Krebs-Ringer? (with Ca’ ‘)
18.5-27
37°C
20.0
0.74-I ~08
-...
“In mM: bicarbonate 25, NaCl I IS, KC1 4.7, KHJ’O, 1.2, MgSO, 1.2 and CaCI, 1.3. tin mM: Na,HPO, 15, NaCl 1.50, KC1 5.8, K&PO, 1.5, MgSO, 1.5 and CaCl, 0.5.
Induction of liver glycogenolysis by pbenylephrine
373
140ymoles glucose/hi+/100 g animal wt in the pertechnique (Fig. 3) and fusion 185 pmoles glu~ose/br~lO~g animal wt in the superfusion technique (Fig. 4A). In the superfusion technique, the rate of glucose release decreases in a strictfy linear manner over a prolonged period of time (Fig. 4). For the perfusion technique, the rates of glucose release are
qO20 MINUTES
Fig. 3. Effect of phenylephrine on the rate of glucose release in the perfusion system. Redrawn from Jakob and Diem (1975), Fig. 5; the arrow shows the addition of phenylephrine (OS PM) to the perfusion medium.
by approx. 80%. This is comparable to the similar increase in the rate of giycogenoIysis effected by phenyiephrine in the perfusion system (Fig. 3). Figure 4B shows that the continuous infusion of phenylephrine, 75 min after the beginning of the experiment, increased the rate of glucose release to the same extent (approx. SOY,). A similar effect has been achieved by starting the infusion of phenylephrine any time within the time span of the experiment (2 hr) (data not shown). This indicates that the liver slices were viable and responsive to hormone throughout the whole experiment. Continuous infusion of 1 mM tolazoline, an cr-adrenergic antagonist, was without effect on the rate of glycogenolysis; continuous infusion of the a-antagonist prior to the application of pllenyiephrine, completely abolished the effect of the a-agonist (data not shown). This clearly indicates that the induction of glycogenolysis by phenyleph~ne is mediated via a-adrenergic receptors. Figure 4C shows that the brief exposure of tissue slices to phenylephrine (30sec), prior to the experiment, followed by a continuous superfusion without effecters, greatly decreased the rate of glycogenolysis. This strong inhibition of glycogenolysis by phenylephrine has not been further examined, although such inhibitory effects have not been reported in the literature on perfusion systems. So far, the above observation serves only to illustrate the potentialities of the superfusion system. DISCUSSION
The above experimental evidence clearly indicates the important similarities between the perfusion and the superfusion techniques. Despite the protocol and experimental differences, the rate of glucose release in the absence of effecters, 20 min after the beginning of (su)perfusion, is comparable for both techniques:
1
0
20
10
60
80
100
120
MINUTES
Fig. 4. Effects of phenylephrine (20 p M) on the rate of glucose release in the superfusion system. (A) Continuous infusion of phenylephrine from the beginning of experiment (0). control: infusion of medium without phenyiephrine (A); animal wt 14.1 g, weight of tissue slices 222.4 mg. (B) Continuous infusion of pheny~ephrine 75min after the beginning of experiment (a), control: infusion of phenylephrine from the beginning of experiment (0); animal 15.1 g, tissue slices 266.7 mg. (C) Brief exposure of slices (30 set) to phenylephrine prior to the experiment, followed by a superfusion without effecters (A), control: infusion of phenylephrine from the beginning of experiment (0); animal 15.4g, tissue slices 185.7 mg.
374
V.
hSKOVAC
reported only for limited periods of time (Fig. 3); nevertheless, the reported literature data indicate that the rate of glucose release probably also decreases in the perfusion technique as well (Jakob and Diem, 1974, 1975). The principal difference between the two techniques should be expected on the grounds of the difference in rates of diffusion of glucose and phenylephrine through the liver tissue. In the perfusion technique, the whole vascular bed is perfused evenly, providing balanced conditions for the diffusion of phenylephrine into the cells, and the diffusion of glucose from the cells into the vascular bed. In the superfusion technique, phenylephrine must reach the deeper cell layers by diffusion from the surface of slices, and glucose must constantly diffuse from the deeper cell layers to the surface of slices. The differences between both techniques are shown in Figs 3 and 4. In the perfusion technique, infusion of phenylephrine rapidly raised the rate of glucose release from the lower to the higher level (Fig. 3). In the superfusion technique, the response to phenylephrine is different. Here, the rate of glucose release is constantly and linearly decreasing; infusion of phenylephrine increased this rate to a new and constant level (Fig. 4B). The two different responses of glucose release to phenylephrine must obviously be attributed to different diffusion routes by which phenylephrine and glucose must reach the cells, in the two different techniques. The perfusion technique is superior to the superfusion technique, since it resembles more closely in
et al. vivo physiological
conditions. However, only liver and few other organs are routinely accessible to perfusion techniques, and most organs and tissues are not. Therefore, in most cases, the potential advantages of superfusion techniques may gain in importance. This communication is restricted to liver tissue, but it is obvious that the successful application of superfusion techniques to other tissues as well requires careful quantitative assessment of the technique itself for each particular tissue and each physiological event in turn. Experimental data presented here indicate that the application of superfusion techniques requires quantitative assessment of each detail in each physiological event, in order to avoid artefacts and reach a proper interpretation of the same. REFERENCES
Bergmeyer H. U. (1971) Methoden der enzymatischen Analyse. Chemie-Verlag, Weinheim. Deheu T. and Walker D. A. (1972) Measurement of photosynthetic oxygen evolution. New Phytol. 71, 201-220. Jakob A. and Diem S. (1974) Activation of glycogenolysis in perfused rat liver by glucagon and metabolic inhibitors. Biochim. biophys. Acta 362, 469-479. Jakob A. and Diem S. (1975) Metabolic response of perfused rat livers to alpha- and beta-adrenergic agonists, glucagon and cyclic AMP. Biochim. biophys. Acta 404, 57-66. Williamson J. R., Browning E. T. and Scholz R. (1969) Control mechanisms of gluconeogenesis and ketogenesis. J. biol. Chem. 244, 46074627.