Digestive Diseases and Sciences, Vol. 36, No. 4 (April 1991), pp. 485-488

CONFERENCE REPORT

oL-Adrenergic Receptors in Liver Regeneration JENNIFER L. CRUISE, PhD

The al-adrenergic receptor mediates the effects of catecholamines on DNA synthesis, as observed in rat liver following a 2/3 partial hepatectomy and in serum-free primary cultures of adult rat hepatocytes exposed to epidermal growth factor. In vitro, norepinephrine action at this receptor heterologously down-regulates epidermal growth factor receptors. In vivo, the otI receptor's effect on DNA synthesis is restricted to early time points after partial hepatectomy. ~1 receptor binding capacity does not vary until 48 hr after liver resection (at which time binding is reduced), but an uncoupling of receptor binding from membrane phosphoinositide turnover occurs between 8 and 16 hr after partial hepatectomy. This change is preceded by a fall in membrane-associated ras p21 detected by radioimmunoassays (46% of control levels by 2 hr after partial hepatectomy). Whether this change represents a loss of p21 protein from membranes or a modification that results in a loss of immunoreactivity is not known. KEY WORDS: ch-adrenergic receptors; liver regeneration; ras p21.

The development of primary culture systems of adult rat hepatocytes has created the opportunity to examine directly the effects of adrenergic agents on DNA synthesis and the interaction of these agents with factors found in serum. Information obtained from such in vitro systems then can be tested in the in vivo model of regenerative DNA synthesis following 2/3 partial hepatectomy. Cultures used to evaluate growth promotion should be composed of adult parenchymal hepatocytes; be quiescent, yet responsive to stimulation by growth factors; and be maintained in the absence of serum, which may contribute uncharacterized

Manuscript received January 9, 1990; accepted August 28, 1990. From the Department of Biology, College of St. Thomas, St. Paul, Minnesota 55105. Presented at the Proceedings of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989, The work described in this manuscript was completed while J.L.C. was affiliated with the Department of Pathology, Duke University Medical Center, Durham, North Carolina. Address for reprint requests: J.L. Cruise, Department of Biology, Mail #4298, College of St. Thomas, 2115 Summit Avenue, St. Paul, Minnesota 55105.

effectors. The short-term primary liver cell cultures used in the following studies met these criteria. The methods used in the work described are identical to those found in the cited references (1, 2, 4-6, 8, 9). Serum from partially hepatectomized rats, when added to primary hepatocyte cultures, was found to stimulate more DNA synthesis than did serum from control animals. When added to control serum, the adrenergic hormones epinephrine and norepinephfine, significantly increased hepatocyte DNA synthesis, approximating the effect of posthepatectomy serum (1). This effect was, however, relatively small, and the variation in activity among batches of serum postponed clear delineation of this adrenergic effect until serum-free culture conditions were established. The addition of norepinephrine (NE) to serumfree hepatocyte culture in the presence of epidermal growth factor (EGF) and insulin was observed to stimulate the incorporation of [3H]thymidine into trichloroacetic acid-precipitable material. Prazosin (an al-adrenergic blocker) reduced nuclear labeling indices to control levels and was

Digestive Diseases and Sciences, Vol. 36, No. 4 (April 1991)

0163-2116/91/0400-048556.50/0 9 1991PlenumPublishingCorporation

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CRUISE 1000-fold more effective than yohimbine (an ot2 antagonist). No significant stimulation of thymidine incorporation was observed with clonidine, a selective a , agonist or with isoproterenol, a J3-adrenergic agonist. Epinephrine produced a dose-dependent stimulation of DNA synthesis similar to that of NE, which was inhibited by prazosin. These data established that the effect of NE on DNA synthesis is mediated via the a~-adrenergic receptor (2).

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The presence of E G F in culture media was required to see the enhancement of DNA synthesis achieved with NE, and the dose-response to NE was enhanced in the presence of insulin. Timecourse experiments illustrated that cells exposed transiently to NE during the first 24 hr in culture showed enhanced responsiveness to the subsequent addition of EGF. This effect led to an investigation of whether NE pretreatment altered hepatocyte responses to EGF at the receptor level. The binding of [125I]EGF binding to control cultures fell during the first 24 hr after plating, reaching 51 - 12% that seen at 0 hr. These data are similar to those of Lin et al (3). At 12 and 24 hr, NE-treated cultures had significantly lower levels of EGF binding than controls. Thus, cultures that responded with enhanced DNA synthesis to EGF addition bound less EGF than matched controls (4). One hour of exposure to NE was sutficient to achieve a maximal reduction of specific EGF binding to a level of 55-65% that of control. NE did not compete directly for binding with EGF. NE EFFECT ON EGF RECEPTOR NUMBERS

Analysis of the saturation binding of [125I]EGF to control cultures indicated a single population of receptor sites, with an estimated Kd of 2.19 nM and a Bmax of 118 pg/105 cells. Preincubation for 1 hr with NE did not affect receptor affinity but reduced by 40% the binding capacity, indicating that NE is capable of heterologous down-regulation of EGF receptors. In Figure 1, a dose-response to NE is plotted, superimposing the stimulation of DNA synthesis on the reduction of EGF receptor number. The threshold response was similar for both effects and the divergence at higher concentrations of NE was due presumably to the great difference between them in time of assay. EGF binding changes were recorded after only an hour of NE treatment, while DNA synthesis experiments ran for 48 hr. These longer

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Fig 1. Dose-response relationship between NE concentration and DNA synthesis (solid line) or inhibition of [1251]EGF binding (dashed line). Cultures were incubated as described (5), then either processed for liquid scintillation counting of [3H]thymidine uptake or assayed for EGF binding. For binding, cultures were preincubated with NE for 1 hr and then assayed in 5 ng/ml (0.835 nM) [125IlEGF (100 p,Ci/~g). Binding data represent the mean _ SEM of data from two independent experiments, each performed with triplicate cultures. DNA synthesis data represent the mean --- SEM of three independent experiments (figure from reference 5).

experiments may require higher initial doses of NE in order to maintain an effective concentration over a more prolonged period of exposure. The ability of a t blockade by prazosin to inhibit the effect of NE on EGF binding suggested that the same population of adrenergic receptors was being used as that which mediated NE enhancement of DNA synthesis. No other receptor subtype appeared to be involved (5). This confusing correlation between a reduction in EGF receptor binding and increased responsiveness to EGF has been found using several stimulators of the phosphatidylinositol signaling pathway--a pathway shared by the al-adrenergic receptor--such as TPA and vasopressin. [We also have found that, like NE, both arg-vasopressin and angiotensin 1I had some stimulatory effect on hepatocyte DNA synthesis in the presence of EGF. (6)] No satisfactory model exists to explain this paradoxical association. It has been suggested that where down-regulation is long-standing, a reduced overall degradation of EGF in media may maintain higher EGF concentrations over time (7); it is also possible that what appears as a reduction in surface receptors reflects a higher receptor turnover rate and thus a greater internalization of EGF over time. Digestive Diseases and Sciences, Vol. 36, No. 4 (April 1991)

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[NE] (uM) Fig 2. Stimulation of inositol phosphate (IP) production by NE after partial hepatectomy (PHX). Cells isolated from control and regenerating rat livers were prelabeled by incubation with [aH]myoinositol and then incubated for 10 min with NE at concentrations ranging from 0.01 to 100 p.M. The labeled, water-soluble inositol phosphates formed were separated by ion-exchange chromatography and quantitated by liquid scintillation. Data are expressed relative to basal IP production in cells unsupplemented by NE. Between 8 and 16 hr after PHX, the dose-dependent stimulation of IP generation by NE was lost. By 24 hr, the el receptor was again coupled to IP production. N = 2-8 independent experiments per point, using triplicate samples (figure from reference 9).

~1 RECEPTORS AND LIVER REGENERATION

Several lines of evidence suggest a role for ~l receptors in early regenerative DNA synthesis following a 2/3 partial hepatectomy. A dramatic increase in plasma catecholamines was observed as early as 2 hr after resection and an increased level was maintained through at least the first two postoperative days. Both surgical hepatic sympathec: tomy and injection with prazosin inhibit the first wave of DNA synthesis seen at 26 hr after hepatectomy. The ability of receptor blockade by prazosin to perturb EGF receptor binding was investigated. Control animals showed a decline in EGF receptor binding after resection, and prazosin treatment interferes with this response (8). Thus it appears that both early regenerative DNA synthesis and NEstimulated DNA synthesis in culture were affected by cxl adrenergic receptor events. oq RECEPTORS AND IP GENERATION

Surface adrenergic receptors were examined during the early stages of liver regeneration. Although no change was observed in oq receptor binding Digestive Diseases and Sciences, Vol. 36, No. 4 (April 1991)

during the prereplicative hours, the coupling of these receptors was altered between 8 and 16 hr after partial hepatectomy. During this period, binding of NE to receptors did not elicit a release of phophatidylinositol metabolites, this receptor's usual signaling mechanism (Figure 2). Coupling returned after 24 hr; by 72 hr, the dose-response of inositol phosphate (IP) generation to NE addition was similar to that of controls, although the maximum response was somewhat diminished. This may be due to a reduced number of etI sites seen at 72 hr

(9). Interestingly, another growth-promoting action of NE is to overcome the inhibitory effects of transforming growth factor-beta (TGF-B) on hepatocytes stimulated in culture by EGF. When hepatocytes are isolated from regenerating livers during the period of uncoupling described above, NE addition has an even greater effect on the resistance to TGF-B than it does on that of control cells (10, and KA Houck, unpublished data). These data suggest that in the prereplicative phase of liver regeneration, NE acts to affect DNA

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CRUISE synthesis, through an a~ receptor that is not coupled to its usual second messenger system. ras

p21 IN LIVER REGENERATION

Changes in the expression of the ras protooncogenes have been described following partial hepatectomy. Messenger RNA for these genes increases concurrently with DNA synthesis (11). The immunoreactive translation products of these genes (p21s) were examined to determine if there were any prereplicative changes that might implicate ras in the early events leading to DNA synthesis. Two types of assays demonstrated a loss of anti-p21 antibody binding in liver membranes soon after partial hepatectomy. In both, an initial drop in p21 immunoreactivity by 2 hr was followed by a partial restoration and a subsequent decrease between 12 and 24 hr. After 24 hr, p21 content increased toward control levels (9). Thus, very soon after partial hepatectomy and prior to the uncoupling of the al receptors, changes in ras protein occur. While this appears as a "loss" of p21, the failure of these proteins to be recognized by antibody may be due to one or more causes. These proteins are normally associated with the cytoplasmic face of the plasma membrane; it is possible that they are translocated to another location within the cell. While initial screening of cytosol failed to demonstrate the appearance of p21 in this fraction, a high dilution in this compartment may require extensive concentration for detection. The identification of p21-associated factors, such as GAP (12), suggests that changes in immunoreactivity may be due to interaction of p21s with an unknown membrane or cytosolic constituent. Any number of other modifications of p21 that affect its reactivity with antibodies are possible. The use of antibodies that are specific to Ha-, K-, or N - r a s p21s should allow us to examine their changes. All previous work was done using the pan-reactive monoclonal Y13-259. It must be stated that currently it is not clear whether the changes in p21 and the uncoupling of the al receptor that follows are linked. The al-adrenergic receptor has been demonstrated to play a role in the enhancement of DNA synthesis in primary cultures of adult rat hepatocytes. I n vitro, this effect correlates with heterologous down-regulation of the EGF receptor. During early liver regeneration, the c~1 receptor also plays a

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stimulatory role, which again appears to be linked to a change in the hepatic EGF receptor. Activation of the am receptor probably is the first or one of the first events that initiates regenerative DNA synthesis following partial hepatectomy. Prereplicative events at the hepatic plasma membrane include an early decline in immunoreactive ras p21 protein, followed by an uncoupling of the a 1 receptor to the generation of inositol phosphates. This uncoupled receptor appears to be the form that is involved in DNA synthesis. REFERENCES 1. Michalopoulos G, Cianciulli HD, Novotny AR, Kligerman AD, Strom SC, Jirtle RL: Liver regeneration studies with rat hepatocytes in primary culture. Cancer Res 42:4673-4682, 1982 2. Cruise JL, Houck KA, Michalopoulos G: Induction of DNA synthesis in cultured rat hepatocytes through stimulation of alpha-1 adrenoreceptor by norepinephrine. Science 227:749751, 1985 3. Lin S-H, Blalsdell J, O'Keefe E, Earp HS: Insulin inhibits the glucocorticoid-mediated increase in hepatocyte EGF binding. J Cell Physiol 119:267-272, 1984 4. Cruise JL, Michalopoulos G: Norepinephrine and epidermal growth factor: dynamics of their interaction in the stimulation of hepatocyte DNA synthesis. J Cell Physiol 125:45-50, 1985 5. Cruise JL, Cotecchia S, Michalopoulas G: Norepinephrine decreases EGF binding in primary rat hepatocyte cultures. J Cell Physiol 127:39-44, 1986 6. Cruise JL, Houck KA, Michalopoulos G: Early events in the regulation of hepatocyte D N A synthesis: The role of alphaadrenergic stimulation. Stand J Gastroenterol 23(suppl 151):19-30, 1988 7. Magun BE, Matrisian LM, Bowden GS: Epidermal growth factor. Ability of tumor promotor to alter its degradation, receptor affinity and receptor number. J Biol Chem 255:6373-6381, 1980 8. Cruise JL, Knechtle SJ, Bollinger RR, Kuhn C, Michalopoulos G: Alpha1 adrenergic effects and liver regeneration. Hepatol 7:1189-1194, 1987 9. Cruise JL, Muga SJ, Lee Y-S, Michalopoulos GK: Regulation of hepatocyte growth: Alpha-1 adrenergic receptor and ras p21 changes in liver regeneration. J Cell Physiol 140:195201, 1989 10. Houck KA, Cruise JL, Michalopoulos G: Norepinephrine modulates the growth-inhibitory elect of transforming growth fator-beta in primary rat hepatocyte cultures. J Cell Physiol 135:551-555, 1988 11. Thompson NL, Mead JE, Braun L, Goyette M, Shank PR, Fausto NL: Sequential protooncogene expression during rat liver regeneration. Cancer Res 46:3111-3117, 1986 12. Adari H, Lowy DR, Willumsen BM, Der CJ, McCormick F: Guanosine triphosphatase activating protein (GAP) interacts with p21 ras effector binding doman. Science 240:518-521, 1988

Digestive Diseases and Sciences, Vol. 36, No. 4 (April 1991)