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Stress-induced regulation of the renal peripheral benzodiazepine receptor: Possible role of the renin-angiotensin system
Psychoneuroendocrinology,Vol. 19, No. 1, pp. 43-54, 1994
0306-4530/94$6.00 + .00 © 1993PergamonPress Ltd.
Printed in the U.S.A.
STRESS-INDUCED REGULATION OF THE RENAL PERIPHERAL BENZODIAZEPINE RECEPTOR: POSSIBLE ROLE OF THE RENIN-ANGIOTENSIN SYSTEM PHILIP V. HOLMES l and ROBERT C. DRUGAN* Department of Psychology, Schrier Research Laboratory, Brown University, Providence, Rhode Island 02912, U.S.A.
(Received 27 April 1993; in final form 23 July 1993)
SUMMARY The etiology of the decrease in renal peripheral benzodiazepine receptor (PBR) binding caused by stress was studied in rats. Prior investigations suggest that the response of the renal PBR to stress occurs independently of the hypothalamo-pituitary-adrenal (HPA) axis and sympathetic nervous system. The present experiments tested the hypothesis that the renin-angiotensin system is involved in regulating the PBR. Eighty min of brief, intermittent tailshocks caused increases in plasma renin activity and decreases in renal PBR binding. The stress-induced decrease in renal PBR binding was reversed by pretreatment with captopril. Acute admininstration of angiotensin II (ANG II) alone caused reductions in PBR binding in kidney, heart, and cerebral cortex. These data suggest that ANG II may be an endogenous factor responsible for regulating the PBR in several tissues during stress.
INTRODUCTION
THE PERIPHERALBENZODIAZEPINEreceptor (PBR) is a relatively small (17-18 kdalton) protein situated predominantly on the outer mitochondrial membrane in both peripheral and central nervous system (CNS) tissues (Anholt, 1986; Antkiewicz-Michaluk et al., 1988; Basile & Skolnick, 1986). The highest densities of PBR are found in the adrenal gland, but PBR densities are also relatively high in the kidney, heart, lung, ovary, and testes (Benavides et al.,1983a; Davies and Huston, 1981; DeSouza et al., 1985; Fares et al., 1987). PBR in the CNS are associated primarily with glial cells, and the highest densities are found in choroid plexus, ependyma, pineal gland, olfactory bulb, and circumventricular organs (Benavides et al., 1983b; Doble et al., 1987). The PBR is thus physically and anatomically distinct from the central benzodiazepine receptor (CBR), which is situated on the postsynaptic GABAA receptor/chloride ionophore complex in the CNS (McCabe & Wamsley, 1986; Mohler & Okada, 1977; Squires & Braestrup, 1977; Ticku, 1983). The recent cloning and sequencing of the DNA encoding the 18 kdalton PBR protein confirms this distinction at the molecular level, suggesting that the PBR is a distinct class of receptor (Krueger et al., 1990; Riond et al., 1991; Sprengel et al., 1989). *Address c o r r e s p o n d e n c e and reprint requests to: Robert C. Drugan, D e p a r t m e n t of Psychology, Schrier R e s e a r c h L a b o r a t o r y , Box 1853, B r o w n University, Providence, R h o d e Island 02912. 1Current Address: Section on Behavioral N e u r o p h a r m a c o l o g y , E x p e r i m e n t a l Therapeutics Branch, N I M H , Bethesda, M D 20892. 43
44
P.V. HOLMES and R. C. DRUGAN
The mitochondrial localization of the PBR and its high density in steroidogenic tissues probably have been the most valuable clues for determining its function. Recent evidence suggests that the PBR regulates steroidogenesis in both the periphery and in the brain (see Ferrarese et al., 1993; Papadopoulos et al., 1991 for review). PBR ligands, including the endogenous peptide diazepam binding inhibitor (DBI) and its fragments, stimulate pregnenolone synthesis in cultured adrenocortical, Leydig, granulosa, and glial cells (Amsterdam & Suh, 1991; Besman et al., 1989; Mukhin et al., 1989; Papadopoulos et al., 1990; Yanagibashi et al., 1989). Experiments demonstrating the responsivity of the PBR to environmental stressors provide further evidence of a regulatory role for the PBR in endocrine systems. Reports from several laboratories reveal that stress generally causes rapid and shortlived alterations in the density (Bmax) of PBR in rats, but this effect is highly dependent on the type of stress employed and the tissues surveyed. For example, brief noise stress causes immediate increases in PBR density in adrenal gland, cerebral cortex, and hippocampus (Ferrarese et al., 1991). PBR density in olfactory bulb and kidney is increased immediately following a brief forced swim (Novas et al., 1987; Rago et al., 1989). Presentation of a fear-conditioned stimulus causes increases in PBR binding in the olfactory bulb (Holmes et al., 1992). Studies in humans reveal a similar type of response in PBR from blood platelets, with increases in the density of PBR occurring immediately following a written examination (Karp et al., 1989). In contrast, more severe stress tends to cause decreases in PBR binding. An 80 min session of 80 tailshocks causes decreases in PBR binding in cerebral cortex, pituitary, heart, and kidney (Drugan et al., 1986). Scatchard analyses in renal membranes has revealed that the reduction in PBR binding seen following 80 min of stress is due to a drop in receptor density (Bmax) and not affinity (Kd). Alterations in PBR binding during stress have thus been observed in a variety of tissues, including those that are not typically regarded as being steroidogenic (i.e., kidney, heart, blood, olfactory bulb). The systems responsible for regulating the PBR may therefore be tissue-specific. Experiments in this laboratory have focused on the stress-induced decrease in PBR density in kidney. Previous attempts to elucidate the physiological mechanisms responsible for modulating renal PBR binding during stress suggest that neither the hypothalamo-pituitary-adrenal (HPA) axis, the neurohypophysis, nor the sympathetic nervous system are directly involved. For example, stress-induced decreases in renal PBR binding persist following hypophysectomy, and are actually enhanced following adrenalectomy (Drugan et al., 1988). Furthermore, neither chemical sympathectomy, adrenergic blockade, nor unilateral renal denervation influence the response of the renal PBR to stress (Drugan et al., 1988; Holmes & Drugan, in press). Though the renin-angiotensin system is closely linked to the HPA axis, neurohypophysis, and sympathetic nervous system, there are several mechanisms for activating this system independently of the others (Kotchen & Roy, 1983; Campbell & Heinrich, 1990). The renin-angiotensin system may therefore function as an independent "stress axis" vis-a-vis the renal PBR. Earlier experiments from this laboratory have demonstrated that the administration of Angiotensin II (ANG II) alone causes dose-dependent reductions in renal PBR density similar to those caused by stress (Holmes & Drugan, 1992). The present experiments further tested the possibility that the alterations in PBR binding in kidney during stress are linked to activation of the renin-angiotensin system.
STRESS, ANGIOTENSIN II, AND PBR IN KIDNEY
45
METHODS
Subjects Male Sprague Dawley-derived rats bred at Hunter Laboratory, Brown University, served as subjects. Rats were group-housed (3-6 per cage), allowed free access to food and water, and weighed between 230-330 g at the time of experimentation. The colony was maintained at 23°C on a normal 12 h light/dark cycle (lights on at 0700 h). All behavioral procedures were conducted between 0900 and 1500 h. Drugs ANG II acetate salt (human), and [2S]-l-[3-Mercapto-2-methylpropionyl]-L-proline (captopril) were purchased from Sigma Chemical Co., St. Louis, MO. ANG II was dissolved in distilled water. Captopril was dissolved in 0.15 M sodium carbonate. Injection volume of ANG II was 0.5 ml/rat. Injection volume of captopril was 1 ml/kg. Ro5-4864 was kindly supplied by Drs. Peter Sorter (Hoffman-LaRoche) and Peter Suzdak (Novo Industri). [3H]-Ro5-4864 (sp. act. 79.7-86.9 Ci/mM) was purchased from NEN, Boston, MA. Stress Procedure The stress paradigm used in the present experiments was identical to that previously described (Drugan et al., 1988) with the exception that rats were habituated to the guillotine for five to seven days prior to stress in an attempt to stabilize the variability in radioligand binding typically observed in naive subjects. Rats were semirestrained in Plexiglas boxes (15.5 x 12 x 17 cm) by taping the tail, which protruded through a small notch in the rear of the box, to a horizontal post (10 x 1 × 1 cm) extending behind. Eighty tailshocks of 5 s duration were delivered with an average intershock interval of 1 min. The intensity of current was incremented from 1 to 1.5 mA after 35 shocks and up to 2 mA at 60 shocks. Shocks were administered by a Lafayette Instruments Model 82400 shock generator through electrodes taped to the tail augmented with electrode paste. Effects of Stress on Plasma Renin Activity and PBR Binding Nineteen rats were randomly divided into either stress or naive conditions and stressed as described above or left in the home cage. Stressed and naive rats were killed by decapitation at approximately the same time in a counterbalanced order immediately following the stress session. Trunk blood was collected in microfuge tubes containing 100 t*l of 2% EDTA. The blood was centrifuged at 1300 × g for I min. Plasma was collected and stored at -20°C. Kidneys were dissected and placed in 0.32 M sucrose solution in 20 ml glass vials and rapidly frozen in a COJacetone slurry and stored at -80°C until assay. Captopril Pretreatment Sixty-eight rats were randomly divided into the following drug conditions: captopril 10, 50, and 100 mg/kg and vehicle. Within each group rats were divided into naive and stress conditions. Rats were injected SC with captopril or vehicle and were stressed 15 min later or left in the home cage. Rats were killed and kidneys dissected as described above immediately following the stress session.
46
P.V. HOLMESand R. C. DRUGAN
Acute Administration of ANG H ANG II was administered over an 80 min period in an attempt to mimic the time course of the stress session. Seventeen rats were randomly divided into drug and vehicle conditions and received three 25/xg injections (SC) of ANG II or vehicle at 0, 30, and 60 min for a cumulative dose of 75/zg. Rats were killed at 80 min. Kidney, heart, adrenal gland, olfactory bulb, hippocampus, and cerebral cortex were dissected and frozen as described above. In Vitro Radioligand Binding Tissues were thawed in a 25°C water bath, disrupted in 50 volumes of 50 mM TrisHC1 buffer (pH --- 7.4), and centrifuged at 20,000 × g for 20 min. The tissues were resuspended in 200 volumes (peripheral tissues) or 100 volumes (CNS tissues) of the same buffer, and the binding of [3H]Ro5-4864 was determined as previously described (Drugan et al., 1986, 1988; Weissman et al., 1984). Previous experiments have employed Scatchard analyses to demonstrate that the effect of both 80 tailshocks and ANG II administration on renal membranes is to decrease the density (Bmax)and not affinity (Kd) of PBR (Drugan et al., 1986; Holmes & Drugan, 1992). A single concentration of radioligand was therefore used for the present experiments. Briefly, 0.1 ml of peripheral tissue or 0.6 ml of CNS tissue (containing approximately 0.04 or 0. l mg protein, respectively) was added to each assay tube containing 0.1 ml of radioligand (final concentration, 1 nM), 0.1 ml of unlabeled Ro5-4864 or Tris-HCl buffer, and additional buffer to yield a total volume of 1 ml. Assays were performed triplicate. The reaction was initiated by the addition of tissue and terminated after incubation at 0-4°C for 60 min by rapid filtration over Schleicher and Schuell #32 glass filter strips using a Brandel M-24R filtering manifold. Samples were washed with two 5 ml aliquots of 0-4°C buffer. The specific binding of [3H]Ro5-4864 was defined as the difference in binding obtained in the presence and absence of unlabeled Ro5-4864 (final concentration, 100 /zM). The radioactivity retained by the filters was measured in a Beckman LS 5000TD liquid scintillation spectrometer using 6 ml of Eco-scint solution as a fluor. Specific binding accounted for approximately 70-90% of total binding. Protein concentration was determined as previously described (Lowry et al., 1951; Miller, 1959). Radioimmunoassay Plasma renin activity was determined using an [125I]angiotensin I radioimmunoassay kit from NEN. Plasma renin activity was determined by quantifying the amount of angiotensin I generated from endogenous angiotensinogen by renin. Briefly, angiotensin I was generated from 500 ml plasma samples by incubating at 37°C for 1 h in the presence of converting enzyme inhibitors. [125I]angiotensin I, angiotensin I antiserum (rabbit), and samples (100 /.d) were incubated for 16 h at 4°C. The antigen-antibody complex was extracted by adding 500 ~1 of antirabbit gamma globulin. Tubes were centrifuged and the radioactivity of pellets was quantified by a Micromedic gamma counter. Data Analysis All data were analyzed by between-subjects one-way or factorial analysis of variance (ANOVA). Between-treatment differences were evaluated by posthoc Newman-Keul's tests with a critical value set at 0.05. Correlation coefficients were calculated for plasma renin activity values and PBR binding values from naive and stressed rats.
47
STRESS, A N G I O T E N S I N I I , A N D PBR IN KIDNEY 9¢
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FIG. l : Mean plasma renin activity (ng Angiotensin I/ml/h) for separate groups of rats receiving either 80 tailshocks or no stress. Vertical bars represent SEM. Number of subjects per group is indicated at the base of each column. * indicates significantly different from naive control (P < 0.05).
RESULTS
Tailshock Stress Causes Increases in Plasma Renin Activity and Decreases in PBR Binding Fig. 1 illustrates the effect of 80 min of tailshocks on plasma renin activity. One-way ANOVA revealed that stress significantly elevated plasma renin activity (P < 0.05). The binding of [~H]Ro5-4864 in renal membranes of rats exposed to 80 tailshocks was reduced compared to naive controls. Fig. 2 illustrates this effect. The effect of stress on renal PBR binding was significant as indicated by one-way ANOVA (P < 0.01). No significant correlations between plasma renin activity and PBR binding were observed in either naive or stressed rats. Captopril Pretreatment Blocks the Stress-Induced Reduction in Renal PBR Binding Fig. 3 illustrates the effect of inhibition of endogenous ANG II formation by captopril on renal PBR binding in stressed rats. Factorial ANOVA revealed that there was a significant effect of stress (P < 0.001) and drug treatment (P < 0.05). The interaction term was also significant (P < 0.05), indicating that the effect of stress depended on drug pretreatment. Posthoc tests revealed that stressed rats pretreated with vehicle or 10 mg/kg of captopril differed significantly from naive/vehicle controls, whereas stressed rats pretreated with either 50 or 100 mg/kg of captopril did not. Acute Administration o f A N G H Decreases PBR Binding PBR binding was reduced in kidney (P < 0.001), heart (P < 0.05), and cerebral cortex (P < 0.01) following the administration of 75/zg of ANG II delivered over an 80 min period (see Table I). No significant alterations in PBR binding were seen in the adrenal gland, hippocampus, or olfactory bulb.
48
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FIG. 3: Mean specific binding of [3H]Ro5-4864 (fmole/mg protein) at a concentration of 1 nM in renal membranes from separate groups of rats receiving SC injections of either vehicle or captopril (Cap: 10, 50, or 100 mg/kg) 15 min prior to stress or no treatment (naive). Number of subjects per group is indicated at the base of each column. * indicates significantly different from naive/ vehicle control (P < 0.01). + indicates significantly different from stress/vehicle-treated rats (P < 0.05).
STRESS, ANGIOTENSIN II, AND PBR IN KIDNEY
49
TABLE I. SPECIFIC [3H]RO5-4864 BINDING (FMOLE/MG PROTEIN; MEAN -- SEM)
Vehicle ANG II
Adrenal
Kidney
Heart
9583.2 + 528 9064.1 + 851
1420.3 + 99 1012.9 + 25*
OIL bulb
Cortex
Hippo
1325.0 + 54 483.4 + 32 59.9 + 3 54.1 + 6 1090.0 + 170t 451.2 + 46 47.2 + 3* 49.5 + 1
*p < .01; tp < .05.
DISCUSSION Results from the present experiments provide preliminary support for the proposal that circulating ANG II is involved in the stress-induced regulation of the renal PBR. As expected, 80 min of intermittent tailshocks caused decreases in renal PBR binding. Tailshock stress also stimulated renin secretion. Pretreating rats with the converting enzyme inhibitor captopril, which blocks the formation of ANG II, reversed the stressinduced decrease in PBR binding. Products of angiotensin I conversion may therefore be necessary for the alterations in renal PBR binding observed in this paradigm. Furthermore, the administration of ANG II alone to naive rats is sufficient to alter PBR binding in a manner similar to stress. The lack of any significant correlation between plasma renin activity and PBR binding suggests that there is no simple linear relationship between these two variables. However, in the present experiments it was not possible to determine the relationship between stress-induced alterations in plasma renin activity and stress-induced alterations in PBR binding from baseline values. The dose-dependent effect of ANG II administration on renal PBR binding previously reported may be a better indication of the relationship between circulating ANG II and the PBR (Holmes & Drugan, 1992). The ability of captopril to attenuate the PBR response to stress may not be due solely to inhibition of ANG II formation. Direct interactions with other enzyme systems, such as those involved in the synthesis and degradation of eicosanoids and bradykinin, cannot be ruled out (Douglas, 1985; Harding et al., 1991; Schwieler & Hjemdahl, 1992). CNS actions of captopril cannot be excluded either. Converting enzyme inhibitors produce anxiolytic- and antidepressant-like effects in the °'learned helplessness" model of anxiety and depression in rats (Martin et al., 1990). Antidepressant-like effects of converting enzyme inhibitors have also been reported in human studies (Testa et al., 1993; Deicken, 1986). The central and/or peripheral mechanism of the antidepressant action, be it the blockade of ANG II formation or bradykinin degradation, is unclear. The inescapable tailshock stress paradigm employed in the present experiments is similar to that of the ~'learned helplessness" model of depression (Drugan et al., 1989). However, further studies are required to determine whether there is a relationship between captopril's ability to reverse stress-induced behaviors and its ability to reverse stress-induced PBR alterations in rats. The effect of stress on the renal PBR can be mimicked by peripheral administration of ANG II. Previous experiments have demonstrated that ANG II, like stress, causes decreases in renal PBR density (Bmax)and not affinity (Kd) (Drugan et al., 1986; Holmes & Drugan, 1992). The present experiments surveyed several tissues revealing a degree of correspondence between tissues sensitive to ANG II and those that were previously reported to be altered by 80 tailshocks. In the original demonstration of stress-induced
50
P . V . HOLMES and R. C. DRUGAN
alterations in PBR binding, reductions were observed in kidney, heart, cerebral cortex, and pituitary but not adrenal or hippocampus (Drugan et al., 1986). In the present experiments, ANG II caused reductions in PBR binding in kidney, heart, and cerebral cortex but not adrenal or hippocampus (pituitary was not surveyed). The mechanism by which ANG II reduces PBR density remains to be established. The dose and time course of ANG II administration used in this study cause elevations in systemic arterial pressure similar to that observed during tailshock stress (Holmes & Drugan, 1992). However, decreases in renal PBR binding do not appear to be a direct consequence of elevated systemic arterial pressure. Elevating arterial pressure with norepinephrine to the levels induced by stress or ANG II does not affect the renal PBR (Holmes & Drugan, 1992). ANG II influences multiple endocrine systems. For example, ANG II stimulates aldosterone release from the adrenal cortex and vasopressin release from the posterior pituitary (Kotchen & Roy, 1983; Phillips, 1987). It is unlikely, however, that either aldosterone or vasopressin are directly involved in regulating the PBR during stress. Although chronic (one to two weeks) manipulation of aldosterone levels alters renal PBR density (Basile et al., 1987), the rapid alterations in PBR binding caused by stress are not diminished, but enhanced, following adrenalectomy (Drugan et al., 1988). Furthermore, aldosterone does not directly interact with the PBR, as indicated by its inability to influence [3H]Ro5-4864 binding in vitro (Deckert & Marangos, 1986). That adrenalectomized rats show enhanced renin secretion, possibly due to a lack of negative feedback by aldosterone (Darlington et al., 1989), may explain the enhanced PBR response to stress in adrenalectomized rats. The failure of hypophysectomy to influence the response of the renal PBR to stress suggests that neurohypophysial vasopressin is not a critical factor either (Drugan et al., 1988). Experiments conducted in this laboratory have indicated that nanomolar to micromolar concentrations of ANG II do not alter in vitro [3H]Ro5-4864 binding in mitochondrial membrane preparations (unpublished observations). ANG II thus does not appear to competively inhibit binding of [3H]Ro5-4864 at the mitochondrial PBR. ANG II may therefore act as an extracellular signal that, through specific extracellular receptor interactions, induces a cascade of intracellular activity that ultimately influences the PBR. Further in vitro studies using intact cells or slice preparations are required to determine whether the effect of ANG II on PBR binding is mediated specifically by extracellular ANG II receptors. Such studies are also critical to determine the identity and nature of second messengers or intracellular mediators that may be involved in regulating the PBR. Possible intracellular regulators include DBI (Alho et al., 1991; Papadopolous et al., 1991), cAMP (Amsterdam & Suh, 1991), and arachidonic acid (Beaumont et al., 1988; Fonlupt et al., 1990). Though interactions between ANG II and adenylate cyclase or arachadonic acid metabolites have been well established (Ardaillou et al., 1985; Dunn, 1983; Langlois et al., 1992), interactions between ANG II and DBI have yet to be examined. As discussed above, the effect of ANG II appears to be specific to those tissues that exhibit decreases in PBR binding in response to the 80 min stress paradigm. The failure of ANG II to modulate the PBR in other tissues that are altered by different types of stress suggest that several, distinct mechanisms exist for regulating the PBR. These mechanisms may be tissue-specific. In other words, ANG II may regulate the PBR in kidney, heart, and cerebral cortex, whereas other factors may regulate the PBR in adrenal gland, blood platelets, olfactory bulb, and hippocampus. Differences in sensitivity to
STRESS, ANGIOTENSIN II, AND PBR IN KIDNEY
51
circulating ANG II may also account for the tissue-specific responses of the PBR to both 80 tailshocks and ANG II treatment. Though only the renal, cardiac, and cerebral cortical PBR responses to ANG II were siginficant, all tissues showed a trend toward decreases in PBR binding. A great deal of evidence linking the PBR to the regulation of steroidogenesis has accumulated recently. This regulation appears to involve the delivery of cholesterol to a cytochrome P-450 of the inner mitochondrial membrane (see Ferrarese et al., 1993; Papadopoulos et al., 1991 for review). This process is the rate-limiting step in steroidogenesis and has been well characterized in Leydig and adrenocortical cells in vitro (Papadopoulos et al., 1990; Yanagibashi et a1.,1989). Furthermore, in vivo experiments have demonstrated alterations in PBR density in the adrenal gland following stress, and this response appears to be related to steroid synthesis (Ferrarese et al., 1991). However, the response of the PBR in the adrenal gland is not as robust and consistently observed across stress paradigms as the response of the PBR in kidney (cf. Drugan et al., 1986, 1988; Ferrarese et al., 1991; Novas et al., 1987; Rago et al., 1989). This apparent discrepancy suggests that either the kidney synthesizes cholesterol-derived products during stress, or the function of the PBR across tissues is not limited to steroidogenesis. Recent molecular pharmacologocial data reveal an association between the PBR, the outer mitochondrial membrane voltage-dependent anion channel, and the inner mitochondrial membrane adenine nucleotide carrier (McEnery et al., 1992). Such a mitochondrial complex has been proposed to be involved in the transport of a variety of substrates, including ATP as well as several ions (Kinnally et al., 1993; McEnery et al., 1992; Verma & Snyder, 1989). The function of the PBR may therefore be rather broad, and the putative role of the PBR in regulating the synthesis of secretory products could be a general phenomenon across endocrine tissues. The PBR may be involved in the synthesis of endocrine, paracrine, or autocrine, factors that share a common dependence on outer mitochondrial transport for their synthesis. According to this interpretation, a possible role of the PBR in kidney could be the regulation of arachidonic acid metabolism and eicosanoid production. Some arachidonic acid metabolites, such as hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acid, are derived from metabolism by a cytochrome P-450 (Campbell & Henrich, 1990). The hypothetical link between between the PBR and the cytochrome P-450 mechanism involved in eicosanoid formation may be similar to that described for steroidogenesis. Specifically, the PBR may be involved in the delivery of arachidonic acid to a cytochrome P-450 in a fashion similar to cholesterol transport. The influence of ANG II on both eicosanoid formation (Ardaillou et al., 1985) and the PBR may thus be related. Though this particular mechanism is highly speculative, the association between the PBR and cellular endocrine physiology should encourage further study of the role of the PBR in regulating hormone synthesis during stress in a variety of tissues. Acknowledgments: All
behavioral procedures were approved by the Brown University Institutional Animal Care and Use Committee. The authors thank Dr. David A. Bereiter and C.T. Graeber for their assistance in conducting the radioimmunoassay. Submitted as partial fulfillment of requirements for Ph.D. (P.V.H.). Research was supported by a PHS grant #MH45475 and an Alfred P. Sloan Research Fellowship to R.C.D.
REFERENCES
Alho H, Harjuntausta T, Schultz R, Pelto-Huikko M, Bovolin P (1991) Immunohistochemistry of diazepam binding inhibitor in the central nervous system and peripheral organs: Its possible role as an endogenous regulator of different types of benzodiazepine receptors. Neuropharmacol 30:1381-1386.
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P.V. HOLMESand R. C. DRUGAN
Amsterdam A, Suh B-S (1991) An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinol 129:503-510. Anholt RRH (1986) Mitochondrial benzodiazepine receptors as potential modulators of intermediary metabolism. Trends Pharm Sci 7:506-511. Antkiewicz-Michaluk L, Guidotti A, Kreuger KE (1988) Molecular characterization and mitochondrial density of a recognition site for peripheral-type benzodiazepine ligands. Mol Pharmacol 34:272-278. Ardaillou N, Hagege J, Nivez M-P, Ardaillou R, Schlondorff D (1985) Vasoconstrictor-evoked prostaglandin synthesis in cultured human mesangial cells. Am J Physiol 17:F240-F246. Basile AS, Ostrowski NL, Skolnick P (1987) Aldosterone-reversible decrease in the density of renal peripheral benzodiazepine receptors in the rat after adrenalectomy. J Pharmacol Exp Ther 240:1006-1013. Basile AS, Skolnick P (1986) Subcellular localization of"peripheral-type" binding sites for benzodiazepines in rat brain. J Neurochem 46:305-308. Beaumont K, Skowronski R, Vaughn DA, Fanstil DD (1988) Interactions of lipids with peripheraltype benzodiazepine receptors. Biochem Pharmacol 37:1009-1014. Benavides J, Quarteronet D, Imbault F, Malgouris C, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G (1983a) Peripheral-type benzodiazepine binding sites in rat adrenals: Binding studies with [3H]PK I 1195 and autoradiographic localization. Arch Int Pharmacodyn 266:38-49. Benavides J, Malgouris C, Imbault F, Begassat F, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G (1983b) Labelling of "peripheral-type" benzodiazepine binding sites in the rat brain using [3H]PK 11195, and isoquinoline carboxamide derivative: Kinetic studies and autoradiographic localization. J Neurochem 41:1744-1750. Besman MJ, Yanagibashi K, Lee TD, Kawamura M, Hall PF, Shively JE (1989) Identification of des-(Gly Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: Stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proc Natl Acad Sci USA 86:4897-4901. Campbell WB, Henrich WL (1990) Endothelial factors in the regulation of renin release. Kidney Internatl 38:612-617. Darlington DN, Keil LC; Dallman MF (1989) Potentiation of hormonal responsesto hemorrhage and fasting, but not hypoglycemia in conscious adrenalectomized rats. Endocrinol 125:1398-1406. Davies LP, Huston V (1981) Peripheral benzodiazepine binding sites in heart and their interaction with dpyridamole. Eur J Pharm 73:209-211. Deckert J, Marangos PJ (1986) Hormonal interactions with the benzodiazepine bindings sites in vitro. Life Sci 39:675-683. Deicken RF (1986) Captopril treatment of depression. Biol Psychiat 21:425. DeSouza EB, Anholt RRH, Murphy KMM, Snyder SH, Kuhar MJ (1985) Peripheral-type benzodiazepine receptors in endocrine organs: Autoradiographic localization in rat pituitary, adrenal and testes. Endocrinol 126:567-573. Doble A Malgouris C, Daniel M, Daniel N, Imbault T, Basbaum A, Uzan A, Gueremy C, Le Fur G (1987) Labelling of peripheral-type benzodiazepine binding sites in human with [3H]PK 11195: Anatomical and subcellular distribution. Brain Res Bull 18:49-61. Douglas WW (1985) Polypeptides: Angiotensin, plasma kinins, and others. In: Gilman AG, Goodman LS, Rall TW, Murad F (Eds) The Pharmocological Basis of Therapeutics, Seventh Edition. Macmillan Publishing Co., New York, pp 639-659. Drugan RC, Basile AS, Crawley JN, Paul SM, Skolnick P (1986) Inescapable shock reduces [3H]Ro5-4864 binding to peripheral type benzodiazepine receptors in rat. Pharmacol Biochem Behav 24:1673-1677. Drugan RC, Basile AS, Crawley JN, Paul SM, Skolnick P (1988) Characterization of stress-induced alterations in [3H]Ro5-4864 binding to peripheral benzodiazepine receptors in rat heart and kidney. Pharmacol Biochem Behav 30:1015-1020. Drugan RC, Skolnick P, Paul SM, Crawley JN (1989) A pretest procedure reliably predicts performance in two animal models of inescapable stress. Pharmacol Biochem Behav 33:649-654. Dunn MJ (1983) Renal prostaglandins. In" Dunn MJ (Ed) Renal Endocrinology. Williams and Wilkins, Baltimore, pp 1-19. Fares F, Bar-Ami S, Brandes JM, Gavish M (1987) Gonadotropin- and estrogen-induced increase of peripheral-type benzodiazepine binding sites in the hypophyseal-genital axis of rats. Eur J Pharmacol 133:97-102.
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Ferrarese C, Mennini T, Pecora N, Pierpaoli C, Frigo M, Marzorati C, Gobbi M, Bizzi A, Codegoni A, Garattini S, Frattoloa L (1991) Diazepam binding inhibitor (DBI) increases after acute stress in rat. Neuropharmacol 30:1445-1452. Fonlupt P, Croset M, Lagarde M (1990) Benzodiazepine analogues inhibit arachidonate-induced aggregation and thromboxane synthesis in human platelets. Br J Pharmacol 101:920-924. Harding P, Stonier C, Aber GM (1991) Effect of antiotensin-converting enzyme inhibitors on glomerular eicosanoid production in normotensive and spontaneously hypertensive rats. Clin Sci 81:491-497. Holmes PV, Drugan RC (1992) Angiotensin II rapidly modulates the renal peripheral benzodiazepine receptor. Eur J Pharmacol 226:189-190. Holmes PV, Drugan RC (1993) Amygdaloid central nucleus lesions and cholinergic blockade attenuate the response of the renal peripheral benzodiazepine receptor to stress. Brain Res 621:1-9. Holmes PV, Stringer AP, Drugan RC (1992) Impact of psychological dynamics of stress on the peripheral benzodiazepine receptor. Pharmacol Biochem Behav 42:437-444. Karp L, Weizman A, Tyano S, Gavish M (1989) Examination stress, platelet peripheral benzodiazepine binding sites and plasma hormone levels. Life Sci 44:1077-1082. Kinnally KW, Zorov DB, Antonenko YN, Snyder SH (1993) Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands. Proc Natl Acad Sci USA 90:1374-1378. Kotchen TA, Roy MW (1983) Renin-angiotensin. In: Dunn MJ (Ed) Renal Endocrinology. Williams and Wilkins, Baltimore, pp 181-204. Krueger KE, Mukhin AG, Antkiewicz-Michaluk L, Santi MR, Grayson DR, Guidotti A, Sprengel R, Werner P, Seeburg PH (1990) Purification, cloning, and expression of a peripheral-type benzodiazepine receptor. In: Biggio G, Costa E (Eds) GABA and Benzodiazepine Receptor Subtypes. Raven Press, New York, pp 1-13. Langlois D, Begeot M, Berthelon M-C, Jaillard C, Saez JM (1992) Angiotensin II potentiates agonist-induced Y,5'-cyclic adenosine monophosphate production by cultured bovine adrenal cell through protein kinase C and calmodulin pathways. Endocrinology 131:2189-2195. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275. Martin P, Massol J, Scalbert E, Puech AJ (1990) Involvement of angiotensin-converting enzyme inhibition in reversal of helpless behavior evoked by perindopril in rats. Eur J Pharmacol 187:165-170. McCabe RT, Wamsley JK (1986) Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex. Life Sci 39:1937-1945. McEnery MW, Snowman AM, Trifiletti RR, Snyder SH (1992) Isolation of the mitochondriai benzodiazepine receptor: Association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc Natl Acad Sci USA 89:3170-3174. Miller G (1959) Protein determination for large numbers of samples. Anal Chem 31:964-967. Mohler H, Okada T (1977) Benzodiazepine receptors: Demonstration in the central nervous system. Science 198:849-851. Mukhin AG, Papadopoulos V, Costa E, Krueger KE (1989) Mitochondrial benzodiazepine receptors regulate steroid biosynthesis. Proc Natl Acad Sci USA 86:9813-9816. Novas ML, Medina JH, Calvo D, De Robertis E (1987) Increase of peripheral type benzodiazepine binding sites in kidney and olfactory bulb in acutely stressed rats. Eur J Pharmacol 135:243246. Papadopoulos V, Berkovich A, Krueger KE (1991) The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: Regulation of steroid biosynthesis. Neuropharmacol 30:1417-1423. Papadopoulos V, Mukhin AG, Costa E, Krueger KE (1990) The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis. J Biol Chem 265:3772-3779. Phillips MI (1987) Functions of angiotensin in the central nervous system. Annu Rev Physiol 49:413-435. Rago L, Kiivet R-A, Harro J, Pold M (1989) Central- and peripheral-type benzodiazepine receptors: Similar regulation by stress and GABA receptor agonists. Pharmacol Biochem Behav 32:879-883. Riond J, Mattei MG, Kaghad M, Dumont X, Guillemot JC, Le Fur G, Caput C, Ferrara P (1991)
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Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor. Eur J Biochem 195:305-311. Schwieler JH, Hjemdahl P (1992) Influence of angiotensin-converting enzyme inhibition on sympathetic neurotransmission: Possible roles ofbradykinin and prostaglandins. J Cardiovasc Pharmacol 20:$39-$46. Sprengel R, Werner P, Seeburg PH, Mukhin AG, Santi MR, Grayson DR, Guidotti A, Krueger KE (1989) Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor. J Biol Chem 264:20415-20421. Squires R, Braestrup C (1977) Benzodiazepine receptors in rat brain. Nature 266:732-734. Testa MA, Anderson RB, Nackley JF and Hollenberg NK (1993) Quality of life and antihypertensive therapy in men. New Eng J Med 328:907-913. Ticku MK (1983) Benzodiazepine-GABA receptor-ionophore complex: Current concepts. Neuropharmacol 22:1459-1470. Verma A, Snyder SH (1989) Peripheral type benzodiazepine receptors. Annu Rev Pharmacol Toxicol 29:307-322. Weissman BA, Bolger GT, Isaac L, Paul SM, Skolnick P (1984) Characterization of the binding of [3HI Ro5-4864, a convulsant benzodiazepine, to guinea pig brain. J Neurochem 42:969-975. Yanagibashi K, Ohno Y, Nakamichi N, Matsui T, Hayashida K, Takamura M, Yamada K, Tou S, Kawamura M (1989) Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria. J Biochem 106:1026-1029.
0306-4530/94$6.00 + .00 © 1993PergamonPress Ltd.
Printed in the U.S.A.
STRESS-INDUCED REGULATION OF THE RENAL PERIPHERAL BENZODIAZEPINE RECEPTOR: POSSIBLE ROLE OF THE RENIN-ANGIOTENSIN SYSTEM PHILIP V. HOLMES l and ROBERT C. DRUGAN* Department of Psychology, Schrier Research Laboratory, Brown University, Providence, Rhode Island 02912, U.S.A.
(Received 27 April 1993; in final form 23 July 1993)
SUMMARY The etiology of the decrease in renal peripheral benzodiazepine receptor (PBR) binding caused by stress was studied in rats. Prior investigations suggest that the response of the renal PBR to stress occurs independently of the hypothalamo-pituitary-adrenal (HPA) axis and sympathetic nervous system. The present experiments tested the hypothesis that the renin-angiotensin system is involved in regulating the PBR. Eighty min of brief, intermittent tailshocks caused increases in plasma renin activity and decreases in renal PBR binding. The stress-induced decrease in renal PBR binding was reversed by pretreatment with captopril. Acute admininstration of angiotensin II (ANG II) alone caused reductions in PBR binding in kidney, heart, and cerebral cortex. These data suggest that ANG II may be an endogenous factor responsible for regulating the PBR in several tissues during stress.
INTRODUCTION
THE PERIPHERALBENZODIAZEPINEreceptor (PBR) is a relatively small (17-18 kdalton) protein situated predominantly on the outer mitochondrial membrane in both peripheral and central nervous system (CNS) tissues (Anholt, 1986; Antkiewicz-Michaluk et al., 1988; Basile & Skolnick, 1986). The highest densities of PBR are found in the adrenal gland, but PBR densities are also relatively high in the kidney, heart, lung, ovary, and testes (Benavides et al.,1983a; Davies and Huston, 1981; DeSouza et al., 1985; Fares et al., 1987). PBR in the CNS are associated primarily with glial cells, and the highest densities are found in choroid plexus, ependyma, pineal gland, olfactory bulb, and circumventricular organs (Benavides et al., 1983b; Doble et al., 1987). The PBR is thus physically and anatomically distinct from the central benzodiazepine receptor (CBR), which is situated on the postsynaptic GABAA receptor/chloride ionophore complex in the CNS (McCabe & Wamsley, 1986; Mohler & Okada, 1977; Squires & Braestrup, 1977; Ticku, 1983). The recent cloning and sequencing of the DNA encoding the 18 kdalton PBR protein confirms this distinction at the molecular level, suggesting that the PBR is a distinct class of receptor (Krueger et al., 1990; Riond et al., 1991; Sprengel et al., 1989). *Address c o r r e s p o n d e n c e and reprint requests to: Robert C. Drugan, D e p a r t m e n t of Psychology, Schrier R e s e a r c h L a b o r a t o r y , Box 1853, B r o w n University, Providence, R h o d e Island 02912. 1Current Address: Section on Behavioral N e u r o p h a r m a c o l o g y , E x p e r i m e n t a l Therapeutics Branch, N I M H , Bethesda, M D 20892. 43
44
P.V. HOLMES and R. C. DRUGAN
The mitochondrial localization of the PBR and its high density in steroidogenic tissues probably have been the most valuable clues for determining its function. Recent evidence suggests that the PBR regulates steroidogenesis in both the periphery and in the brain (see Ferrarese et al., 1993; Papadopoulos et al., 1991 for review). PBR ligands, including the endogenous peptide diazepam binding inhibitor (DBI) and its fragments, stimulate pregnenolone synthesis in cultured adrenocortical, Leydig, granulosa, and glial cells (Amsterdam & Suh, 1991; Besman et al., 1989; Mukhin et al., 1989; Papadopoulos et al., 1990; Yanagibashi et al., 1989). Experiments demonstrating the responsivity of the PBR to environmental stressors provide further evidence of a regulatory role for the PBR in endocrine systems. Reports from several laboratories reveal that stress generally causes rapid and shortlived alterations in the density (Bmax) of PBR in rats, but this effect is highly dependent on the type of stress employed and the tissues surveyed. For example, brief noise stress causes immediate increases in PBR density in adrenal gland, cerebral cortex, and hippocampus (Ferrarese et al., 1991). PBR density in olfactory bulb and kidney is increased immediately following a brief forced swim (Novas et al., 1987; Rago et al., 1989). Presentation of a fear-conditioned stimulus causes increases in PBR binding in the olfactory bulb (Holmes et al., 1992). Studies in humans reveal a similar type of response in PBR from blood platelets, with increases in the density of PBR occurring immediately following a written examination (Karp et al., 1989). In contrast, more severe stress tends to cause decreases in PBR binding. An 80 min session of 80 tailshocks causes decreases in PBR binding in cerebral cortex, pituitary, heart, and kidney (Drugan et al., 1986). Scatchard analyses in renal membranes has revealed that the reduction in PBR binding seen following 80 min of stress is due to a drop in receptor density (Bmax) and not affinity (Kd). Alterations in PBR binding during stress have thus been observed in a variety of tissues, including those that are not typically regarded as being steroidogenic (i.e., kidney, heart, blood, olfactory bulb). The systems responsible for regulating the PBR may therefore be tissue-specific. Experiments in this laboratory have focused on the stress-induced decrease in PBR density in kidney. Previous attempts to elucidate the physiological mechanisms responsible for modulating renal PBR binding during stress suggest that neither the hypothalamo-pituitary-adrenal (HPA) axis, the neurohypophysis, nor the sympathetic nervous system are directly involved. For example, stress-induced decreases in renal PBR binding persist following hypophysectomy, and are actually enhanced following adrenalectomy (Drugan et al., 1988). Furthermore, neither chemical sympathectomy, adrenergic blockade, nor unilateral renal denervation influence the response of the renal PBR to stress (Drugan et al., 1988; Holmes & Drugan, in press). Though the renin-angiotensin system is closely linked to the HPA axis, neurohypophysis, and sympathetic nervous system, there are several mechanisms for activating this system independently of the others (Kotchen & Roy, 1983; Campbell & Heinrich, 1990). The renin-angiotensin system may therefore function as an independent "stress axis" vis-a-vis the renal PBR. Earlier experiments from this laboratory have demonstrated that the administration of Angiotensin II (ANG II) alone causes dose-dependent reductions in renal PBR density similar to those caused by stress (Holmes & Drugan, 1992). The present experiments further tested the possibility that the alterations in PBR binding in kidney during stress are linked to activation of the renin-angiotensin system.
STRESS, ANGIOTENSIN II, AND PBR IN KIDNEY
45
METHODS
Subjects Male Sprague Dawley-derived rats bred at Hunter Laboratory, Brown University, served as subjects. Rats were group-housed (3-6 per cage), allowed free access to food and water, and weighed between 230-330 g at the time of experimentation. The colony was maintained at 23°C on a normal 12 h light/dark cycle (lights on at 0700 h). All behavioral procedures were conducted between 0900 and 1500 h. Drugs ANG II acetate salt (human), and [2S]-l-[3-Mercapto-2-methylpropionyl]-L-proline (captopril) were purchased from Sigma Chemical Co., St. Louis, MO. ANG II was dissolved in distilled water. Captopril was dissolved in 0.15 M sodium carbonate. Injection volume of ANG II was 0.5 ml/rat. Injection volume of captopril was 1 ml/kg. Ro5-4864 was kindly supplied by Drs. Peter Sorter (Hoffman-LaRoche) and Peter Suzdak (Novo Industri). [3H]-Ro5-4864 (sp. act. 79.7-86.9 Ci/mM) was purchased from NEN, Boston, MA. Stress Procedure The stress paradigm used in the present experiments was identical to that previously described (Drugan et al., 1988) with the exception that rats were habituated to the guillotine for five to seven days prior to stress in an attempt to stabilize the variability in radioligand binding typically observed in naive subjects. Rats were semirestrained in Plexiglas boxes (15.5 x 12 x 17 cm) by taping the tail, which protruded through a small notch in the rear of the box, to a horizontal post (10 x 1 × 1 cm) extending behind. Eighty tailshocks of 5 s duration were delivered with an average intershock interval of 1 min. The intensity of current was incremented from 1 to 1.5 mA after 35 shocks and up to 2 mA at 60 shocks. Shocks were administered by a Lafayette Instruments Model 82400 shock generator through electrodes taped to the tail augmented with electrode paste. Effects of Stress on Plasma Renin Activity and PBR Binding Nineteen rats were randomly divided into either stress or naive conditions and stressed as described above or left in the home cage. Stressed and naive rats were killed by decapitation at approximately the same time in a counterbalanced order immediately following the stress session. Trunk blood was collected in microfuge tubes containing 100 t*l of 2% EDTA. The blood was centrifuged at 1300 × g for I min. Plasma was collected and stored at -20°C. Kidneys were dissected and placed in 0.32 M sucrose solution in 20 ml glass vials and rapidly frozen in a COJacetone slurry and stored at -80°C until assay. Captopril Pretreatment Sixty-eight rats were randomly divided into the following drug conditions: captopril 10, 50, and 100 mg/kg and vehicle. Within each group rats were divided into naive and stress conditions. Rats were injected SC with captopril or vehicle and were stressed 15 min later or left in the home cage. Rats were killed and kidneys dissected as described above immediately following the stress session.
46
P.V. HOLMESand R. C. DRUGAN
Acute Administration of ANG H ANG II was administered over an 80 min period in an attempt to mimic the time course of the stress session. Seventeen rats were randomly divided into drug and vehicle conditions and received three 25/xg injections (SC) of ANG II or vehicle at 0, 30, and 60 min for a cumulative dose of 75/zg. Rats were killed at 80 min. Kidney, heart, adrenal gland, olfactory bulb, hippocampus, and cerebral cortex were dissected and frozen as described above. In Vitro Radioligand Binding Tissues were thawed in a 25°C water bath, disrupted in 50 volumes of 50 mM TrisHC1 buffer (pH --- 7.4), and centrifuged at 20,000 × g for 20 min. The tissues were resuspended in 200 volumes (peripheral tissues) or 100 volumes (CNS tissues) of the same buffer, and the binding of [3H]Ro5-4864 was determined as previously described (Drugan et al., 1986, 1988; Weissman et al., 1984). Previous experiments have employed Scatchard analyses to demonstrate that the effect of both 80 tailshocks and ANG II administration on renal membranes is to decrease the density (Bmax)and not affinity (Kd) of PBR (Drugan et al., 1986; Holmes & Drugan, 1992). A single concentration of radioligand was therefore used for the present experiments. Briefly, 0.1 ml of peripheral tissue or 0.6 ml of CNS tissue (containing approximately 0.04 or 0. l mg protein, respectively) was added to each assay tube containing 0.1 ml of radioligand (final concentration, 1 nM), 0.1 ml of unlabeled Ro5-4864 or Tris-HCl buffer, and additional buffer to yield a total volume of 1 ml. Assays were performed triplicate. The reaction was initiated by the addition of tissue and terminated after incubation at 0-4°C for 60 min by rapid filtration over Schleicher and Schuell #32 glass filter strips using a Brandel M-24R filtering manifold. Samples were washed with two 5 ml aliquots of 0-4°C buffer. The specific binding of [3H]Ro5-4864 was defined as the difference in binding obtained in the presence and absence of unlabeled Ro5-4864 (final concentration, 100 /zM). The radioactivity retained by the filters was measured in a Beckman LS 5000TD liquid scintillation spectrometer using 6 ml of Eco-scint solution as a fluor. Specific binding accounted for approximately 70-90% of total binding. Protein concentration was determined as previously described (Lowry et al., 1951; Miller, 1959). Radioimmunoassay Plasma renin activity was determined using an [125I]angiotensin I radioimmunoassay kit from NEN. Plasma renin activity was determined by quantifying the amount of angiotensin I generated from endogenous angiotensinogen by renin. Briefly, angiotensin I was generated from 500 ml plasma samples by incubating at 37°C for 1 h in the presence of converting enzyme inhibitors. [125I]angiotensin I, angiotensin I antiserum (rabbit), and samples (100 /.d) were incubated for 16 h at 4°C. The antigen-antibody complex was extracted by adding 500 ~1 of antirabbit gamma globulin. Tubes were centrifuged and the radioactivity of pellets was quantified by a Micromedic gamma counter. Data Analysis All data were analyzed by between-subjects one-way or factorial analysis of variance (ANOVA). Between-treatment differences were evaluated by posthoc Newman-Keul's tests with a critical value set at 0.05. Correlation coefficients were calculated for plasma renin activity values and PBR binding values from naive and stressed rats.
47
STRESS, A N G I O T E N S I N I I , A N D PBR IN KIDNEY 9¢
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RESULTS
Tailshock Stress Causes Increases in Plasma Renin Activity and Decreases in PBR Binding Fig. 1 illustrates the effect of 80 min of tailshocks on plasma renin activity. One-way ANOVA revealed that stress significantly elevated plasma renin activity (P < 0.05). The binding of [~H]Ro5-4864 in renal membranes of rats exposed to 80 tailshocks was reduced compared to naive controls. Fig. 2 illustrates this effect. The effect of stress on renal PBR binding was significant as indicated by one-way ANOVA (P < 0.01). No significant correlations between plasma renin activity and PBR binding were observed in either naive or stressed rats. Captopril Pretreatment Blocks the Stress-Induced Reduction in Renal PBR Binding Fig. 3 illustrates the effect of inhibition of endogenous ANG II formation by captopril on renal PBR binding in stressed rats. Factorial ANOVA revealed that there was a significant effect of stress (P < 0.001) and drug treatment (P < 0.05). The interaction term was also significant (P < 0.05), indicating that the effect of stress depended on drug pretreatment. Posthoc tests revealed that stressed rats pretreated with vehicle or 10 mg/kg of captopril differed significantly from naive/vehicle controls, whereas stressed rats pretreated with either 50 or 100 mg/kg of captopril did not. Acute Administration o f A N G H Decreases PBR Binding PBR binding was reduced in kidney (P < 0.001), heart (P < 0.05), and cerebral cortex (P < 0.01) following the administration of 75/zg of ANG II delivered over an 80 min period (see Table I). No significant alterations in PBR binding were seen in the adrenal gland, hippocampus, or olfactory bulb.
48
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FIG. 3: Mean specific binding of [3H]Ro5-4864 (fmole/mg protein) at a concentration of 1 nM in renal membranes from separate groups of rats receiving SC injections of either vehicle or captopril (Cap: 10, 50, or 100 mg/kg) 15 min prior to stress or no treatment (naive). Number of subjects per group is indicated at the base of each column. * indicates significantly different from naive/ vehicle control (P < 0.01). + indicates significantly different from stress/vehicle-treated rats (P < 0.05).
STRESS, ANGIOTENSIN II, AND PBR IN KIDNEY
49
TABLE I. SPECIFIC [3H]RO5-4864 BINDING (FMOLE/MG PROTEIN; MEAN -- SEM)
Vehicle ANG II
Adrenal
Kidney
Heart
9583.2 + 528 9064.1 + 851
1420.3 + 99 1012.9 + 25*
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Cortex
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*p < .01; tp < .05.
DISCUSSION Results from the present experiments provide preliminary support for the proposal that circulating ANG II is involved in the stress-induced regulation of the renal PBR. As expected, 80 min of intermittent tailshocks caused decreases in renal PBR binding. Tailshock stress also stimulated renin secretion. Pretreating rats with the converting enzyme inhibitor captopril, which blocks the formation of ANG II, reversed the stressinduced decrease in PBR binding. Products of angiotensin I conversion may therefore be necessary for the alterations in renal PBR binding observed in this paradigm. Furthermore, the administration of ANG II alone to naive rats is sufficient to alter PBR binding in a manner similar to stress. The lack of any significant correlation between plasma renin activity and PBR binding suggests that there is no simple linear relationship between these two variables. However, in the present experiments it was not possible to determine the relationship between stress-induced alterations in plasma renin activity and stress-induced alterations in PBR binding from baseline values. The dose-dependent effect of ANG II administration on renal PBR binding previously reported may be a better indication of the relationship between circulating ANG II and the PBR (Holmes & Drugan, 1992). The ability of captopril to attenuate the PBR response to stress may not be due solely to inhibition of ANG II formation. Direct interactions with other enzyme systems, such as those involved in the synthesis and degradation of eicosanoids and bradykinin, cannot be ruled out (Douglas, 1985; Harding et al., 1991; Schwieler & Hjemdahl, 1992). CNS actions of captopril cannot be excluded either. Converting enzyme inhibitors produce anxiolytic- and antidepressant-like effects in the °'learned helplessness" model of anxiety and depression in rats (Martin et al., 1990). Antidepressant-like effects of converting enzyme inhibitors have also been reported in human studies (Testa et al., 1993; Deicken, 1986). The central and/or peripheral mechanism of the antidepressant action, be it the blockade of ANG II formation or bradykinin degradation, is unclear. The inescapable tailshock stress paradigm employed in the present experiments is similar to that of the ~'learned helplessness" model of depression (Drugan et al., 1989). However, further studies are required to determine whether there is a relationship between captopril's ability to reverse stress-induced behaviors and its ability to reverse stress-induced PBR alterations in rats. The effect of stress on the renal PBR can be mimicked by peripheral administration of ANG II. Previous experiments have demonstrated that ANG II, like stress, causes decreases in renal PBR density (Bmax)and not affinity (Kd) (Drugan et al., 1986; Holmes & Drugan, 1992). The present experiments surveyed several tissues revealing a degree of correspondence between tissues sensitive to ANG II and those that were previously reported to be altered by 80 tailshocks. In the original demonstration of stress-induced
50
P . V . HOLMES and R. C. DRUGAN
alterations in PBR binding, reductions were observed in kidney, heart, cerebral cortex, and pituitary but not adrenal or hippocampus (Drugan et al., 1986). In the present experiments, ANG II caused reductions in PBR binding in kidney, heart, and cerebral cortex but not adrenal or hippocampus (pituitary was not surveyed). The mechanism by which ANG II reduces PBR density remains to be established. The dose and time course of ANG II administration used in this study cause elevations in systemic arterial pressure similar to that observed during tailshock stress (Holmes & Drugan, 1992). However, decreases in renal PBR binding do not appear to be a direct consequence of elevated systemic arterial pressure. Elevating arterial pressure with norepinephrine to the levels induced by stress or ANG II does not affect the renal PBR (Holmes & Drugan, 1992). ANG II influences multiple endocrine systems. For example, ANG II stimulates aldosterone release from the adrenal cortex and vasopressin release from the posterior pituitary (Kotchen & Roy, 1983; Phillips, 1987). It is unlikely, however, that either aldosterone or vasopressin are directly involved in regulating the PBR during stress. Although chronic (one to two weeks) manipulation of aldosterone levels alters renal PBR density (Basile et al., 1987), the rapid alterations in PBR binding caused by stress are not diminished, but enhanced, following adrenalectomy (Drugan et al., 1988). Furthermore, aldosterone does not directly interact with the PBR, as indicated by its inability to influence [3H]Ro5-4864 binding in vitro (Deckert & Marangos, 1986). That adrenalectomized rats show enhanced renin secretion, possibly due to a lack of negative feedback by aldosterone (Darlington et al., 1989), may explain the enhanced PBR response to stress in adrenalectomized rats. The failure of hypophysectomy to influence the response of the renal PBR to stress suggests that neurohypophysial vasopressin is not a critical factor either (Drugan et al., 1988). Experiments conducted in this laboratory have indicated that nanomolar to micromolar concentrations of ANG II do not alter in vitro [3H]Ro5-4864 binding in mitochondrial membrane preparations (unpublished observations). ANG II thus does not appear to competively inhibit binding of [3H]Ro5-4864 at the mitochondrial PBR. ANG II may therefore act as an extracellular signal that, through specific extracellular receptor interactions, induces a cascade of intracellular activity that ultimately influences the PBR. Further in vitro studies using intact cells or slice preparations are required to determine whether the effect of ANG II on PBR binding is mediated specifically by extracellular ANG II receptors. Such studies are also critical to determine the identity and nature of second messengers or intracellular mediators that may be involved in regulating the PBR. Possible intracellular regulators include DBI (Alho et al., 1991; Papadopolous et al., 1991), cAMP (Amsterdam & Suh, 1991), and arachidonic acid (Beaumont et al., 1988; Fonlupt et al., 1990). Though interactions between ANG II and adenylate cyclase or arachadonic acid metabolites have been well established (Ardaillou et al., 1985; Dunn, 1983; Langlois et al., 1992), interactions between ANG II and DBI have yet to be examined. As discussed above, the effect of ANG II appears to be specific to those tissues that exhibit decreases in PBR binding in response to the 80 min stress paradigm. The failure of ANG II to modulate the PBR in other tissues that are altered by different types of stress suggest that several, distinct mechanisms exist for regulating the PBR. These mechanisms may be tissue-specific. In other words, ANG II may regulate the PBR in kidney, heart, and cerebral cortex, whereas other factors may regulate the PBR in adrenal gland, blood platelets, olfactory bulb, and hippocampus. Differences in sensitivity to
STRESS, ANGIOTENSIN II, AND PBR IN KIDNEY
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circulating ANG II may also account for the tissue-specific responses of the PBR to both 80 tailshocks and ANG II treatment. Though only the renal, cardiac, and cerebral cortical PBR responses to ANG II were siginficant, all tissues showed a trend toward decreases in PBR binding. A great deal of evidence linking the PBR to the regulation of steroidogenesis has accumulated recently. This regulation appears to involve the delivery of cholesterol to a cytochrome P-450 of the inner mitochondrial membrane (see Ferrarese et al., 1993; Papadopoulos et al., 1991 for review). This process is the rate-limiting step in steroidogenesis and has been well characterized in Leydig and adrenocortical cells in vitro (Papadopoulos et al., 1990; Yanagibashi et a1.,1989). Furthermore, in vivo experiments have demonstrated alterations in PBR density in the adrenal gland following stress, and this response appears to be related to steroid synthesis (Ferrarese et al., 1991). However, the response of the PBR in the adrenal gland is not as robust and consistently observed across stress paradigms as the response of the PBR in kidney (cf. Drugan et al., 1986, 1988; Ferrarese et al., 1991; Novas et al., 1987; Rago et al., 1989). This apparent discrepancy suggests that either the kidney synthesizes cholesterol-derived products during stress, or the function of the PBR across tissues is not limited to steroidogenesis. Recent molecular pharmacologocial data reveal an association between the PBR, the outer mitochondrial membrane voltage-dependent anion channel, and the inner mitochondrial membrane adenine nucleotide carrier (McEnery et al., 1992). Such a mitochondrial complex has been proposed to be involved in the transport of a variety of substrates, including ATP as well as several ions (Kinnally et al., 1993; McEnery et al., 1992; Verma & Snyder, 1989). The function of the PBR may therefore be rather broad, and the putative role of the PBR in regulating the synthesis of secretory products could be a general phenomenon across endocrine tissues. The PBR may be involved in the synthesis of endocrine, paracrine, or autocrine, factors that share a common dependence on outer mitochondrial transport for their synthesis. According to this interpretation, a possible role of the PBR in kidney could be the regulation of arachidonic acid metabolism and eicosanoid production. Some arachidonic acid metabolites, such as hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acid, are derived from metabolism by a cytochrome P-450 (Campbell & Henrich, 1990). The hypothetical link between between the PBR and the cytochrome P-450 mechanism involved in eicosanoid formation may be similar to that described for steroidogenesis. Specifically, the PBR may be involved in the delivery of arachidonic acid to a cytochrome P-450 in a fashion similar to cholesterol transport. The influence of ANG II on both eicosanoid formation (Ardaillou et al., 1985) and the PBR may thus be related. Though this particular mechanism is highly speculative, the association between the PBR and cellular endocrine physiology should encourage further study of the role of the PBR in regulating hormone synthesis during stress in a variety of tissues. Acknowledgments: All
behavioral procedures were approved by the Brown University Institutional Animal Care and Use Committee. The authors thank Dr. David A. Bereiter and C.T. Graeber for their assistance in conducting the radioimmunoassay. Submitted as partial fulfillment of requirements for Ph.D. (P.V.H.). Research was supported by a PHS grant #MH45475 and an Alfred P. Sloan Research Fellowship to R.C.D.
REFERENCES
Alho H, Harjuntausta T, Schultz R, Pelto-Huikko M, Bovolin P (1991) Immunohistochemistry of diazepam binding inhibitor in the central nervous system and peripheral organs: Its possible role as an endogenous regulator of different types of benzodiazepine receptors. Neuropharmacol 30:1381-1386.
52
P.V. HOLMESand R. C. DRUGAN
Amsterdam A, Suh B-S (1991) An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells. Endocrinol 129:503-510. Anholt RRH (1986) Mitochondrial benzodiazepine receptors as potential modulators of intermediary metabolism. Trends Pharm Sci 7:506-511. Antkiewicz-Michaluk L, Guidotti A, Kreuger KE (1988) Molecular characterization and mitochondrial density of a recognition site for peripheral-type benzodiazepine ligands. Mol Pharmacol 34:272-278. Ardaillou N, Hagege J, Nivez M-P, Ardaillou R, Schlondorff D (1985) Vasoconstrictor-evoked prostaglandin synthesis in cultured human mesangial cells. Am J Physiol 17:F240-F246. Basile AS, Ostrowski NL, Skolnick P (1987) Aldosterone-reversible decrease in the density of renal peripheral benzodiazepine receptors in the rat after adrenalectomy. J Pharmacol Exp Ther 240:1006-1013. Basile AS, Skolnick P (1986) Subcellular localization of"peripheral-type" binding sites for benzodiazepines in rat brain. J Neurochem 46:305-308. Beaumont K, Skowronski R, Vaughn DA, Fanstil DD (1988) Interactions of lipids with peripheraltype benzodiazepine receptors. Biochem Pharmacol 37:1009-1014. Benavides J, Quarteronet D, Imbault F, Malgouris C, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G (1983a) Peripheral-type benzodiazepine binding sites in rat adrenals: Binding studies with [3H]PK I 1195 and autoradiographic localization. Arch Int Pharmacodyn 266:38-49. Benavides J, Malgouris C, Imbault F, Begassat F, Uzan A, Renault C, Dubroeucq MC, Gueremy C, Le Fur G (1983b) Labelling of "peripheral-type" benzodiazepine binding sites in the rat brain using [3H]PK 11195, and isoquinoline carboxamide derivative: Kinetic studies and autoradiographic localization. J Neurochem 41:1744-1750. Besman MJ, Yanagibashi K, Lee TD, Kawamura M, Hall PF, Shively JE (1989) Identification of des-(Gly Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: Stimulation of cholesterol delivery is mediated by the peripheral benzodiazepine receptor. Proc Natl Acad Sci USA 86:4897-4901. Campbell WB, Henrich WL (1990) Endothelial factors in the regulation of renin release. Kidney Internatl 38:612-617. Darlington DN, Keil LC; Dallman MF (1989) Potentiation of hormonal responsesto hemorrhage and fasting, but not hypoglycemia in conscious adrenalectomized rats. Endocrinol 125:1398-1406. Davies LP, Huston V (1981) Peripheral benzodiazepine binding sites in heart and their interaction with dpyridamole. Eur J Pharm 73:209-211. Deckert J, Marangos PJ (1986) Hormonal interactions with the benzodiazepine bindings sites in vitro. Life Sci 39:675-683. Deicken RF (1986) Captopril treatment of depression. Biol Psychiat 21:425. DeSouza EB, Anholt RRH, Murphy KMM, Snyder SH, Kuhar MJ (1985) Peripheral-type benzodiazepine receptors in endocrine organs: Autoradiographic localization in rat pituitary, adrenal and testes. Endocrinol 126:567-573. Doble A Malgouris C, Daniel M, Daniel N, Imbault T, Basbaum A, Uzan A, Gueremy C, Le Fur G (1987) Labelling of peripheral-type benzodiazepine binding sites in human with [3H]PK 11195: Anatomical and subcellular distribution. Brain Res Bull 18:49-61. Douglas WW (1985) Polypeptides: Angiotensin, plasma kinins, and others. In: Gilman AG, Goodman LS, Rall TW, Murad F (Eds) The Pharmocological Basis of Therapeutics, Seventh Edition. Macmillan Publishing Co., New York, pp 639-659. Drugan RC, Basile AS, Crawley JN, Paul SM, Skolnick P (1986) Inescapable shock reduces [3H]Ro5-4864 binding to peripheral type benzodiazepine receptors in rat. Pharmacol Biochem Behav 24:1673-1677. Drugan RC, Basile AS, Crawley JN, Paul SM, Skolnick P (1988) Characterization of stress-induced alterations in [3H]Ro5-4864 binding to peripheral benzodiazepine receptors in rat heart and kidney. Pharmacol Biochem Behav 30:1015-1020. Drugan RC, Skolnick P, Paul SM, Crawley JN (1989) A pretest procedure reliably predicts performance in two animal models of inescapable stress. Pharmacol Biochem Behav 33:649-654. Dunn MJ (1983) Renal prostaglandins. In" Dunn MJ (Ed) Renal Endocrinology. Williams and Wilkins, Baltimore, pp 1-19. Fares F, Bar-Ami S, Brandes JM, Gavish M (1987) Gonadotropin- and estrogen-induced increase of peripheral-type benzodiazepine binding sites in the hypophyseal-genital axis of rats. Eur J Pharmacol 133:97-102.
STRESS, ANGIOTENSIN II, AND PBR IN KIDNEY
53
Ferrarese C, Mennini T, Pecora N, Pierpaoli C, Frigo M, Marzorati C, Gobbi M, Bizzi A, Codegoni A, Garattini S, Frattoloa L (1991) Diazepam binding inhibitor (DBI) increases after acute stress in rat. Neuropharmacol 30:1445-1452. Fonlupt P, Croset M, Lagarde M (1990) Benzodiazepine analogues inhibit arachidonate-induced aggregation and thromboxane synthesis in human platelets. Br J Pharmacol 101:920-924. Harding P, Stonier C, Aber GM (1991) Effect of antiotensin-converting enzyme inhibitors on glomerular eicosanoid production in normotensive and spontaneously hypertensive rats. Clin Sci 81:491-497. Holmes PV, Drugan RC (1992) Angiotensin II rapidly modulates the renal peripheral benzodiazepine receptor. Eur J Pharmacol 226:189-190. Holmes PV, Drugan RC (1993) Amygdaloid central nucleus lesions and cholinergic blockade attenuate the response of the renal peripheral benzodiazepine receptor to stress. Brain Res 621:1-9. Holmes PV, Stringer AP, Drugan RC (1992) Impact of psychological dynamics of stress on the peripheral benzodiazepine receptor. Pharmacol Biochem Behav 42:437-444. Karp L, Weizman A, Tyano S, Gavish M (1989) Examination stress, platelet peripheral benzodiazepine binding sites and plasma hormone levels. Life Sci 44:1077-1082. Kinnally KW, Zorov DB, Antonenko YN, Snyder SH (1993) Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands. Proc Natl Acad Sci USA 90:1374-1378. Kotchen TA, Roy MW (1983) Renin-angiotensin. In: Dunn MJ (Ed) Renal Endocrinology. Williams and Wilkins, Baltimore, pp 181-204. Krueger KE, Mukhin AG, Antkiewicz-Michaluk L, Santi MR, Grayson DR, Guidotti A, Sprengel R, Werner P, Seeburg PH (1990) Purification, cloning, and expression of a peripheral-type benzodiazepine receptor. In: Biggio G, Costa E (Eds) GABA and Benzodiazepine Receptor Subtypes. Raven Press, New York, pp 1-13. Langlois D, Begeot M, Berthelon M-C, Jaillard C, Saez JM (1992) Angiotensin II potentiates agonist-induced Y,5'-cyclic adenosine monophosphate production by cultured bovine adrenal cell through protein kinase C and calmodulin pathways. Endocrinology 131:2189-2195. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275. Martin P, Massol J, Scalbert E, Puech AJ (1990) Involvement of angiotensin-converting enzyme inhibition in reversal of helpless behavior evoked by perindopril in rats. Eur J Pharmacol 187:165-170. McCabe RT, Wamsley JK (1986) Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex. Life Sci 39:1937-1945. McEnery MW, Snowman AM, Trifiletti RR, Snyder SH (1992) Isolation of the mitochondriai benzodiazepine receptor: Association with the voltage-dependent anion channel and the adenine nucleotide carrier. Proc Natl Acad Sci USA 89:3170-3174. Miller G (1959) Protein determination for large numbers of samples. Anal Chem 31:964-967. Mohler H, Okada T (1977) Benzodiazepine receptors: Demonstration in the central nervous system. Science 198:849-851. Mukhin AG, Papadopoulos V, Costa E, Krueger KE (1989) Mitochondrial benzodiazepine receptors regulate steroid biosynthesis. Proc Natl Acad Sci USA 86:9813-9816. Novas ML, Medina JH, Calvo D, De Robertis E (1987) Increase of peripheral type benzodiazepine binding sites in kidney and olfactory bulb in acutely stressed rats. Eur J Pharmacol 135:243246. Papadopoulos V, Berkovich A, Krueger KE (1991) The role of diazepam binding inhibitor and its processing products at mitochondrial benzodiazepine receptors: Regulation of steroid biosynthesis. Neuropharmacol 30:1417-1423. Papadopoulos V, Mukhin AG, Costa E, Krueger KE (1990) The peripheral-type benzodiazepine receptor is functionally linked to Leydig cell steroidogenesis. J Biol Chem 265:3772-3779. Phillips MI (1987) Functions of angiotensin in the central nervous system. Annu Rev Physiol 49:413-435. Rago L, Kiivet R-A, Harro J, Pold M (1989) Central- and peripheral-type benzodiazepine receptors: Similar regulation by stress and GABA receptor agonists. Pharmacol Biochem Behav 32:879-883. Riond J, Mattei MG, Kaghad M, Dumont X, Guillemot JC, Le Fur G, Caput C, Ferrara P (1991)
54
P.V. HOLMESand R. C. DRUGAN
Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor. Eur J Biochem 195:305-311. Schwieler JH, Hjemdahl P (1992) Influence of angiotensin-converting enzyme inhibition on sympathetic neurotransmission: Possible roles ofbradykinin and prostaglandins. J Cardiovasc Pharmacol 20:$39-$46. Sprengel R, Werner P, Seeburg PH, Mukhin AG, Santi MR, Grayson DR, Guidotti A, Krueger KE (1989) Molecular cloning and expression of cDNA encoding a peripheral-type benzodiazepine receptor. J Biol Chem 264:20415-20421. Squires R, Braestrup C (1977) Benzodiazepine receptors in rat brain. Nature 266:732-734. Testa MA, Anderson RB, Nackley JF and Hollenberg NK (1993) Quality of life and antihypertensive therapy in men. New Eng J Med 328:907-913. Ticku MK (1983) Benzodiazepine-GABA receptor-ionophore complex: Current concepts. Neuropharmacol 22:1459-1470. Verma A, Snyder SH (1989) Peripheral type benzodiazepine receptors. Annu Rev Pharmacol Toxicol 29:307-322. Weissman BA, Bolger GT, Isaac L, Paul SM, Skolnick P (1984) Characterization of the binding of [3HI Ro5-4864, a convulsant benzodiazepine, to guinea pig brain. J Neurochem 42:969-975. Yanagibashi K, Ohno Y, Nakamichi N, Matsui T, Hayashida K, Takamura M, Yamada K, Tou S, Kawamura M (1989) Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria. J Biochem 106:1026-1029.