68, 216-223 (1987)
Effect on Cyclic Adenosine 3’:5’-Monophosphate Level in Isolated Catfish Hepatocytesip2
LUIGI BRIGHENTI, A. CRISTINA PUVIANI, M. EMILIA GAVIOLI, ELENA FABBRI, AND CELESTINA OTTOLENGHI Institute of General Physiology, University of Ferrara, Via L. Borsari 46, 44100 Ferrara, Italy Accepted June 8, 1987 The effect of catecholamines (epinephrine, norepinephrine, isoproterenol, and phenylephrine) on cyclic adenosine 3’:5’-monophosphate (CAMP) level in isolated catfish (Zctalurus me/us) liver cells was studied in the presence or absence of 01(phentolamine) and B (propranolol)-receptor antagonists. All catecholamines increased the hepatocyte CAMP level; the rank of their potency was epinephrine = isoproterenol > norepinephrine > phenylephrine. Propranolol completely blocked the catecholamine effect; phentolamine was ineffective. Results confirm previous findings (L. Brighenti, A. C. Puviani, M. E. Gavioli, and C. Ottolenghi, 1987, Gen. Camp. Endocrinol. 66,306-313) that epinephrine and norepinephrine act via B-receptor activation. However, the comparison of the effects of isoproterenol and phenylephrine on CAMP with those on phosphorylase a and on glycogen breakdown suggests that a more complex mechanism is possibly involved in the catecholamine effect on catfish glycogenolysis. 0 1987 Academic Press. Inc.
The different physiological responses in target tissues induced by catecholamines are due to their interaction with (Y- and /3adrenoreceptors (Ahlquist, 1948). The stimulation of B-receptors induces an increase in the level of cyclic adenosine 3’:5’-monophosphate (CAMP), which is considered to be the intracellular messenger for B-adrenergic effects (see Robison et al., 1971). With regard to the effect of catecholamines on carbohydrate metabolism in liver, as already discussed by Hornbrook (1970), it is not easy to state if the responses to catecholamines are mediated by CL- or B-receptors. In rabbit and dog livers the catecholamine effect results in an increase in CAMP (see Robison et al., 1971), indicating a P-mediated action. In guinea pig liver a &-subtype is involved in catecholamine-stimulated glycogenolysis (Arinze and Kawai, 1983). On the other r This work was supported by a grant for Scientific Research from the Minister0 della Pubblica Istruzione, Rome. 2 A part of this work was presented at the 13th Conference of European Comparative Endocrinologists, Belgrade, 1986. 216 0016-6480187 $1.50 Copyright All rights
Q 1987 by Academic Press, Inc. of reproduction in any form reserved.
hand, Exton and co-workers (Hutson et al., 1976; Cherrington et al., 1976) have demonstrated that epinephrine-induced glycogenolysis in rat hepatocytes is not influenced by propranolol, which is usually considered to be a B-blocking agent. Moreover, it was found that some a-adrenergic antagonists inhibited the glycogen breakdown in rat (Sherline et al., 1972; Tolbert et al., 1973; Exton, 1979). It seems therefore that, at least in rat liver, glycogenolysis is mediated principally by a,-adrenergic receptors. There are great differences in the mechanisms through which catecholamines act on liver carbohydrate metabolism in various animal species (Mayer et al., 1961; Sherline et al., 1972). Moreover, the catecholamine mechanism may be influenced by the age and sex of the animals, due to the transformation of receptors from p to o( type (Blair et al., 1979; Studer and Borle, 1982; Kunos et al., 1985). Until now, little research has been done on these catecholamine effects in lower vertebrates, and in particular in fish. In a previous work (Brighenti et al., 1987) we studied the effects of epinephrine, norepi-
AND CAMP IN CATFISH
TABLE 1 CAMP LEVEL IN ISOLATED CATFISH HEPATOCYTES UNDER BASAL CONDITIONS Treatment
None Phentolamine (lo-4 M) Propranolol (1O-4 M)
9 7 7
CAMP (pmol/mg protein) 16.43 r 0.66 17.19 c 0.76 15.30 f 1.19
Note. Values are means i SEM of n experiments. Incubation time, 10 min.
nephrine, isoproterenol, and phenylephrine and of the CE-and P-inhibitors phentolamine and propranolol on phosphorylase activity, glycogen level, and glucose release in isolated catfish hepatocytes. In order to better explain the mechanism involved, we have now studied the effect of the above-mentioned catecholamines and inhibitors on CAMP levels in the same liver cell preparations. MATERIALS
Animals. Adult catfish, Ictalurus melas, weighing 200-300 g, were purchased from a local dealer. Fish were placed in groups of 10 in tanks containing 200 liters of well-aerated, dechlorinated, and continuously depurated tap water at the environmental temperature (18-24”). Animals were maintained without food for some days until the experiments commenced. Hepatocyte incubation. The hepatocyte preparation was performed essentially with the technique de-
scribed by Seglen (1972). as reported in a previous work (Ottolenghi et al., 1984). Hepatocytes (150 ~1, corresponding to about 20 mg wet wt and to 3-4 mg protein) were suspended in 1 ml incubating mixture (teleost Ringer bicarbonate, pH 7.4, containing 2% albumin, and 5 mM theophylline as a phosphodiesterase inhibitor). in test tubes and -were gassed with O,/CO, 95%/5% (v/v) mixture. Cell incubation was carried out in a shaking bath at 30” ascording to the experimental time schedule; in general, catecholamines were added after a 5-min preincubation, and incubation lasted 5 min more. If an inhibitor was added, this was done at the beginning of preincubation. Catecholamine and inhibitor concentrations were 10d5 and 10e4, respectively, unless otherwise indicated. According to Ferretti et al. (1976), at thhe end of incubation in order to remove the CAMP released in the medium, cell suspensions were rapidly cooled on ice and centrifuged (10 min, 2OOQg, 4”. and then 1 ml 6% cold trichloroacetic acid (TCA) was added to the pellets. CAMP analysis. TCA suspensions were centrifuged and extracted four times, each time with 5 ml watersaturated ethyl ether. The final aqueous solution was tested for CAMP by the competitive, protein-binding assay according to Brown et al. (1971). Results were expressed as picomoles of CAMP per milligram protein, as evaluated by the method of Lowry ef al. (1951). Statistical analysis was carried out using the paired Student t test. Materials. The binding protein for CAMP analysis was prepared from beef adrenals according to Brown et al. (1971). Reagents for teleost Ringer solution and buffers for cell preparation and incubation, theophylline and 2-mercaptoethanol for the CAMP test, were supplied by Merck (Darmstadt, FRG). [3HjcAMP was a product of Amersham International plc (Amersham,
TABLE 2 EFFECT OF THEOPHYLLINE ON CAMP LEVEL IN ISOLATED CATFISH HEPATOCYTES Incubation time (min) 0 0 0.5 0.5 5 5
epinephrine” + + +
10.2 26.8 9.1 76.2 10.9 61.0
t 1.0 t 3.2’ I 0.6 IC_2.8-.9$ 2 0.9 i 4.7”~s
+ 17.1 33.8 19.6 161.8 22.2 248.4
-?3 1.0” + 2.0*[email protected]
i l.l** -+ 9.2”+§ rt 0.F i 11 .2”“,“.(i#
Note. Values are means f SEM of three experiments. a 10-5 M. b 5 mM. Levels of significance (paired Student’s t test): *P < 0.05 and **P <: 0.01 as compared with samples in the absence of theophylline; l P < 0.05 and l *P < 0.01 as compared with samples in the absence of epinephrine at the same time of incubation; §P < 0.05 and §§P < 0.01 as compared with samples at zero time in the presence of epinephrine.
UK). The liquid scintillation solution, Atomlight, was a product of New England Nuclear (Boston, MA). Collagenase and all other reagents for cell isolation and tests, catecholamines, and antagonists were from Sigma Chemical Co. (St. Louis, MO).
The basal CAMP levels after a lo-min incubation in control samples and in propranololand phentolamine-treated samples are reported in Table 1. The small changes observed were not significant. In Table 2 the effect of theophilline added to the incubation medium is shown. In the presence of this phosphodiesterase inhibitor the hepatocyte CAMP level is always significantly higher, either in the absence or in the presence of epinephrine. The dose-effect curves of the tested catecholamines on CAMP level, reported in Fig. 1, indicate that the effect increases continuously with the agonist concentration: epinephrine and isoproterenol gave the greatest response, while norepineph-
rine was less effective, and phenylephrine even less so. Figure 2 shows the time course for the catecholamine effect on CAMP level. Epinephrine and isoproterenol show their maximal effect after a 30-min incubation, norepinephrine and phenylephrine after a lo-min incubation, and then the values decrease slightly. Figure 3 shows the effect of catecholamines on hepatocyte CAMP level in the presence or in the absence of the w and pblockers phentolamine and propranolol. Epinephrine and isoproterenol greatly increased (about 15 times) the CAMP level, whereas norepinephrine caused a significant but smaller increase (about 8 times). Phenylephrine induced the smallest effect (twofold increase) in the CAMP level. Pro-
FIG. 1. Dose effect of catecholamine on CAMP level in isolated catfish hepatocytes. For incubation conditions see Materials and Methods. Values are means 2 SEM of three experiments.
min 2. Time course for the catecholamine (lO-5 M) effect on CAMP level in isolated catfish hepatocytes. For incubation conditions see Materials and Methods. Values are means i SEM of (n) experiments. Levels of significance (paired Student’s f test): for epinephrine, isoproterenol, norepinephrine, all values were significant (F’ C 0.01) as compared with controls at the same incubation time; for phenylephrine, only values at 5- and IO-min incubations were significant (P < 0.05) as compared with controls. FIG.
AND CAMP IN CATFISH EPIN.
FIG. 3. Effects of catecholamines i (1O-5 M), propranolol(l0 -4 M), and phentolamine (10m4 M) on CAMP level in isolated catfish hepatocytes. For incubation conditions see Materials and Methods. Values are means i SEM of n experiments, as indicated in the columns. Levels of significarkce (paired Student’s t test): (00) P < 0.01 as compared with controls; (xx) P < 0.01 as compared with samples incubated without inhibitor.
pranolol completely blocked the catecholof events has been extensively documented amine-induced effect, while phentolamine (for references, see Berridge, 1975). Morewas ineffective. over, Exton (1979) demonstrated that er-adIn Fig. 4 the dose effect of propranolol renergic stimulation of glycogenolysis in on catecholamine-induced action is shown. rat liver involves the mobilization of Ca2” Fifty percent inhibition was reached at a ions from the mitochondria and the stimu10M7 M concentration for norepinephrine lation of phosphorylase kinase by the reand phenylphrine and at about 10W6 M for sulting increase in cytosolic Ca2+ concenepinephrine and isoproterenol. tration . In this study we have evaluated the inDISCUSSION tracellular CAMP content in isolated catfish The extensive studies of Sutherland and hepatocytes in response to adrenal cateassociates (see Robison et al., 1971) have cholamines and to two catecholaminergic shown that the primary action of catecholdrugs (isoproterenol and phenylephrine). It amines on the liver is the stimulation of ad- is noticeable that the basal levels of CAMP, enylate cyclase and the formation of CAMP, as reported in Table 1 and in Fig. 1, are which starts the cascade of reactions higher than those reported in mammals leading to the glycogen phosphorylase acti- (Christoffersen and Berg, 1974; Blair et al. I vation. However, catecholamines may 1979; Tolbert et al., 1973) and amphibians exert their glycogenolytic effect on the (Janssens et al., 1983; Janssens and Crigg, liver independently of CAMP formation. 1984), but in tilapia, Sarotherodon mdsSherline et al. (1972) showed that in the rat sambicms, Carrillo et al. (1980) found liver the glycogenolytic action of epinephrine CAMP levels of 5-2.5 pmol/mg protein accannot be related to a P-receptor-mediated cording to the photoperiod, values not too increase in the hepatic CAMP level. On the far from ours. Moreover, Pit and Djabali other hand, the role of the calcium ion as (1982) found values of the same order in the an intracellular messenger in a whole series gill of mullet (Mugil cupito). In any case,
FIG. 4. Dose effect of propranolol on the catecholamine (1O-5 k&induced action on CAMP level in isolated catfish hepatocytes. For incubation conditions see Materials and Methods. Values are means and ranges of two experiments.
we think that our results indicate the true level of CAMP formed in catfish hepatocytes, since our method includes theophylline addition. In fact, as it is seen in Table 2, theophylline, which inhibits the phosphodiesterase activity, prevents the decay of CAMP. The doses of catecholamines and inhibitors were chosen after preliminary experiments on the effect of epinephrine. Figure 1 shows that 10e5 M epinephrine produced a considerable effect in increasing the CAMP level (about 15 times), although the plateau of response has not yet been reached. On the other hand, doses of 10e5 and lop6 M were used by other authors (Birnbaum et al., 1976; Negishi et al.,
1982; Herman and Brown, 1983). From Fig. 1 it can be seen that levels of epinephrine (1O-8-1O-7 M) which come close to the hormone level after stress in some fish (Nakano and Tomlinson, 1967; Dashow et al., 1982; Plisetskaya et al., 1984) caused significant increases in CAMP level. Figures 1, 2, and 3 show that epinephrine and isoproterenol have a more potent effect in increasing the CAMP concentration than norepinephrine and phenylephrine. The effect of phenylephrine is by far the lowest. The sequence of catecholamine effects via P-receptor is (1) interaction with membrane receptor, followed by stimulation of adenylate cyclase and increase in CAMP level; (2) activation of the enzymatic cascade that results in the activation of glycogen phosphorylase and in the increased breakdown of stored glycogen; and (3) increase of the glucose output. Therefore the increase in CAMP level is one of the earlier effects. The time courses of CAMP level (Fig. 2) and of glucose output (Brighenti et al., 1987) agree with the above sequence of events. The high levels of CAMP found at zero time in catecholamine-treated samples (Fig. 2) are possibly due to the time lag between agonist addition and the cooling of cells; during this time, even if limited to 15-20 set, catecholamines may activate CAMP synthesis and, in addition, the presence of theophylline inhibits the CAMP decay due to phosphodiesterase activity (Table 2). The rapidity of the catecholamine action is demonstrated by the slope of the catecholamine-tested curve (Fig. 2), which is always steep for 5 min of incubation and then becomes less steep. Later, the epinephrine and isoproterenol curves increase slowly until 30 min; those of norepinephrine and phenylephrine increase until 10 min. Thereafter a small decrease in CAMP content occurs. This decrease is possibly due to a long-term phosphodiesterase activation by catecholamines, as supposed by Solomon (1975). The distinction between the (Y- and P-re-
AND CAMP IN CATFISH
ceptor-mediated action of a variety of agonists is related to the order of their potency, as well as to the blockade by (x- and B-pharmacological antagonists. The catecholamines tested by us on hepatocyte CAMP concentration showed a stimulatory action with the following order of potency: epinephrine = isoproterenol > norepinephrine > phenylephrine. The antagonist propranolol blocked the effect of all substances tested, while the a-antagonist phentolamine was ineffective. The classical order of potency is isoproterenol > epinephrine > norepinephrine > phenylephrine for B-receptor-mediated responses and, epinephrine > norepinephrine > phenylephrine > isoproterenol for cx-receptor-mediated responses (Exton, 1980). Thus, the descending rank of potency in catecholamine activity we observed could indicate a B-adrenergic action. With regard to the effect of propranolol concentration on catecholamine-induced CAMP increase (Fig. 3), we found that LOP4 M propranolol brought the CAMP present in the liver cell back to control level in the presence of all 10m5 h4 catecholamines. But the 50% inhibition varied according to the catecholamine tested. Epinephrine and isoproterenol (the catecholamines that had the greatest effect) needed a propranolol concentration 10 times greater than that of norepinephrine and phenylephrine. This fact would indicate that epinephrine and isoproterenol bound more tightly to their p-receptors. A comparison of the effects of (Y- and pantagonists on norepinephrine and phenylephrine, which are generally regarded as 01agonists, indicates that their responses are inhibited by the B-antagonist propranolol, and not by phentolamine. Therefore, in catfish hepatocytes these catecholamines also seem to interact at P-receptor sites. These results are the opposite of those found for glycogenolysis in rat hepatocytes (Hutson et al., 1976), but they agree with the finding obtained for phenylephrine by
Arinze and Kawai (1983) in guinea pig hepatocytes, by Rufo et al. (1981) in rabbit liver, and by Janssens et al. (1983) and Janssens and Grigg (1984) in amphibian cultured livers. The comparison of the effects of catecholamines on catfish hepatocyte CAMP concentration with the effects of the same catecholamines on the glycogen phosphorylase activity, glycogen level, and glucose release, reported in the previous work (Brighenti et al., 1987), confirm that the effects of epinephrine and norepinephrine are due to a. B-adrenergic action. More uncertain and difficult to explain is the phenylephrine action. In fact, whereas its effect on CAMP was small and much lower than that of all other catecholamines, its effect on phosphorylase a activity and on glycogen content (but not on the glucose output) was almost the same as that for epinephrine. These results may indicate, as already discussed by us (Brighenti et ai,, 1987), that an a-receptor-mediated action could be present, but as propranoiol blunts this effect and phentolamine does not, it is possible that in catfish hepatocytes propranolol may also act directly or indirectly on the a-mechanism, and/or phenylephrine may also have a small but well-defined paction. A similar finding was reported by Janssens et aE. (1983). With regard to isoproterenol, propran0101 completely blocked its induced increase in CAMP level, but was less effective in inhibiting’the isoproterenol effect on phosphorylase a activity, on glycogen level, and on glucose release (Brighenti el’ al., 1987). This may indicate that isoproterenol could have a small a-action, which, however, is not blocked by phe~tolami~e. Then, the interpretation may be that some other mechanisms are involved together with adenylate cyclase system activation. REFERENCES Ahlquist, R. P. (1948). Study of adrenotropic ceptors. Amer. J. Physiol. 153, 586-600.
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AND CAMP IN CATFISH
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Sherline, P., Linch, A., and Glinsman, W. H. (1972). Cyclic AMP and adrenergic receptor control of rat liver metabolism. Endocrinology 91, 680-690. Solomon, S. S. (1975). Effect of insulin and lypolytic hormones on cyclic AMP phosphodiesterase activity in normal and diabetic rat adipose tissue. Endocrinology 96, 1366- 1373. Studer, R. K., and Borle, A. B. (1982). Differences between male and female rats in regulation of hepatic glycogenolysis. J. Biol. Gem. 257, 7987-7993. Tolbert, M. E. M., Butcher, F. R., and Fain, J. N. (1973) Lack of correlation between catecholamine effects on cyclic adenosine 3’:5’-monophosphate and gluconeogenesis in isolated rat liver cells, J. Biol. Chern. 248, 5685-5692.