3 13 (1987)
Mechanisms involved in Catecholamine Effect on Glycogenolysis in Catfish Isolated Hepatocytes1v2 LUIGI
PUVIANI, M. EMILIA OTTOLENGHI
A. CRISTINA CELESTINA University
Via L. Borsari
Accepted December 14, 1986 Isolated catfish hepatocytes were treated with epinephrine, norepinephrine, isoproterenol, and phenylephrine in the presence or in the absence of propranolol or phentolamine as beta and alpha inhibitors, respectively. Glycogen phosphorylase a activity and glycogen content, as well as glucose released from cells, were tested. Phosphorylase activity was stimulated by all the catecholamines and was accompanied by a decrease of glycogen content in cells and by an increase in glucose output into the medium. Whereas phentolamine did not affect the catecholamine action on any parameter considered, propranolol inhihited the effect of epinephrine, norepinephrine, and phenylephrine, but hardly altered that of isoproterenol. The effect of epinephrine and norepinephrine, as modified by propranolol and not by phentolamine, is consistent with a beta action of these catecholamines. The fact that propranolol and not phentolamine inhibited the phenylephrine effect indicates that in catfish hepatocytes phenylephrine behaves as a beta agonist and/or that propranolol may also bind to alpha receptors. Results also indicate that in catfish liver cells isoproterenol, whose effect is scarcely influenced by propranolol, is not a pure beta agonist. Q 19x7 ACTdemic Press, Inc.
The stimulatory effect of catecholamines on the activity of hepatic glycogen phosphorylase and the consecutive glycogenolysis is a well-known phenomenon in mammals (Sutherland and Cori, 1951). In fish, the response of glycogen metabolism to catecholamines is related to the animal species. Hepatocytes isolated from goldfish (Carassius auratus) respond to epinephrine treatment by increasing glucose release to the medium via the glycogenolytic pathway (Birnbaum et al., 1976). On the other hand, the effect of epinephrine on liver slices of rainbow trout (Sulmo guirdneri) is an enhancement of the glucose output with no
i This work was supported by a grant for scientific research (60%) from the Minister0 della Pubblica Istruzione. * A part of this work has been presented at the Joint Meeting of The Physiological Society and the Societa Italiana di Fisiologia, Oxford, 1985 (Brighenti et al., 1985).
decrease, and sometimes with an increase, in the glycogen content (Morata et al., 1982b). In previous works (Ottolenghi et al., 1985, 1986) we found that catfish liver slices incubated in the presence of epinephrine revealed a glycogen phosphorylase activity exceeding that of the controls, with a decrease in glycogen content accompanied by an increase in glucose output into the incubation medium. It is known that the physiological responses of target tissues to cateeholamines are mediated by alpha and/or beta adrenoreceptors (Ahlquist, 1948). While there is much evidence that aIpha receptors are important in the glycogenolytic action of catecholamines in rat liver (Sherline et al., 1972; Exton, 1979), the picture is not clear in other species, and little research has been done on the mechanism of action of catecholamines in lower vertebrates. To verify the mechanism involved in the epinephrine-mediated effect in catfish liver, 306
0016~6480/87 $1.50 Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.
we test the glycogen phosphorylase activity, the glycogen decrease, and the glucose output in isolated hepatocytes in the presence of epinephrine, norepinephrine, isoproterenol, phenylephrine, and of the alpha and beta antagonists, phentolamine and propranolol , MATERIALS
Animals. Adult cattish (Ictalurus melas) weighing approximately 200 g were purchased from a local dealer. Fish were placed in a group of 10 in tanks containing 200 liters of well-aerated, dechlorinated, and continuously depurated tap water, at the environmental temperature (18-24”). Animals were fasted for some days until the experiment. Hepatocyte preparation and incubation. The hepatocyte preparation was performed essentially with the Seglen (1972) technique, as reported in a previous work (Ottolenghi et al., 1984). Hepatocytes (about 100 mg wet wt, corresponding to about 2 x IO6 cells) were suspended in 2.5 ml of teleost Ringer solution, pH 7.4; the antagonists (1O-4 M, were added 5 min prior to the addition of catecholamines (1O-5 M). Incubation was carried out according to the experimental time schedule in a shaking bath at 25” in Erlenmeyer flasks gassed with OJCO,, 95%:5% (v/v) mixture. At the end of incubation the samples were centrifuged (10 min, 5Og, at 4”). Aliquots were taken from the supematant for glucose assay. Then, hepatocytes were immediately homogenized in 10 vol of ice-cold buffer and assayed for phosphorylase a activity. Other samples incubated in the same way were used for glycogen determination. Analysis. Glucose in the supernatant was determined by the glucose oxidase peroxidase method (Bergmeyer and Bernt, 1963). Glycogen content in cells was tested as glucose after enzymatic hydrolysis with amyloglucosidase (Carr and Neff, 1984). Glycogen phosphorylase a activity was assayed with the method described by Umminger and Benziger (1975). Statistical analysis was carried out using the paired Student’s t test. Materials. Reagents for teleost-Ringer solution and buffers were supplied by Merck (Darmstadt, FRG); for the glucose test a kit of Boehringer-Mannheim (Mannheim, FRG) was used. All other reagents and enzymes for cell isolation and tests, as well as catecholamines and antagonists, were from Sigma Chemical Co. (St. Louis, MO).
RESULTS During cell incubation, the phosphorylase activity, as well as the glucose output,
increased, whereas the glycogen content decreased (Table 1). Propranolol prevented the changes of phosphorylase activity and, to a lesser extent, the glucose release variations, but was ineffective on the glycogen level of the hepatocytes. Phentolamine had no effect on any of these changes. Table 2 shows the effect of catecholamines on phosphorylase a activity. After 60 min incubation all tested catecholamines induced a very significant increase in the enzyme activity, epinephrine being the most active. Propranolol completely blocked epinephrine- , norepinephrine- , and phenylephrine-stimulated phosphorylase activity, and had a smaller effect on the isoproterenol-increased enzyme activity. Phentolamine did not show any effect on catecholamine activation of phosphorylase. The effects of catecholamines on glycogen content of isolated hepatocytes are reported in Table 3. Catecholamines induced a significant decrease of the cell polysaccharide level, norepinephrine being the most active. Propranolol prevented the glycogen decrease induced by epinephrine, norepinephrine, and phenylephrine. In isoproterenol-treated cells propranolol showed a small and insignificant effect. Phentolamine did not affect the glycogen decay caused by all catecholamines. The glucose release induced by catecholamines is shown in Table 4. Results indicate that the glucose release was highly and significantly stimulated by all the catecholamines tested, the increase with respect to the incubated control being greatest for epinephrine, followed in decreasing order by norepinephrine, phenylephrine, and isoproterenol. Propranolol blocked the effect of epinephrine, norepinephrine, and phenylephrine, while it had much less influence on the glucose output stimulated by isoproterenol. Phentolamine was ineffective as an antagonist of all catecholamines on the glucose output. In Fig. 1 is shown the time course of the epinephrine effect on glucose release, which increased linearly in the first
ET AL. I
PHOSPHORYLASE~ ACTIVITY, GLYCOGENCONTENT,ANDGLUCOSEOUTPUTOF~SOLATEDCATFISH HEPATOCYTESUNDEKBASALCONDITIONS
Propranolol Phentolamine (IO-4 M) (10-4M) n 60 mitt 60 min ---.I__-_-.. Phosphorylase ua 21 6.28 +- 0.40 6.81 + 0.42W 21 6.29 ” 0.67 7.82 + 0.70” Glycogenb 22 65.03 k 6.65 61.08 i 6.40*” 17 52.73 ? 5.47 47.02 + 5.64** Glucoseb 29 2.18 5 0.30 5.54 c 0.53tt 29 2.76 -c 0.40 9.03 + 1.23** ---__ Note. Values are means 2 SEM of n experiments. Levels of significance (paired Student’s r test): *P < 0.05 and **P < 0.01 as compared with control at 0 min; WP < 0.01 as compared with control at 60 min. 0 pm01 P,lg cells/min. b mg/g cells. Control 0 min
Control 60 min ..- -___ l_-8.96 + 0.48*” 8.37 it 0 6.5*” 59.00 t 5:88** 48.11 i 5.16** 6.79 t 0.65** 7.66 i 0.82**
hour of incubation; then the rate of in- lated liver cells in this study. Nevertheless, creasewas lower. the results of epinephrine effect on phosphorylase a activity in hepatocytesagree DISCUSSION substantially with those that we obtained The suspension of viable hepatocytes from liver slices (Ottolenghiet at., 1986).In provides a system in vitro well suited for that work we observedthat phosphorylase the experimental study of complex bio- a activity in incubated controls was lower chemical phenomenain fish, including gly- than that in slices at zero time, whereasthe cogenolysis.Liver slices are indeed a non- experimentsperformed with isolated hepauniform system with an external surface tocytes showedan increaseof about35% in containing mechanically damaged cells, phosphorylasea activity after 60 min of inand for this reasonwe decided to use iso- cubation (‘Ittble 1). It was assumedthat in TABLE 2 EFFECTOFCATECHOLAMINES,PROPRANOLOL,ANDPHENTOLAM~NEONPHOSPHORYLASE ISOLATEDCATFISHHEFATOC~~ES
Catechoiamine (10-T M) Epinephrine Norepinephrine Isoproterenol Phenylephrine
Control 0 min
Control 60 min
Catecholamine 60 mm
6.30 + 0.87 (11) 6.32 e 0.36 (14) 7.06 k 0.92
9.00 r 0.66 (11) 8.51 r 0.49 (14) 9.30 -+ 1.13
11.24 rt 0.47** (11) 10.05 -c 0.38* (14) 10.38 + 1.07’
5.14 -c 0.63 (3
(12) 8.47 f 0.50 (5)
(12) 10.45 k 0.75* (5)
Catechoiamine propranolol (10-d h!f) 60 min 5.54 f 0.89tt
(6) 4.45 2 0.43tt
(8) 9.43 -c 1.21t (7) 6.11 + 0.57tt (3
a ACTIVITYIN Catecholamine + phentofamine (10-d M) 60 min 10.11 -c 0.74
(6) 10.37 ” 0.48
(8) 8.73 -e 1.96 (5) 10.66 ?c 0.85 (5)
Note. Values are means f SEM of n experiments. Pbospborylase a activity expressed as JWIQ~Pig celh&in. Levels of significance (paired Student’s t test):~*P < 0.05 and **P < 0.01 as cotmpared with control at 60 min; tP < 0.05 and ftP < 0.01 as compared with catechdamine-treated samples at 60 min.
EFFECT OF CATECHOLAM~NES, PROPRANOLOL, AND PHENTOLAMINE ON GLYCOGEN CONTENT OF ISOLATED CATFISH HEPATOCYTES
Catecholamine (10-T M)
Control 0 min
Control 60 min
Catecholamine 60 min
74.75 + 6.71 (17) 57.59 ” 5.31 (9) 62.40 2 8.49
66.79 t 5.86 (17) 53.26 f 5.16 (9) 58.00 2 8.79
51.41 2 6.67* (17) 34.29 t 5.59* (9) 38.33 k 5.25*
40.70 2 4.53
36.13 -t 3.77
25.10 2 3.71*
Catecholamine propranolol (10-d kq 60 min 73.21 5 9.30t (10) 52.85 f 7.20t
Catecholamine + phentolamine (IO-4b!f) 60 min 42.66 ‘- 8.69
27.65 2 7.22
47.19 t 8.85 (7) 36.97 5 4.26t (7)
33.28 2 5.46 (7) 24.75 r 5.14 (7)
Note. Values are means -t SEM of n experiments. Glycogen content expressed as mg/g cells. Levels of significance (paired Student’s t test): *P < 0.01 as compared with control at 60 min; tP < 0.01 as compared with catecholamine-treated samples at 60 min.
fish the stress of handling and killing may cause a catecholamine release, which would stimulate the activation of phosphorylase in liver slices (Morata et al., 1982a; Ottolenghi et al., 1985). The thorough washing of the liver in the course of preparation of isolated hepatocytes removes catecholamines from the system, preventing their activating effect on phosphorylase. The activation of the enzyme in control cells during incubation is possibly depen-
dent on the activation of phosphorylase kinase due to an increase in the cytosolic Ca2+ (Exton 1979). As regards’ the effect of propranolol and phentolamine alone on the considered parameters, it is remarkable that, while phentolamine is practically without any effect, propranolol prevents the phosphorylase activity elevation during the incubation period; but it is less effective on the glucose output, and it does not change the glycogen
EFFECT OF CATECHOLAMINES, PROPRANOLOL, AND PHENTOLAMINE ON GLUCOSE OUTPUT FROM ISOLATED CATFISH HEPATOCYTES
Catecholamine (10-5 M) Epinephrine Norepinephrine Isoproterenol Phenylephrine
Control 60 min
Catecholamine 60 min
4.39 2 0.55 (14) 6.12 2 0.64 (15) 9.46 2 1.11 (17) 7.28 f 0.84 (14)
26.64 f 1.65** (14) 26.83 r 1.93** (15) 23.98 f 1.60** (17) 25.79 + 2.22** (14)
Catecholamine propranolol (10-d M) 60 min 7.18 + 1.31tt
(8) 5.34 2 0.73tt (14) 20.42 5 2.20t (11) 6.65 f 0.8W (13)
Catecholamine + phentolamine (10-4 M) 60 min 26.68 k (7) 27.88 2 (9) 22.28 2 (10) 23.32 2
2.09 1.36 1.64 2.40
Note. Values are means + SEM of n experiments. Glucose expressed as mg/g cells. Levels of significance (paired Student’s t test): **P < 0.01 as compared with control at 60 min; tP < 0.05 and ttP < 0.01 as compared with catecholamine-treated samples at 60 min.
FIG. glucose Values Levels < 0.05 0 min.
glucose output was not maximal (see Fig. 1). The doses of agonists and inhibitors were chosen taking into account the results obtained in a previous work (Ottolenghi cjl al., 1986) regarding the effect of epinephrine on phosphorylase activity in liver slices. Moreover, doses of 10e5 and 10 -6 M were used by other authors (Stimpson, 196.5; Birnbaum et al., 1976; Pit and Djabali, 1982; Nagishi et (A., 1982; Herman and Brown, 1983). Figure 2 shows that where phosphorylase u activity is stimulated, the glycogen level falls and the glucose release increases, and vice versa. Catecholamines exhibit their physiolog-
1. Time course of the epinephrine effect on the output from isolated catfish hepatocytes. are the means -t SEM of three experiments. of significance (paired Student’s t test): (e) P and f8&) P < 0.01 as compared with control at
decrease. Propranolol could perhaps interfere in the Ca2+ movements, inhibiting the spontaneous activation of phosphorylase. A hyperglycemic and/or glycogenolytic effect of epinephrine has been reported in vivo in skates (Plisetskaya et al., 1964), in goldfish (Young and Chavin, l%S), in scorpion fish (Plisetskaya and Leibson, 1966), in dogfish and ratfish (Patent, 1970), in eel (Larsson, 1973), and in isolated goldfish hepatocytes (Birnbaum et al., 1976). The response of isolated hepatocytes to epinephrine supports the belief that hepatic glycogenolysis contributes substantially to the epinephrine-induced hyperglycemic response of lower vertebrates. In this study, we have tested the effects of epinephrine, norepinephrine, isoproterenol, and phenylephrine on phosphorylase activity, glycogen level, and glucose output. The cells were incubated for 1 hr, during which the epinephrine effect on the
:: z e
200 100 :
FIG. 2. Diagram summarizing the effects of catecholamines and inhibitor treatment on glycogen @ho+ phorylase a activity, glycogen content, and glucose released of isolated catfish hepatocytes. Values are the means f SEM as a percentage ofcontral values at 60 min incubation, and were obtained from the data of Tables 2, 3, and 4.
ical action via their interaction with a va- cally relevant catecholamines, which can riety of cellular receptors. Ahlquist (1948) act as alpha and beta agonists; the synoriginally classified these receptors on the thetic drugs isoproterenol and phenylephbasis of the relative potency of various ago- rine are considered beta and alpha agonists in stimulating responses through these nists, respectively (Weiner, 1985). Phentolreceptors. amine and propranolol are generally Alpha adrenergic receptors are important regarded as alpha and beta adrenergic mediators of catecholamine action on liver blocking agents. Nevertheless recent metabolism in several species (Exton, studies emphasize that there is not a well1979). Sherline et al. (1972) were the first to defined division between alpha and beta repropose the existence of a CAMP-indepenceptors, even in the same animal species dent mechanism mediated by alpha re- according to the cellular differentiation ceptors for catecholamine stimulation of (Kunos et al., 1985; Nakamura et al., 1985; glycogenolysis in rat liver. Many studies Tsujimoto et al., 1986). suggest that an alpha adrenergic mechaThe characteristic rank of potency for nism is operative in dog (Mayer et al., beta-2 adrenergic receptors is isoproterenol 1961), cat (Ellis et al., 1967), and guinea pig > epinephrine > norepinephrine (Lands et (Arinze and Kawai, 1983). It is well docu- al., 1967; Lefkowitz and Hoffman, 1980). mented (Exton, 1979) that alpha adrenergic Our results in the catfish hepatocyte stimulation of glycogenolysis in rat liver in- system indicate that epinephrine had the volves the mobilization of Ca2+ ions from greatest effect on phosphorylase a activity mitochondria, resulting in an increase in and on glucose output; norepinephrine, cytosolic Ca2 + concentration, which stimu- phenylephrine, and isoproterenol followed lates the phosphorylase kinase. The inter- closely. This descending order of potency action of an agonist with beta adrenergic in the activity of catecholamines could inreceptors stimulates the membrane-bound dicate that in cattish the classical beta adadenylate cyclase; this action leads to the renergic action does not exist. Moreover, it intracellular accumulation of CAMP and to is difficult to explain why norepinephrine the activation of CAMP-dependent protein had the greatest effect only in the case of kinase (Stiles et al., 1984). Epinephrine ap- hepatocyte glycogen. pears to act predominantly via beta adrenoThe effect of the inhibitors propranolol receptors in adult rabbit (Rufo et al., 1981) and phentolamine on catecholamine-stimuand man (Rizza et al., 1980). The finding lated activities requires some considerthat either alpha or beta receptors mediate ations. Propranolol completely blocked the the regulation of catecholamine action in action of epinephrine and norepinephrine the liver of different animals must be re- on phosphorylase activity, glycogen level, lated to the density of alpha and/or beta re- and glucose output, whereas phentolamine ceptors in this organ. These receptors have was ineffective. Propranolol was less effecbeen quantified only in rat liver (Schmelck tive in inhibiting the isoproterenol action and Hanoune, 1980). on the three parameters studied. This sugClassification of the hepatic adrenergic gests that isoproterenol in catfish liver is receptors as alpha and beta has been dis- not a pure beta agonist. The effects of puted because of the variability of the re- phenylephrine, generally regarded as an sponses and the lack of the specificity of alpha agonist, were blocked by propranolol some of the blocking agents used to estab- and not by phentolamine, and so it is poslish the nature of the receptor (Hornbrook, sible that in catfish propranolol may also 1970; Kraus-Friedmann, 1984). Epinephbind to alpha receptors, or that phenylephrine and norepinephrine are the physiologirine in this fish is a beta agonist. Effects of
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