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Activation of renal adenylate cyclase by forskolin: Assessment of enzymatic activity in animal models of the secondary hyperparathyroid state
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 225, No. 2, September, pp. 898-905, 1983
Vol.
Activation of Renal Adenylate Cyclase by Forskolin: Assessment Enzymatic Activity in Animal Models of the Secondary Hyperparathyroid State LEONARD
of
R. FORTE
M-523 iifedical Science Building, Department of Pha rmaeology, School of Medicine, University and Harry S Truman Memorial Veterans Hospital, Columbia, Mtisauri 65212
of Missouri,
Received December 13, 1982, and in revised form May 19, 1983
The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptormediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.
Forskolin, a diterpene that was isolated from the roots of the Indian plant, Coleus forshcohlii (l), has been shown to be a potent cardiovascular drug (2, 3). Forskolin is a powerful activator of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.11 in both intact cells and tissue and also in membrane preparations (4-7). Unlike the activation found with cholera toxin, fluoride, and guanylnucleotides, the forskolin-stimulated adenylate cyclase is readily reversible when the drug is removed from the system (5-7). Although the mechanism of forskolin activation of ad0003-9861183 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
enylate cyclase is unknown, it has been proposed that the drug directly interacts with the catalytic subunit of this enzyme (8). Moreover, forskolin activates the adenylate cyclase catalytic subunit when the nucleotide regulatory protein (N protein) has been removed from the enzyme by chromatographic techniques (9, 10). The purpose of the present study was to evaluate forskolin as an experimental tool for measuring the total enzymatic activity of kidney adenylate cyclase in animal models that exhibit a marked decrease in hormone-dependent enzyme activity. Rats 898
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or chickens raised on diets deficient in either vitamin D or calcium exhibit downregulation of the renal parathyroid hormone (PTH)‘-dependent adenylate cyclase activity (11-15). There is direct evidence that the loss of tissue responsiveness to PTH is due to a substantial reduction in membrane receptors for the hormone (15). Of physiologic significance is the finding that vitamin D-deficient rats exhibit a refractory phosphaturic response to PTH in viva (11). Reported here are the results of studies designed to assess the use of forskolin-stimulated increases in kidney slice cyclic AMP accumulation and forskolinstimulated adenylate cyclase activity of renal membranes as a means of determining whether the forskolin-stimulated activity of renal adenylate cyclase is affected by diets deficient in either vitamin D or calcium. EXPERIMENTAL
PROCEDURE
Aniwmls and dietary procedure. White Leghorn cockerels were obtained at 1 day of age from Colonial Poultry Farms, Pleasant Hill, Missouri. They were housed in our animal facility using commercial brooders maintained at 32°C (Brower Manufacturing, Quincy, Ill.). The light was devoid of uv irradiation and maintained on a 12/12 light/dark cycle. Chicks were allowed free access to food and deionized water. The chicks were fed one of two synthetic diets obtained from Teklad, Madison, Wisconsin. A diet deficient in vitamin D (-D), containing 1.1% Ca, 0.6% P, and 0.07% Mg (No. 170245) by our analysis of the wet-ashed diet, was fed to chicks beginning at 1 day of age. Controls birds (+D) received the same diet and were given 70 IU of vitamin Da in 50 pl propylene glyeol by mouth every third day at 0800 h during the course of the experiment. The second experimental diet (No. 80378) was also deficient in vitamin D and was low in Ca, 0.01% (-Ca). The P and Mg values were the same in the -Ca as in the -D diet. Chicks fed the -Ca diet received vitamin Da as described above. The control birds (+D, +Ca) were the same control animals for both -D and -Ca groups. Chicks were fed these diets for 14 days and then killed by decapitation to obtain kidneys for the analytical procedures (15). i Abbreviations used: PTH, parathyroid hormone; -D, vitamin D-deficient diet; +D, control diet; -Ca, low-calcium diet; +Ca, control; MIX, methylisobutylxanthine; nlPTH(l-34), 8,18 norleucine, 34 tyrosinebovine PTH(l-34)amide.
CYCLASE
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Male, 21-day-old weanling rats obtained from Holtzman Company, Madison, Wisconsin, were fed a vitamin D-deficient diet (0.47% Ca, 0.36% P, and 0.048% Mg) and maintained in a room devoid of uv light (11). Control (+D) animals received the same diet and were pair-fed with the D-deficient rats, but also received oral doses of 70 IU vitamin Da twice weekly. Rats were killed by decapitation after 4 weeks of the experimental diets. Renal cyclic AMP content. Thin slices (0.5 mm) of chicken or rat kidney weighing approximately 25 mg were prepared using a Stadie-Riggs microtome (Arthur Thomas, Philadelphia, Pa.). They were placed in ice-cold 0.9% NaCl until weighed and then transferred to Krebs-Ringer bicarbonate buffer that had been adjusted to pH 7.4 by bubbling 95% Oa-5% COP through the medium. One slice was preincubated for 15 min in 1 ml of this medium containing 10 mM glucose and 2 mM methylisobutylxanthine (MIX). The temperature was maintained at 37°C using a Dubnoff metabolic incubator. After the preincubation, slices were transferred to the same medium containing either vehicle or synthetic bovine PTH-(1-34). At the end of 10 min of incubation, which was the peak cyclic AMP response to PTH, the slices were transferred to 50 rnM sodium acetate (pH 4.0) maintained at 100°C. The slices were then homogenized in this solution, and the homogenate was centrifuged at 2OOOg for 20 min. The supernatant was removed for cyclic AMP determination by radioimmunoassay using the method of Steiner et al (16) as modified by Harper and Brooker (17). Preparatkm of renal membranes. Plasma membranes were isolated from the pooled kidneys of rats according to the method of Fitzpatrick et al (18), and membranes were prepared from chick kidney by the Fitzpatrick et aL (18) technique as modified for chick kidney (19). Membranes were stored in a buffer containing 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCI (pH 7.4) at -70C until used for radioreceptor or adenylate cyclase assays. Membrane protein was measured using the Lowry procedure (20) with bovine albumin as the reference standard. Assay of adenylate cyclase activity. The conditions for assay of this enzyme have been previously described (11,15). In brief, the reaction mixture (75 ~1) contained 50 mM Tris-HCl (pH 7.5), 6.7 mM MgC12, 12 mM creatine phosphate, 0.1 mrd cyclic AMP, 16 mM caffeine, 1.2 mM [a-q]ATP (5 X 10” cpm/mol), 266 pg/ml of bovine serum albumin, 13.3 U/ml creatine phosphokinase, and 50-100 pg of membrane protein. Incubation was at 30°C for 15 min. Under these experimental conditions, the formation of product was linear for at least 20 min. Cyclic m& was separated from other q-labeled compounds by the method of Saloman et al. (21). PTH-receptor binding assay. Iodination of 8,18 norleucine, 34 tyrosine-bovine PTH(l-34) amide [I?-
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nlPTH(l-34)] and purification of the radioligand was by a procedure that has been previously described in detail (15). Binding of ‘%I-nlPTH-(1-34) to renal membranes was carried out in a total incubation volume of 250 pl at 20°C for 90 min. The incubation contained 50 mM Tris-HCl (pH 7.5), 6.7 mM MgClc, 16 mM caffeine, 0.1 mM cyclic AMP, 1.2 mM ATP, 0.8% bovine albumin, 1 X lo5 cpm ‘251-nlPTH-(1-34), and approximately 75-150 pg membrane protein. Unlabeled nlPTH-(1-34) was added at various concentrations between 10-l’ and 10-r M. Bound 1251-nlPTH-(134) was separated from free hormone using 0.2-pm cellulose acetate filters (Millipore, Bedford, Mass.). Each filter was pretreated with 1 ml 50% human serum. At the end of the incubation, 2 ml of ice-cold wash buffer containing 25 mM Tris-HCl (pH 7.5), 2 mM MgClr, 125 mM NaCl, and 0.1% bovine albumin was added to each tube and immediately filtered. The filter was then washed with 5 ml of the same buffer for a total of four washes. Radioactivity was measured in a y-scintillation spectrometer. Binding of ‘%InlPTH-(1-34) to chick kidney membranes was proportional to membrane protein between 50 and 200 pg/tube. Nonspecific binding was measured as the binding of ‘%I-nlPTH-(1-34) in the presence of 0.1 pM unlabeled nlPTH-(1-34) and was 2% of total radioactivity in the incubation. These levels of radioactivity were subtracted from total binding of the radioligand to yield the percentage of specific binding. Specific binding of 1zI-nlPTH-(1-34) to chick renal membranes (-150 pg) under these conditions was 8-9% of total radioactivity in the incubation tube. Mute&k. Cyclic rH]AMP (38 Ci/mmol) and [aq]ATP (lo-30 Ci/mmol) were purchased from New England Nuclear, Boston, Massachusetts Parathyroid hormone (bovine synthetic PTH-(l-34)-tetratriacontapeptide) and [Nle’, Nle”, Tyr”]bPTH-(1-34) amide were obtained from Peninsula Laboratories, San Carlos, California. Forskolin was obtained from Calbiochem-Behring Corporation, La Jolla, California. RESULTS
Slices of rat kidney cortex were incubated with l-300 PM forskolin in the presence of 2 mM MIX in order to assess the concentration-response relationship for forskolin stimulation of tissue cyclic AMP accumulation (Fig. 1). The cyclic AMP response to forskolin appeared to be maximal at 300 pM, and the concentration of forskolin required for the half-maximal increase of cyclic AMP (Et!&,) was about 20 PM. Qualitatively similar results were found in experiments with kidney slices from g-week-old chickens that were incubated under the same conditions. In this
R. FORTE 60
Rat
Kidney
.
10 .
A
I/ basal
6
4
5 -Log
CForskalinl
M
FIG. 1. Effect of forskolin on cyclic AMP accumulation in cortical slices of rat kidney. The data are expressed as the mean of three slices at each condition. Cyclic AMP content of the slices is expressed as pmol/ mg wet wt.
species, forskolin (100 PM) increased renal cyclic AMP from 0.7 f 0.1 (mean * SE) to 75.3 + 5.0 pmol cyclic AMP/mg slice (n = 8). When weanling rats or newly hatched chicks were raised on diets that are deficient in vitamin D (-D), the PTH-dependent adenylate cyclase activity of highly purified plasma membranes is markedly depressed, whereas fluoride-stimulated enzyme activity is unaffected by vitamin D deficiency (11,15). We examined the effect of forskolin on the adenylate cyclase activity of kidney plasma membranes isolated from rats (Fig. 2) and chickens raised on +D or -D diets (Fig. 3). Forskolin concentration-response curves for activation of adenylate cyclase were similar in renal membranes from +D and -D rats or chickens although membranes from -D rats were activated by the diterpene slightly less than that of renal membranes from -tD rats. Maximal activation of adenylate cyclase was observed at 100 PM forskolin and the EC&, for forskolin was about 10 PM in membranes from both species and dietary groups. These data suggest that the enzymatic activity per se in renal membranes from -D rats or chickens is either not affected or minimally reduced relative to the forskolin-stimulated ade-
ACTIVATION
OF
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ADENYLATE
1 I
6
5 -Log
[ Forrkolin
4 ]
M
FIG. 2. Activation of rat kidney adenylate eyclase by forskolin. The data are expressed as the mean of triplicate assays of a plasma membrane preparation isolated from control (+D) or vitamin D-deficient (-D) rats (see Experimental Procedure).
nylate cyclase activity of control (+D) membranes. An interesting pharmacologic characteristic of forskolin is the marked potentiation produced by the diterpene when studied in the presence of hormones that activate adenylate cyclase (6, 7, 21, 22). Augmentation of hormone-stimulated adenylate cyclase activity occurs at low concentrations of forskolin and is most apparent in intact cells or tissue in comparison to broken cell preparations. The effects of forskolin on PTH-stimulated cyclic AMP accumulation were examined in kidney slices from chicks raised on either +D or -D diets or a diet that was vitamin Dreplete but deficient in calcium (-Ca). Both the -D and -Ca diets induce hypocalcemia and a progressive reduction in the renal cyclic AMP response to PTH which appears to be associated with the secondary hyperparathyroid state (11-15). Data in Fig. 4 confirmed that either the -D or the -Ca diets caused a marked decrease in the cylic AMP response of chicken kidney slices to PTH in vitro. This peptide hormone produced a greater than lo-fold increase in cyclic AMP content at 0.25 PM PTH in slices from +D chicken kidney. In contrast, PTH elicited only a two- to threefold increase in cyclic AMP accumulation in the kidney
CYCLASE
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slices from either -D or -Ca chickens. When 1 PM forskolin was included in the incubation medium, only a slight increase in cyclic AMP content was observed in the absence of PTH (Fig. 5). In marked contrast, forskolin potentiated the PTH-stimulated accumulation of cyclic AMP in a dramatic fashion. Kidney slices from +D chickens had a lo-fold greater content of cyclic AMP in the presence of 0.25 PM PTH plus 1 PM forskolin (Fig. 5) compared to PTH alone (Fig. 4). Potentiation of the renal cyclic AMP response to PTH for forskolin was also found in kidney slices from both the -D and -Ca animals. However, the maximal response to PTH in the presence of 1 PM forskolin of renal slices from either -D or -Ca birds remained lower than that observed in tissue from +D chickens. Thus, PTH-receptor-mediated activation of adenylate cyclase in the tissue from -D or -Ca birds is reduced both in the presence of the diterpene and when forskolin is absent. Although the effect of forskolin to potentiate the cyclic AMP response to PTH was not studied in renal slices from -D or -Ca rats, we examined this effect of forskolin in +D rat kidney (Fig. 6). Forskolin at 1 I.LM produced a qualitatively similar potentiation of the kidney slice cyclic AMP response to PTH. At 0.25
. + 0 Chlch 0 - 0 ChiCl 22.000
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of chicken kidney adenylate See Fig. 3 and Experimental
cyPro-
902
LEONARD
eo;o1‘. b
i -Log
[PTH
f I-341
M
FIG. 4. Renal slice cyclic AMP accumulation response to PTH in tissue from chickens raised on control (+D+Ca), vitamin D-deficient (-D+Ca), or calcium-deficient (+D-Ca) diets. The diets were fed for 14 days from the time of hatching. Serum total calcium at the time birds were killed was +D+Ca, 11.0 f 0.1; -D+Ca, 7.9 f 0.4 (P < 901); +D-Ca, 8.4 + 0.2 (P < 901). Data are expressed as the mean f SE of two slices assayed in duplicate at each point and are slice cyclic AMP content, pmol/mg wet wt.
PTH plus 1 PM forskolin we found about a ninefold increase in cyclic AMP over that observed with PTH alone. Thus, the cyclic AMP response to the combination of PTH and forskolin was markedly greater than that caused by either PTH or forskolin alone in both rat and chicken kidney. We next studied the effects of forskolin on the PTH-dependent adenylate cyclase of plasma membranes from chicken kidney (+D) in order to further characterize the interaction between forskolin and PTH (Fig. 7). The data from concentration-response curves for PTH-mediated activation of renal adenylate cyclase in the absence or presence of 0.1, 1.0, and 10 PM forskolin revealed much less potentiation of the receptor-mediated activation of adenylate cyclase by forskolin in plasma membranes than that observed in the intact tissue (Fig. 5 and 6). The activation of adenylate cyclase by a combination of forskolin and PTH was, however, greater than the sum of the activation achieved by forskolin alone and PTH alone. These data support PM
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the concept that forskolin potentiates the PTH-receptor-mediated activation of renal adenylate cyclase but does not explain the quantitative difference in the efficacy of forskolin for potentiating the PTH-mediated effects on cyclic AMP formation in the intact tissue versus broken-cell preparations. The effect of forskolin on the binding of a radiolabeled PTH agonist to PTH receptors was examined using chicken kidney plasma membranes in order to determine whether the diterpene influenced the hormone-receptor interaction. The radioligand (agonist) that we used is the 8,18 norleucine, 34 ‘%I-tyrosine PTH (l-34)amide derivative of synthetic bovine PTH (l-34). The data summarized in Fig. 8 shows that 1 j.&M forskolin had no effect on the binding of this PTH analog to renal receptors. Therefore, the potentiation of PTH-dependent adenylate cyclase activity by forskolin is apparently not due to an action of forskolin on the binding of PTH to its receptor sites in renal plasma membranes. DISCUSSION
We found that forskolin was a useful probe of the renal adenylate cyclase system. Specifically, forskolin-stimulated adenylate cyclase activity was used as an es-
es;ol-
9
9 -LOO
FIG.
sponse
5. Forskolin of chicken
CPTH
potentiates kidney slices
r 1-341
M
the cyclic AMP to PTH. See Fig.
re4.
ACTIVATION
OF
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ADENYLATE
CYCLASE
BY
903
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Chicken
4p 60 0 G
Kidney
0 0
40 20
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/--,--
-r---7---+---4
FIG. 6. sponse of sented as of kidney expressed
8
7 -LOQ
(bPTH
l-34)
M
Forskolin potentiates the cyclic AMP rerat kidney slices to PTH. The data are prethe mean + SE (vertical bars) of three slices cortex for such condition. Cyclic AMP is as pmol/wg wet wt.
timate of total enzymatic activity in situations where it has been documented that the PTH-dependent adenylate cyclase is markedly downregulated (11-15). In experimental models of the secondary hyperparathyroid state (-D, -Ca diets) it has been shown that the reduction in PTHdependent enzyme activity is correlated with a specific reduction in PTH receptors (15). Activity of adenylate cyclase in the 24j
basal
Chicken
Kidney
8
IFS,
7 -LOP
tbPTH
1.0
uM FSK
0.1
uM FSK
6 l-341 1-341
M
FIG. ‘7. Forskolin potentiates the activation of renal adenylate cyclase by PTH. Data are the mean of triplicate assays of adenylate cyclase activity at each condition. Plasma membranes were prepared from chicken kidney as described under Experimental Procedures. Qualitatively similar data were obtained with rat kidney plasma membranes (data not shown).
Bo’
1’0
6 -Log
i3 tnl
bPTH
l-341
i M
FIG. 8. Lack of effect of forskolin on PTH-receptor binding. Plasma membranes were prepared from 1% month-old hens. The data are expressed as the percentage of specific binding which is the percentage of the total radioactivity in the incubation (see Experimental Procedures). Bo is the binding of iwInlbPTH(l-34) in the absence of unlabeled peptide (nlbPTH(l-34)). A representative experiment is presented with each point being the mean of triplicate assays.
presence of fluoride (11, 12, 15) or guanynucleotides (unpublished) was not affected by either vitamin D or calcium deficiency. However, fluoride and guanylnucleotides activate the adenylate cyclase via a mechanism that involves the nucleotide regulatory subunit (23), whereas forskolin has been postulated to directly interact with and activate the catalytic subunit (8). Ross (9) and Pfeuffer and Metzger (10) have provided the strongest data supporting that hypothesis. Using different techniques, these investigators separated the catalytic and nucleotide regulatory subunits and demonstrated that forskolin activated the resolved catalytic subunit. Therefore, forskolin-mediated stimulation of adenylate cyclase does not appear to require the nucleotide regulatory subunit. Accordingly, the forskolin-dependent enzymatic activity may be used as an estimate of total catalytic activity. Therefore, data presented here indicate that the renal adenylate cyclase activity per se is rela-
904
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tively unchanged in -D rats or chicks compared to the corresponding +D control. This provides additional evidence that the molecular mechanism which accounts for the reduction in PTH-dependent enzyme activity in rats or chicks raised on either -D or -Ca diets is a specific downregulation of PTH receptor sites (15). It should be pointed out that the activation of adenylate cyclase by PTH affects only a portion of total adenylate cyclase of kidney, whereas forskolin should activate both PTH-dependent and PTH-independent forms of the enzyme (i.e., total adenylate cyclase). A second prominent characteristic of the pharmacologic action of forskolin is that the diterpene potentiates the activation of adenylate cyclase by PTH. This effect of forskolin has been documented with other agonists and tissues and is considerably more dramatic in the intact compared to broken-cell preparations (6, ‘7, 21, 22, 24). We found that 1 /IM forskolin markedly potentiated the cyclic AMP accumulation response to PTH in kidney slices from rats and chicks even though this concentration of forskolin in the absence of hormone had only a small effect on cyclic AMP levels. Moreover, forskolin augmented the cyclic AMP response to PTH in tissue from both -D and -Ca chicks. However, the cyclic AMP response to PTH in the presence of 1 PM forskolin remained lower in tissue from either -D or -Ca birds compared to control slices. Forskolin did not restore full responsiveness of the adenylate cyclase to PTH, which is consistent with the hypothesis that the molecular alteration in renal membranes is restricted to the hormone-receptor component of the adenylate cyclase system in these secondary hyperparathyroid animal models. Our data clearly demonstrated that forskolin does not affect the binding of PTH to renal receptors. On the other hand, potentiation of hormone-mediated activation of adenylate cyclase by forskolin suggests that the diterpene may exert actions on this system at a site separate and distinct from the catalytic subunit. Such a site of action is suggested by the report that forskolin does not increase cyclic AMP levels in the intact viable sperm, nor activate adenylate
R. FORTE
cyclase from either cytosolic or membrane fractions of mammalian sperm (25). The adenylate cyclase of sperm is thought to be devoid of functionally active receptors for hormones and the nucleotide regulatory subunit (26-28). Therefore, it may be proposed that forskolin influences the adenylate cyclase by interacting with a component or components of the enzyme system which influences both the catalytic subunit activity and that which is mediated by the nucleotide regulatory subunit. The potency and efficacy of forskolin in activating adenylate cyclase in vitro has been useful in studying cyclic AMP-mediated events of physiologic significance (22,24,29,30). In renal cortical slices from the rat, it has been demonstrated that forskolin increases the synthesis of 1,25-dihydroxycholecalciferol and decreases 24,25-dihydroxycholecalciferol formation similar to the effects of PTH (31). Moreover, PTH plus forskolin produced an increase in 1,25-dihydroxycholecalciferol formation which was no greater than that caused by maximally effective concentrations of either agonist alone. This suggests that both PTH and forskolin influence the renal 25-hydroxycholecalciferol hydroxylase system via cyclic AMP. Forskolin has also been shown to mimic PTH in the embryonic chick tibiae where the diterpene enhanced calcium efflux (32). Thus, forskolin appears to be a useful tool for examination of the mechanism involved in regulation of adenylate cyclase per se and for investigating cyclic AMP-mediated regulation of cell function in target tissues for PTH. ACKNOWLEDGMENTS This research was supported by the Veterans ministration Medical Research Service and by National Institutes of Health Grant AM 14’787. technical assistance of Mr. Richard Poelling and Sammy Langeluttig and the preparation of manuscript by Mrs. Marie Johnson are gratefully knowledged.
Adthe The Mrs. this ac-
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2. LINDNER, D., DOHADWALLA, A. N., AND BHA’ITACHARYA, B. K. (1978) Awneiwz. Forsch/Lkug Res. 28,284-289. 3. DUBEY, M. P., SRIMAL, R. C., NITYANAND, S., AND DHAWAN, B. N. (1981) J. Ethnopharmacol. 3, l13. 4. METZGER, H., AND LINDNER, E. (1981) IRCS Med sci 9, 99. 5. SEAMON, K. B., PADGEIT, W., AND DALY, J. W. (1981) Proc. NatL Acd Sci. USA 78,3363-3367. 6. SEAMON, K. B., AND DALY, J. W. (1981) J. C&ic Nucl. Res. 7, 201-224. 7. DARFLER, F. J., MAHAN, L. C., KOACHMAN, A. M., AND INSEL, P. A. (1982) J. Biol. Chem 257, 11901-11907. 8. SEAMON, K., AND DALY, J. W. (1981) J. Biol. Chem 256, 9799-9801. 9. Ross, E. M. (1982) J. Bid Chem. 257,10751-1058. 10. PFEUFFER, T., AND METZGER, H. (1982) FEBSLett 146,369-375. 11. FORTE, L. R., NICKOLS, G. A., AND ANAST, C. S. (1976) J. Clin Invest. 57, 559-568. 12. CARNES, D. L., ANAST, C. S., AND FORTE, L. R. (1980) Biochim Biophys. Acta 629, 546-552. 13. CARNES, D. L., NICKOLS, G. A., ANAST, C. S., AND FORTE, L. R. (1980) Amer. J. Physiid 239, E396E400. 14. KAKUTA, S., SATO, C., SUDA, T., KIMURA, N., AAKI, N., ONO, Y., AND NAGATA, N. (1978) B&him. Biophys. Acta 539, 173-180. 15. FORTE, L. R., LANGELU~IG, S., POELLING, R. E., AND THOMAS, M. L. (1982) Amer. J. PhysioL 242, E154-E163. 16. STEINER, A. L., KIPNIS, D. M., UTIGER, R., AND PARKER, C. (1969) Proc. Natl. Acad Sci. USA 64, 367-373. 17. HARPER, J. F., AND BROOKER, G. (1975) J. Cyclic Nut. Res. 1. 207-218.
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18. FITZPATRICK, D. F., DAVENPORT, G. R., FORTE, L. R., AND LANDON, E. J. (1969) J. BioL Chew. 244, 3561-3569. 19. NISSENSON, R. A., AND ARNAUD, C. D. (1979) J. Biol Chem. 254,1469-1475. 20. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem, 193, 265-275. 21. DALY, J. W., PADGE~, W., AND SEAMON, K. B. (1982) J. Neurochem 38, 532-544. 22. BURNS, T. W., LANGLEY, P. E., TERRY, B. E., BYLUND, D. B., AND FORTE, L. R. (1982) Life Sci. 31, 815-821. 23. LIMBIRD, L. E. (1981) Biochem J. 185, 1-13. 24. SIEGL, A. M., DALY, J. W., AND SMITH, J. B. (1982) Mol. Phamw.col. 21, 680-687. 25. FORTE, L. R., BOUND, D. B., AND ZAHLER, W. L. (1983) Mel Pharmmol. 24,42-47. 26. HERMAN, C. A., ZAHLER, W. L., DOAKS, G. A., AND CAMPBELL, B. J. (1976) Arch Biochem Biophys. 177, 622-629. 27. BRAUN, T., AND DODS, R. F. (1975) Proc Natl. Ad Sci. USA 72, 1097-1101. 28. GARBERS,D. L., AND KOPF, G. S. (1980) in Advances in Cyclic Nucleotide Research, Vol. 13, pp. 251306, Raven Press, New York. 29. LITOSCH, I., HUDSON, T. H., MILLS, I., LI, S.-Y., AND FAIN, J. N. (1982) Mel Pkmmmxd 22,109115. 30. MORIWAKI, K., ITOH, Y., IIDA, S., AND ICHIHARA, K. (1982) Lz$e Sci 30, 2235-2240. 31. ARMBRECHT, H. J., FORTE, L. R., WONGSURAWAT, N., ZENSER, T. V. AND DAVIS, B. B. (1983) Clin Res. 31, 380A. 32. MARTZ, A. AND THOMAS, M. L. (1983) Biochem Phmmnd, in press.
Vol.
Activation of Renal Adenylate Cyclase by Forskolin: Assessment Enzymatic Activity in Animal Models of the Secondary Hyperparathyroid State LEONARD
of
R. FORTE
M-523 iifedical Science Building, Department of Pha rmaeology, School of Medicine, University and Harry S Truman Memorial Veterans Hospital, Columbia, Mtisauri 65212
of Missouri,
Received December 13, 1982, and in revised form May 19, 1983
The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptormediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.
Forskolin, a diterpene that was isolated from the roots of the Indian plant, Coleus forshcohlii (l), has been shown to be a potent cardiovascular drug (2, 3). Forskolin is a powerful activator of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.11 in both intact cells and tissue and also in membrane preparations (4-7). Unlike the activation found with cholera toxin, fluoride, and guanylnucleotides, the forskolin-stimulated adenylate cyclase is readily reversible when the drug is removed from the system (5-7). Although the mechanism of forskolin activation of ad0003-9861183 $3.00 Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
enylate cyclase is unknown, it has been proposed that the drug directly interacts with the catalytic subunit of this enzyme (8). Moreover, forskolin activates the adenylate cyclase catalytic subunit when the nucleotide regulatory protein (N protein) has been removed from the enzyme by chromatographic techniques (9, 10). The purpose of the present study was to evaluate forskolin as an experimental tool for measuring the total enzymatic activity of kidney adenylate cyclase in animal models that exhibit a marked decrease in hormone-dependent enzyme activity. Rats 898
ACTIVATION
OF
RENAL
ADENYLATE
or chickens raised on diets deficient in either vitamin D or calcium exhibit downregulation of the renal parathyroid hormone (PTH)‘-dependent adenylate cyclase activity (11-15). There is direct evidence that the loss of tissue responsiveness to PTH is due to a substantial reduction in membrane receptors for the hormone (15). Of physiologic significance is the finding that vitamin D-deficient rats exhibit a refractory phosphaturic response to PTH in viva (11). Reported here are the results of studies designed to assess the use of forskolin-stimulated increases in kidney slice cyclic AMP accumulation and forskolinstimulated adenylate cyclase activity of renal membranes as a means of determining whether the forskolin-stimulated activity of renal adenylate cyclase is affected by diets deficient in either vitamin D or calcium. EXPERIMENTAL
PROCEDURE
Aniwmls and dietary procedure. White Leghorn cockerels were obtained at 1 day of age from Colonial Poultry Farms, Pleasant Hill, Missouri. They were housed in our animal facility using commercial brooders maintained at 32°C (Brower Manufacturing, Quincy, Ill.). The light was devoid of uv irradiation and maintained on a 12/12 light/dark cycle. Chicks were allowed free access to food and deionized water. The chicks were fed one of two synthetic diets obtained from Teklad, Madison, Wisconsin. A diet deficient in vitamin D (-D), containing 1.1% Ca, 0.6% P, and 0.07% Mg (No. 170245) by our analysis of the wet-ashed diet, was fed to chicks beginning at 1 day of age. Controls birds (+D) received the same diet and were given 70 IU of vitamin Da in 50 pl propylene glyeol by mouth every third day at 0800 h during the course of the experiment. The second experimental diet (No. 80378) was also deficient in vitamin D and was low in Ca, 0.01% (-Ca). The P and Mg values were the same in the -Ca as in the -D diet. Chicks fed the -Ca diet received vitamin Da as described above. The control birds (+D, +Ca) were the same control animals for both -D and -Ca groups. Chicks were fed these diets for 14 days and then killed by decapitation to obtain kidneys for the analytical procedures (15). i Abbreviations used: PTH, parathyroid hormone; -D, vitamin D-deficient diet; +D, control diet; -Ca, low-calcium diet; +Ca, control; MIX, methylisobutylxanthine; nlPTH(l-34), 8,18 norleucine, 34 tyrosinebovine PTH(l-34)amide.
CYCLASE
BY
FORSKOLIN
899
Male, 21-day-old weanling rats obtained from Holtzman Company, Madison, Wisconsin, were fed a vitamin D-deficient diet (0.47% Ca, 0.36% P, and 0.048% Mg) and maintained in a room devoid of uv light (11). Control (+D) animals received the same diet and were pair-fed with the D-deficient rats, but also received oral doses of 70 IU vitamin Da twice weekly. Rats were killed by decapitation after 4 weeks of the experimental diets. Renal cyclic AMP content. Thin slices (0.5 mm) of chicken or rat kidney weighing approximately 25 mg were prepared using a Stadie-Riggs microtome (Arthur Thomas, Philadelphia, Pa.). They were placed in ice-cold 0.9% NaCl until weighed and then transferred to Krebs-Ringer bicarbonate buffer that had been adjusted to pH 7.4 by bubbling 95% Oa-5% COP through the medium. One slice was preincubated for 15 min in 1 ml of this medium containing 10 mM glucose and 2 mM methylisobutylxanthine (MIX). The temperature was maintained at 37°C using a Dubnoff metabolic incubator. After the preincubation, slices were transferred to the same medium containing either vehicle or synthetic bovine PTH-(1-34). At the end of 10 min of incubation, which was the peak cyclic AMP response to PTH, the slices were transferred to 50 rnM sodium acetate (pH 4.0) maintained at 100°C. The slices were then homogenized in this solution, and the homogenate was centrifuged at 2OOOg for 20 min. The supernatant was removed for cyclic AMP determination by radioimmunoassay using the method of Steiner et al (16) as modified by Harper and Brooker (17). Preparatkm of renal membranes. Plasma membranes were isolated from the pooled kidneys of rats according to the method of Fitzpatrick et al (18), and membranes were prepared from chick kidney by the Fitzpatrick et aL (18) technique as modified for chick kidney (19). Membranes were stored in a buffer containing 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCI (pH 7.4) at -70C until used for radioreceptor or adenylate cyclase assays. Membrane protein was measured using the Lowry procedure (20) with bovine albumin as the reference standard. Assay of adenylate cyclase activity. The conditions for assay of this enzyme have been previously described (11,15). In brief, the reaction mixture (75 ~1) contained 50 mM Tris-HCl (pH 7.5), 6.7 mM MgC12, 12 mM creatine phosphate, 0.1 mrd cyclic AMP, 16 mM caffeine, 1.2 mM [a-q]ATP (5 X 10” cpm/mol), 266 pg/ml of bovine serum albumin, 13.3 U/ml creatine phosphokinase, and 50-100 pg of membrane protein. Incubation was at 30°C for 15 min. Under these experimental conditions, the formation of product was linear for at least 20 min. Cyclic m& was separated from other q-labeled compounds by the method of Saloman et al. (21). PTH-receptor binding assay. Iodination of 8,18 norleucine, 34 tyrosine-bovine PTH(l-34) amide [I?-
900
LEONARD
nlPTH(l-34)] and purification of the radioligand was by a procedure that has been previously described in detail (15). Binding of ‘%I-nlPTH-(1-34) to renal membranes was carried out in a total incubation volume of 250 pl at 20°C for 90 min. The incubation contained 50 mM Tris-HCl (pH 7.5), 6.7 mM MgClc, 16 mM caffeine, 0.1 mM cyclic AMP, 1.2 mM ATP, 0.8% bovine albumin, 1 X lo5 cpm ‘251-nlPTH-(1-34), and approximately 75-150 pg membrane protein. Unlabeled nlPTH-(1-34) was added at various concentrations between 10-l’ and 10-r M. Bound 1251-nlPTH-(134) was separated from free hormone using 0.2-pm cellulose acetate filters (Millipore, Bedford, Mass.). Each filter was pretreated with 1 ml 50% human serum. At the end of the incubation, 2 ml of ice-cold wash buffer containing 25 mM Tris-HCl (pH 7.5), 2 mM MgClr, 125 mM NaCl, and 0.1% bovine albumin was added to each tube and immediately filtered. The filter was then washed with 5 ml of the same buffer for a total of four washes. Radioactivity was measured in a y-scintillation spectrometer. Binding of ‘%InlPTH-(1-34) to chick kidney membranes was proportional to membrane protein between 50 and 200 pg/tube. Nonspecific binding was measured as the binding of ‘%I-nlPTH-(1-34) in the presence of 0.1 pM unlabeled nlPTH-(1-34) and was 2% of total radioactivity in the incubation. These levels of radioactivity were subtracted from total binding of the radioligand to yield the percentage of specific binding. Specific binding of 1zI-nlPTH-(1-34) to chick renal membranes (-150 pg) under these conditions was 8-9% of total radioactivity in the incubation tube. Mute&k. Cyclic rH]AMP (38 Ci/mmol) and [aq]ATP (lo-30 Ci/mmol) were purchased from New England Nuclear, Boston, Massachusetts Parathyroid hormone (bovine synthetic PTH-(l-34)-tetratriacontapeptide) and [Nle’, Nle”, Tyr”]bPTH-(1-34) amide were obtained from Peninsula Laboratories, San Carlos, California. Forskolin was obtained from Calbiochem-Behring Corporation, La Jolla, California. RESULTS
Slices of rat kidney cortex were incubated with l-300 PM forskolin in the presence of 2 mM MIX in order to assess the concentration-response relationship for forskolin stimulation of tissue cyclic AMP accumulation (Fig. 1). The cyclic AMP response to forskolin appeared to be maximal at 300 pM, and the concentration of forskolin required for the half-maximal increase of cyclic AMP (Et!&,) was about 20 PM. Qualitatively similar results were found in experiments with kidney slices from g-week-old chickens that were incubated under the same conditions. In this
R. FORTE 60
Rat
Kidney
.
10 .
A
I/ basal
6
4
5 -Log
CForskalinl
M
FIG. 1. Effect of forskolin on cyclic AMP accumulation in cortical slices of rat kidney. The data are expressed as the mean of three slices at each condition. Cyclic AMP content of the slices is expressed as pmol/ mg wet wt.
species, forskolin (100 PM) increased renal cyclic AMP from 0.7 f 0.1 (mean * SE) to 75.3 + 5.0 pmol cyclic AMP/mg slice (n = 8). When weanling rats or newly hatched chicks were raised on diets that are deficient in vitamin D (-D), the PTH-dependent adenylate cyclase activity of highly purified plasma membranes is markedly depressed, whereas fluoride-stimulated enzyme activity is unaffected by vitamin D deficiency (11,15). We examined the effect of forskolin on the adenylate cyclase activity of kidney plasma membranes isolated from rats (Fig. 2) and chickens raised on +D or -D diets (Fig. 3). Forskolin concentration-response curves for activation of adenylate cyclase were similar in renal membranes from +D and -D rats or chickens although membranes from -D rats were activated by the diterpene slightly less than that of renal membranes from -tD rats. Maximal activation of adenylate cyclase was observed at 100 PM forskolin and the EC&, for forskolin was about 10 PM in membranes from both species and dietary groups. These data suggest that the enzymatic activity per se in renal membranes from -D rats or chickens is either not affected or minimally reduced relative to the forskolin-stimulated ade-
ACTIVATION
OF
RENAL
ADENYLATE
1 I
6
5 -Log
[ Forrkolin
4 ]
M
FIG. 2. Activation of rat kidney adenylate eyclase by forskolin. The data are expressed as the mean of triplicate assays of a plasma membrane preparation isolated from control (+D) or vitamin D-deficient (-D) rats (see Experimental Procedure).
nylate cyclase activity of control (+D) membranes. An interesting pharmacologic characteristic of forskolin is the marked potentiation produced by the diterpene when studied in the presence of hormones that activate adenylate cyclase (6, 7, 21, 22). Augmentation of hormone-stimulated adenylate cyclase activity occurs at low concentrations of forskolin and is most apparent in intact cells or tissue in comparison to broken cell preparations. The effects of forskolin on PTH-stimulated cyclic AMP accumulation were examined in kidney slices from chicks raised on either +D or -D diets or a diet that was vitamin Dreplete but deficient in calcium (-Ca). Both the -D and -Ca diets induce hypocalcemia and a progressive reduction in the renal cyclic AMP response to PTH which appears to be associated with the secondary hyperparathyroid state (11-15). Data in Fig. 4 confirmed that either the -D or the -Ca diets caused a marked decrease in the cylic AMP response of chicken kidney slices to PTH in vitro. This peptide hormone produced a greater than lo-fold increase in cyclic AMP content at 0.25 PM PTH in slices from +D chicken kidney. In contrast, PTH elicited only a two- to threefold increase in cyclic AMP accumulation in the kidney
CYCLASE
BY
901
FORSKOLIN
slices from either -D or -Ca chickens. When 1 PM forskolin was included in the incubation medium, only a slight increase in cyclic AMP content was observed in the absence of PTH (Fig. 5). In marked contrast, forskolin potentiated the PTH-stimulated accumulation of cyclic AMP in a dramatic fashion. Kidney slices from +D chickens had a lo-fold greater content of cyclic AMP in the presence of 0.25 PM PTH plus 1 PM forskolin (Fig. 5) compared to PTH alone (Fig. 4). Potentiation of the renal cyclic AMP response to PTH for forskolin was also found in kidney slices from both the -D and -Ca animals. However, the maximal response to PTH in the presence of 1 PM forskolin of renal slices from either -D or -Ca birds remained lower than that observed in tissue from +D chickens. Thus, PTH-receptor-mediated activation of adenylate cyclase in the tissue from -D or -Ca birds is reduced both in the presence of the diterpene and when forskolin is absent. Although the effect of forskolin to potentiate the cyclic AMP response to PTH was not studied in renal slices from -D or -Ca rats, we examined this effect of forskolin in +D rat kidney (Fig. 6). Forskolin at 1 I.LM produced a qualitatively similar potentiation of the kidney slice cyclic AMP response to PTH. At 0.25
. + 0 Chlch 0 - 0 ChiCl 22.000
0_,,_.... ..--.- . . .. . . ..
I
/
,. ,..’ ,./ ,....’
../,...‘. 0 ,.2,..’
x., ‘.., i
:i ...’ ,..’ 0 ,..’
,..i’ ,..’ ,_..”
/
1
2*oooL 6
-Log
3. Activation clase by forskolin. cedure. FIG.
5
4 [ Forokolin]
M
of chicken kidney adenylate See Fig. 3 and Experimental
cyPro-
902
LEONARD
eo;o1‘. b
i -Log
[PTH
f I-341
M
FIG. 4. Renal slice cyclic AMP accumulation response to PTH in tissue from chickens raised on control (+D+Ca), vitamin D-deficient (-D+Ca), or calcium-deficient (+D-Ca) diets. The diets were fed for 14 days from the time of hatching. Serum total calcium at the time birds were killed was +D+Ca, 11.0 f 0.1; -D+Ca, 7.9 f 0.4 (P < 901); +D-Ca, 8.4 + 0.2 (P < 901). Data are expressed as the mean f SE of two slices assayed in duplicate at each point and are slice cyclic AMP content, pmol/mg wet wt.
PTH plus 1 PM forskolin we found about a ninefold increase in cyclic AMP over that observed with PTH alone. Thus, the cyclic AMP response to the combination of PTH and forskolin was markedly greater than that caused by either PTH or forskolin alone in both rat and chicken kidney. We next studied the effects of forskolin on the PTH-dependent adenylate cyclase of plasma membranes from chicken kidney (+D) in order to further characterize the interaction between forskolin and PTH (Fig. 7). The data from concentration-response curves for PTH-mediated activation of renal adenylate cyclase in the absence or presence of 0.1, 1.0, and 10 PM forskolin revealed much less potentiation of the receptor-mediated activation of adenylate cyclase by forskolin in plasma membranes than that observed in the intact tissue (Fig. 5 and 6). The activation of adenylate cyclase by a combination of forskolin and PTH was, however, greater than the sum of the activation achieved by forskolin alone and PTH alone. These data support PM
R. FORTE
the concept that forskolin potentiates the PTH-receptor-mediated activation of renal adenylate cyclase but does not explain the quantitative difference in the efficacy of forskolin for potentiating the PTH-mediated effects on cyclic AMP formation in the intact tissue versus broken-cell preparations. The effect of forskolin on the binding of a radiolabeled PTH agonist to PTH receptors was examined using chicken kidney plasma membranes in order to determine whether the diterpene influenced the hormone-receptor interaction. The radioligand (agonist) that we used is the 8,18 norleucine, 34 ‘%I-tyrosine PTH (l-34)amide derivative of synthetic bovine PTH (l-34). The data summarized in Fig. 8 shows that 1 j.&M forskolin had no effect on the binding of this PTH analog to renal receptors. Therefore, the potentiation of PTH-dependent adenylate cyclase activity by forskolin is apparently not due to an action of forskolin on the binding of PTH to its receptor sites in renal plasma membranes. DISCUSSION
We found that forskolin was a useful probe of the renal adenylate cyclase system. Specifically, forskolin-stimulated adenylate cyclase activity was used as an es-
es;ol-
9
9 -LOO
FIG.
sponse
5. Forskolin of chicken
CPTH
potentiates kidney slices
r 1-341
M
the cyclic AMP to PTH. See Fig.
re4.
ACTIVATION
OF
RENAL
ADENYLATE
CYCLASE
BY
903
FORSKOLIN
Chicken
4p 60 0 G
Kidney
0 0
40 20
P Ll QJ 80
/--,--
-r---7---+---4
FIG. 6. sponse of sented as of kidney expressed
8
7 -LOQ
(bPTH
l-34)
M
Forskolin potentiates the cyclic AMP rerat kidney slices to PTH. The data are prethe mean + SE (vertical bars) of three slices cortex for such condition. Cyclic AMP is as pmol/wg wet wt.
timate of total enzymatic activity in situations where it has been documented that the PTH-dependent adenylate cyclase is markedly downregulated (11-15). In experimental models of the secondary hyperparathyroid state (-D, -Ca diets) it has been shown that the reduction in PTHdependent enzyme activity is correlated with a specific reduction in PTH receptors (15). Activity of adenylate cyclase in the 24j
basal
Chicken
Kidney
8
IFS,
7 -LOP
tbPTH
1.0
uM FSK
0.1
uM FSK
6 l-341 1-341
M
FIG. ‘7. Forskolin potentiates the activation of renal adenylate cyclase by PTH. Data are the mean of triplicate assays of adenylate cyclase activity at each condition. Plasma membranes were prepared from chicken kidney as described under Experimental Procedures. Qualitatively similar data were obtained with rat kidney plasma membranes (data not shown).
Bo’
1’0
6 -Log
i3 tnl
bPTH
l-341
i M
FIG. 8. Lack of effect of forskolin on PTH-receptor binding. Plasma membranes were prepared from 1% month-old hens. The data are expressed as the percentage of specific binding which is the percentage of the total radioactivity in the incubation (see Experimental Procedures). Bo is the binding of iwInlbPTH(l-34) in the absence of unlabeled peptide (nlbPTH(l-34)). A representative experiment is presented with each point being the mean of triplicate assays.
presence of fluoride (11, 12, 15) or guanynucleotides (unpublished) was not affected by either vitamin D or calcium deficiency. However, fluoride and guanylnucleotides activate the adenylate cyclase via a mechanism that involves the nucleotide regulatory subunit (23), whereas forskolin has been postulated to directly interact with and activate the catalytic subunit (8). Ross (9) and Pfeuffer and Metzger (10) have provided the strongest data supporting that hypothesis. Using different techniques, these investigators separated the catalytic and nucleotide regulatory subunits and demonstrated that forskolin activated the resolved catalytic subunit. Therefore, forskolin-mediated stimulation of adenylate cyclase does not appear to require the nucleotide regulatory subunit. Accordingly, the forskolin-dependent enzymatic activity may be used as an estimate of total catalytic activity. Therefore, data presented here indicate that the renal adenylate cyclase activity per se is rela-
904
LEONARD
tively unchanged in -D rats or chicks compared to the corresponding +D control. This provides additional evidence that the molecular mechanism which accounts for the reduction in PTH-dependent enzyme activity in rats or chicks raised on either -D or -Ca diets is a specific downregulation of PTH receptor sites (15). It should be pointed out that the activation of adenylate cyclase by PTH affects only a portion of total adenylate cyclase of kidney, whereas forskolin should activate both PTH-dependent and PTH-independent forms of the enzyme (i.e., total adenylate cyclase). A second prominent characteristic of the pharmacologic action of forskolin is that the diterpene potentiates the activation of adenylate cyclase by PTH. This effect of forskolin has been documented with other agonists and tissues and is considerably more dramatic in the intact compared to broken-cell preparations (6, ‘7, 21, 22, 24). We found that 1 /IM forskolin markedly potentiated the cyclic AMP accumulation response to PTH in kidney slices from rats and chicks even though this concentration of forskolin in the absence of hormone had only a small effect on cyclic AMP levels. Moreover, forskolin augmented the cyclic AMP response to PTH in tissue from both -D and -Ca chicks. However, the cyclic AMP response to PTH in the presence of 1 PM forskolin remained lower in tissue from either -D or -Ca birds compared to control slices. Forskolin did not restore full responsiveness of the adenylate cyclase to PTH, which is consistent with the hypothesis that the molecular alteration in renal membranes is restricted to the hormone-receptor component of the adenylate cyclase system in these secondary hyperparathyroid animal models. Our data clearly demonstrated that forskolin does not affect the binding of PTH to renal receptors. On the other hand, potentiation of hormone-mediated activation of adenylate cyclase by forskolin suggests that the diterpene may exert actions on this system at a site separate and distinct from the catalytic subunit. Such a site of action is suggested by the report that forskolin does not increase cyclic AMP levels in the intact viable sperm, nor activate adenylate
R. FORTE
cyclase from either cytosolic or membrane fractions of mammalian sperm (25). The adenylate cyclase of sperm is thought to be devoid of functionally active receptors for hormones and the nucleotide regulatory subunit (26-28). Therefore, it may be proposed that forskolin influences the adenylate cyclase by interacting with a component or components of the enzyme system which influences both the catalytic subunit activity and that which is mediated by the nucleotide regulatory subunit. The potency and efficacy of forskolin in activating adenylate cyclase in vitro has been useful in studying cyclic AMP-mediated events of physiologic significance (22,24,29,30). In renal cortical slices from the rat, it has been demonstrated that forskolin increases the synthesis of 1,25-dihydroxycholecalciferol and decreases 24,25-dihydroxycholecalciferol formation similar to the effects of PTH (31). Moreover, PTH plus forskolin produced an increase in 1,25-dihydroxycholecalciferol formation which was no greater than that caused by maximally effective concentrations of either agonist alone. This suggests that both PTH and forskolin influence the renal 25-hydroxycholecalciferol hydroxylase system via cyclic AMP. Forskolin has also been shown to mimic PTH in the embryonic chick tibiae where the diterpene enhanced calcium efflux (32). Thus, forskolin appears to be a useful tool for examination of the mechanism involved in regulation of adenylate cyclase per se and for investigating cyclic AMP-mediated regulation of cell function in target tissues for PTH. ACKNOWLEDGMENTS This research was supported by the Veterans ministration Medical Research Service and by National Institutes of Health Grant AM 14’787. technical assistance of Mr. Richard Poelling and Sammy Langeluttig and the preparation of manuscript by Mrs. Marie Johnson are gratefully knowledged.
Adthe The Mrs. this ac-
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ACTIVATION
OF RENAL
ADENYLATE
2. LINDNER, D., DOHADWALLA, A. N., AND BHA’ITACHARYA, B. K. (1978) Awneiwz. Forsch/Lkug Res. 28,284-289. 3. DUBEY, M. P., SRIMAL, R. C., NITYANAND, S., AND DHAWAN, B. N. (1981) J. Ethnopharmacol. 3, l13. 4. METZGER, H., AND LINDNER, E. (1981) IRCS Med sci 9, 99. 5. SEAMON, K. B., PADGEIT, W., AND DALY, J. W. (1981) Proc. NatL Acd Sci. USA 78,3363-3367. 6. SEAMON, K. B., AND DALY, J. W. (1981) J. C&ic Nucl. Res. 7, 201-224. 7. DARFLER, F. J., MAHAN, L. C., KOACHMAN, A. M., AND INSEL, P. A. (1982) J. Biol. Chem 257, 11901-11907. 8. SEAMON, K., AND DALY, J. W. (1981) J. Biol. Chem 256, 9799-9801. 9. Ross, E. M. (1982) J. Bid Chem. 257,10751-1058. 10. PFEUFFER, T., AND METZGER, H. (1982) FEBSLett 146,369-375. 11. FORTE, L. R., NICKOLS, G. A., AND ANAST, C. S. (1976) J. Clin Invest. 57, 559-568. 12. CARNES, D. L., ANAST, C. S., AND FORTE, L. R. (1980) Biochim Biophys. Acta 629, 546-552. 13. CARNES, D. L., NICKOLS, G. A., ANAST, C. S., AND FORTE, L. R. (1980) Amer. J. Physiid 239, E396E400. 14. KAKUTA, S., SATO, C., SUDA, T., KIMURA, N., AAKI, N., ONO, Y., AND NAGATA, N. (1978) B&him. Biophys. Acta 539, 173-180. 15. FORTE, L. R., LANGELU~IG, S., POELLING, R. E., AND THOMAS, M. L. (1982) Amer. J. PhysioL 242, E154-E163. 16. STEINER, A. L., KIPNIS, D. M., UTIGER, R., AND PARKER, C. (1969) Proc. Natl. Acad Sci. USA 64, 367-373. 17. HARPER, J. F., AND BROOKER, G. (1975) J. Cyclic Nut. Res. 1. 207-218.
CYCLASE
BY FORSKOLIN
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18. FITZPATRICK, D. F., DAVENPORT, G. R., FORTE, L. R., AND LANDON, E. J. (1969) J. BioL Chew. 244, 3561-3569. 19. NISSENSON, R. A., AND ARNAUD, C. D. (1979) J. Biol Chem. 254,1469-1475. 20. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem, 193, 265-275. 21. DALY, J. W., PADGE~, W., AND SEAMON, K. B. (1982) J. Neurochem 38, 532-544. 22. BURNS, T. W., LANGLEY, P. E., TERRY, B. E., BYLUND, D. B., AND FORTE, L. R. (1982) Life Sci. 31, 815-821. 23. LIMBIRD, L. E. (1981) Biochem J. 185, 1-13. 24. SIEGL, A. M., DALY, J. W., AND SMITH, J. B. (1982) Mol. Phamw.col. 21, 680-687. 25. FORTE, L. R., BOUND, D. B., AND ZAHLER, W. L. (1983) Mel Pharmmol. 24,42-47. 26. HERMAN, C. A., ZAHLER, W. L., DOAKS, G. A., AND CAMPBELL, B. J. (1976) Arch Biochem Biophys. 177, 622-629. 27. BRAUN, T., AND DODS, R. F. (1975) Proc Natl. Ad Sci. USA 72, 1097-1101. 28. GARBERS,D. L., AND KOPF, G. S. (1980) in Advances in Cyclic Nucleotide Research, Vol. 13, pp. 251306, Raven Press, New York. 29. LITOSCH, I., HUDSON, T. H., MILLS, I., LI, S.-Y., AND FAIN, J. N. (1982) Mel Pkmmmxd 22,109115. 30. MORIWAKI, K., ITOH, Y., IIDA, S., AND ICHIHARA, K. (1982) Lz$e Sci 30, 2235-2240. 31. ARMBRECHT, H. J., FORTE, L. R., WONGSURAWAT, N., ZENSER, T. V. AND DAVIS, B. B. (1983) Clin Res. 31, 380A. 32. MARTZ, A. AND THOMAS, M. L. (1983) Biochem Phmmnd, in press.