The Last Conversation With Dr. Earl W. Sutherland, Jr.: The Feedback Regulation of Cyclic Nucleotides Ren-Jye Ho, Thomas
Russell, and Takeo Asakawa*
I
T WAS February of 1974. Dr. Sutherland had not been well for weeks, he was considering taking a sick leave, yet science was always turning him on, and we still had frequent scientific discussions either at the Keys or at his Coral Gables home or by telephone. I (RJH) spoke to him on February 25, 1974; our conversation focused on a few manuscripts on feedback regulation of cyclic nucleotides. It was the last conversation between us and was one of many brief influential talks by Earl in his last days. He was hospitalized 2 days later and died on March 9, 1974. From the day of the relocation of his research team to his death, we had been at the University of Miami exactly 6 months and 9 days. Within this short half-year, under his dynamic leadership, our research had progressed at a rapid pace. It was my good fortune to have such an excellent teacher and friend in my life. He had acquired recognition for his discovery of CAMP and its role in hormone action. He was even happier just before his death with the results which suggested the possible mechanism of lowering the stimulated adenylate cyclase activity: a feedback regulation process. We are writing this brief discussion in memory of Dr. Sutherland’s great and consistent interest in cyclic nucleotide research. It is a review as well as a preview of some of the results observed in this laboratory. Cellular CAMP Levels
One of the characteristics of CAMP-mediated hormone action is the elevation of CAMP levels in the target cells, which preceeds the hormone response.’ It is well established that CAMP mediates the action of lipolytic hormones in adipocytes. The quantitative difference between the hormone-stimulated rise of CAMP levels in incubated adipose tissue and isolated adipocytes has been recognized; however, this difference has not yet been explained. In isolated incubated rat epididymal fat pads a twofold stimulation of CAMP levels was observed when the tissue was incubated in the presence of 1 mM caffeine and 23 PM epinephrine.2 In the rat adipocytes, however, a 50-lOO-fold increase in this cyclic nucleotide was found in the presence of 0.6 PM epinephrine and 1 mM caffeine at 3 min (Fig. 1). This 3-min reaction time is the peak time of CAMP levels in adipocytes observed in vitro. After this peak, the CAMP level decreases precipitously in spite of the continuous presence of caffeine and *T. Asakawa
is a Career Investigator
Fellow of the American
Heart Association.
From the E. W. Sutherland Research Laboratories. Department of Biochemistry. University of Miami School of Medicine. Miami. Fla. Received for publication October I I, 1974. Reprint requests should be addressed to Ren-Jye Ho, E. W. Sutherland Research Laboratories, Biochemistry, UMED. P.O. Box 520875, Miami, Fla. 33152. o 1975 by Grune & Strarlon. Inc. Mefabolism, Vol. 24, No. 3 (March), 1975
257
258
HO, RUSSELL,AND ASAKAWA
EPINEPHRINE CAFFEINE
30 z \ z g
20
I c 3
10
0
0
z
0
9
18
27
INCUBATION
36
45
TIME (MINI
54
63
Fig. 1. CAMP levels following single and repeated exposure to caffeine and epinephrine. Adipocytesof epididymal ddipose tissue of rat were incuba+ in a KrebsRinger bicarbonate-albumin medium with epinephrine (0.6 pM) and caffeine (1 mm) for O-63 min at 37-C. Arrows indicate times of additional stimulations by epinephrine and caffeine. CAMP levels am expressed as nmoles/g of adipocyte lipid. (Ho and Sutherland, unpublished observations.)
the elevation and declination of epinephrine.3 In adipose tissue, however, CAMP levels following hormone stimulation were not rapid. This is probably due to the necessity for diffusion of the hormone from the periphery of the tissue to the deep-seated cells. Therefore, the onset of hormone action corresponded to the hormone diffusion rate, and, presumably, the biphasic time course for the whole tissue is an average of a series of many biphasic curves with different onset times and peak times. The CAMP peak and after-peak drop were absent, a characteristic of the incubated tissue. In the perfused adipose tissue this diffusion phenomenon does not take place due to the rapid distribution of perfusion medium through the vascular beds. During the period of the after-peak decrease, the cells were unresponsive to a second or third addition of both caffeine and epinephrine (Fig. 1). This unresponsiveness of the adipocytes was not the result of (1) a metabolic inhibitor derived from hormone degradation or (2) an increase in phosphodiesterase activity, since there was no response to multiple additions of caffeine (and epinephrine). Such unresponsiveness could last for 63 min of incubation. The increase and decrease in CAMP levels in the incubation system is no doubt due to a shift in the rates of synthesis and degradation of CAMP. The net decrease in CAMP levels during the after-peak decrease takes place intracellularly. The differential decrease to added ‘H-CAMP and CAMP in the incubation medium indicated that the degradation of CAMP takes place in a compartment other than the extracellular medium.3 If an increase in phosphodiesterase activity could not account for the net decrease in CAMP levels, then either the synthesis of CAMP was not at a constant rate during hormone action, or other unknown mechanisms to hydrolyze the CAMP were activated. Evidence supports the adenylate cyclase theory. The responsiveness of adipocytes to hormones was recovered when the incubation medium was replaced by fresh buffer. When insensitive cells were washed five times, each with 50 ml of fresh incubation medium, the response was completely recovered.3
FEEDBACK
REGULATION
OF CYCLIC NUCLEOTIDES
259
The restoration of hormone sensitivity of adipocytes by exchanging the incubation medium suggested the existence of an inhibitor in the incubation medium. Addition of medium from incubations of cells with hormone to incubations containing fresh cells diminished the hormone-stimulated rise of CAMP. A factor was isolated from the incubation medium of adipocytes of rats following treatment with epinephrine and caffeine. 3 This factor essentially retained the inhibitory activity. The unresponsiveness of adipocytes was not only initiated by lipolytic hormones but was also initiated by phosphodiesterase inhibitors such as caffeine. The formation and release of feedback regulator (FR) was stimulated either by adenylate cyclase activators,3 phosphodiesterase inhibitors,4 or CAMP.’ The formation of this factor appeared to be a CAMPmediated phenomenon. The formation of FR stimulated by CAMP is strong evidence for the role of CAMP in FR formation. Precautions should be taken in order to demonstrate this phenomenon without assay difficulties. When CAMP or dibutyryl CAMP are used as stimulators for the formation of FR, the partially purified FR will be contaminated with cyclic nucleotide, and the degree of contamination could upset the determination for CAMP in the assay system. It is therefore advisable to use 14C-adenine-labeled adipocytes as a tool to counter this problem. 14C-CAMP formed in the assay system can be easily determined by measuring the radioactivity in the isolated CAMP samples. This system is convenient and also sensitive, and it is especially useful for this purpose.’ Release of the FR and Its Relationship to CAMP Levels The formation and subsequent release of FR from adipocytes following hormone stimulation is time dependent. The elevation of CAMP levels preceeds the release of FR, and the release of FR is best recognized at the period of postpeak decrease of CAMP, during which time the unresponsiveness of adipocytes to further stimulation of hormone was observed.3 When adipocytes from hamster, rabbit, or human are used, a longer duration of the biphasic curve was observed following a specific hormone stimulation. In these cases, the formation and release of FR was also observed at later time periods, correlating with the later peak CAMP levels.5 In all cases the intracellular FR content appeared earlier than its release into the incubation medium. FR was formed or activated during hormone action, presumably in response to an increased level of CAMP. The discovery of FR in the circulating blood6 suggested that FR may be formed in and released from tissues other than adipocytes. The site of degradation or inactivation of FR is not yet clear. FR may be inactivated in the same tissue in which it was formed. Unpublished observations of degradation or inactivation of FR by adipose tissue cells are consistent with the requirements of FR as a biological regulator. The formation and release of FR by incubated adipose tissue was also different from that of adipocytes (Ho and Sutherland, unpublished observation). The degree of ease with which FR is released from the more deeply seated cells to the incubation medium may result in an important difference in this respect. The recent report of Schimmel’ should be reexamined along these lines.
HO, RUSSELL,
260
L
I
T
_L
FR -
-
COWT Inhibition of Adenylate
EPI
ACTH
AND ASAKAWA
6LucAcow
Fig. 2. Effect of epinephrincstimulated feedback regulator on the CAMP levels in adipocytes elc epinophrine, voted by ACTH, or glucagon.3
Cyclase as a Negative Feedback E#ect of FR
FR is formed during hormone activity and as a result of an increased CAMP level. The formation of FR caused the unresponsiveness of adipocytes to further hormone stimulation. The relationship is due to a decreased maximum stimulation of adenylate cyclase activity, i.e., adenylate cyclase is in an inhibited state due to the action of endogenous FR. The biphasic time course of CAMP levels during the action of lipolytic hormone is therefore understandable. Using partially purified FR, an inhibition of hormone-stimulated rise of CAMP and of hormone-sensitive adenylate cyclase of plasma membrane of rat adipocytes was demonstrated. (Figs, 2 and 3). It was found that the inhibitory action of FR was not hormone specific; it seems to be at the site beyond the hormone-specific receptors. Also, the inhibitory action is reversible. It lowered the V,,, of adenylate cyclase without effecting the apparent K, for ATP or K, for epinephrine.8 The inhibitory action of FR was partially overcome by in(Fig. 4) and seems to require the transferable creased Mg2+ concentrations phosphate group of ATP at the y--position, since the action of FR was greater when ATP rather than APP(NH)P was used as a substrate. Furthermore, FR stimulated the self-phosphorylation of plasma membrane of rat adipocytes.’ The link between phosphorylation of plasma membrane and inhibition of adenylate cyclase remains to be studied. Stimulation of Protein Kinase and Phosphorylase Eflect of FR
b Kinase as a Positive Forward
FR stimulated rabbit or bovine skeletal CAMP-dependent protein kinase activity. The phosphorylation of substrate histone was increased either with or without the addition of CAMP (Fig. 5). It increased the V,, without effecting the apparent K, for CAMP. It is therefore believed that the FR effect is independent of CAMP. FR increased phosphorylation of histone when the catalytic subunit was used.9 In addition to protein kinase, FR also stimulated the phosphorylation of
FEEDBACK
REGULATION
OF CYCLIC NUCLEOTIDES
Fig. 3. Effect of feedback regulator on the stimulation of phosphorylase b kinose and inhibition of adenylate cyclase. The phorphorylase kinase of rabbit skeletal muscle was incubated in a system containing 30 mM potassium phosphate buffer, pH 6.8, 7 mM Mg’+, 2 mM ATP, approximately 1 x 10’ cpm ATP-y-‘*P, and varying amounts of partially purified FR.” The volume was 70 pl. The reaction was started with the kinase and incubated at 30-C for 15 min. The rwction was stopped, and phosphorylated protein was measured. The results are expressed as fold stimulation. The activity assayed in the absence of FR was onefold. Each data point was a mean of three values. Adenylate cyclase assay curve (solid line) is also shown for the purpose of comparison. The adenylate cyclase of adipocyte plasma membrane was measured with varied amounts of FR. The incubation system consisted of
0
I FEEDBACK
3
2
UNIT
REGULATOR.
2 mM ATP, 4 mM Mgx+, 6 mM theophylline, 25 JL~ epinephrine, 50 mM glycyl glycine, pH 7.4, and 28 pg membrane protein, with or without FR. The final volume was 500 pl. The incubation was carried out at 30-C for 15 min. The rwction was stopPed by the addition of perchloric acid to a Anal concentration of 0.3 M. Results are expressed as percent inhibition which was calculated as follows: [(rate of CAMP formation, without FR - mte of CAMP formation, with FR) x (rate of CAMP formation, without FR)-‘1 x 100. The unit of FR was daRned as that amount of FR which caused a 50% decrease in hormone-stimulated adenylate cyclase activity in this assay system. Each data point is a mean of four incubations.’
1 0.5
v
0.4
Fi
4. Effect of concentrations on the inhibitory action of feedback regulator on adenylate cyclase activity. The incubation system was the same as that described in Fig. 3 except that ,,a+ concentmtions werr, varied. Each value was a mean of four incubations. Figure 4 is a lineweaver-Burk plot, l/S = mM_‘. (Ho and Sutherland, artitle in preparation.)
4
i
.
HO,
RUSSELL,
AND
ASAKAWA
r
/ / 1’
/
0
0
1
l % /
0 0
0
2
FEEDBACK REGULATOR,
3 UNIT
‘+
CAMP
Fig. 5. Effect of feedback regulator on CAMP-dependent protein kinase activity. CAMPdependent protein kinase from rabbit skeletal muscle was assayed with varying amounts of partially purified FR” in the resence and absence of B IO- M CAMP. Results were plotted as percent stimulation versus the unit of FR added. Percent stimulation was calculated as followr: [(J”‘P incorpomted, with FR - ‘P incur 8?without FR) x ( P mted, incorporated, without FR)-’ x 100. Solid curve, -CAMP; dash curve, +cAMP, 10e6M. Each point is the mean of two observed values. (Ho, Sodarling, and Sutherland, article in preparation.)
partially purified phosphorylase b kinase (Fig. 3).’ We use these properties of FR as an assay method for estimation of FR content in fractions eluted from chromatographic columns. Using a combination of several properties of FR, the separation of FR from other adenylate cyclase inhibitors, such as adenosinerOv” and long-chain acyl CAMP,” or CAMP-lowering agents, such as PGE,,‘3*‘4 is becoming less tedious. We have found that FR does not have maximal absorbtion at 260 nM, and PGEr does not inhibit adipocytes plasma membrane adenylate cyclase; also, adenosine does not stimulate protein kinase. A personal communication by Dr. John Fain advises that adenosine has little effect on protein kinase activity of fat-cell homogenates or on adenylate cyclase at concentrations below 5 PM.
Inhibition of PDE Activity by FR as a “Bufering” E$ect on CAMP Levels It was thought that a stimulation of CAMP PDE activity by FR might be one of the causes of the postpeak decrease of CAMP levels in adipocytes. This thought was worthwhile to test, especially following the report by Zinman et al.,15 Manganiello et al.,16 and D’Armiento et al.,” that CAMP-elevating hormones or agents or CAMP alone can cause an increase in PDE activity. In the report by Zinman et al.,u’ epinephrine could cause an increase in PDE in adipocytes by 157&20%. This effect of FR on PDE was therefore tested and reported. ” PDE from adipocyte plasma membrane, as well as PDE from 10,OOOg supernatant of rat epididymal adipose tissue, was inhibited by FR. FR appeared to increase the apparent K, for CAMP and decrease the V,,,. It inhibited supernatant PDE more than PDE from plasma membrane. The concentration of FR required for 50% inhibition of membrane-bound PDE was approximately five to ten times higher than that for membrane-bound adenylate cyclase activity. The inhibition of CAMP synthesis by FR is in concert with its inhibition on PDE activity. This is consistent with the thought that a reinforcement of the hormone action by FR is through stabilization of cellular CAMP
FEEDBACK REGULATION OF CYCLIC NUCLEOTIDES
263
PHYSIOLOGICAL RESPONSE - - -*
INACTIVE
Fig. 6. A working hypothesis concoming FR. (Ho and Sutherland, unpublished mults.)
FR
levels. This effect of FR is apparently different from PDE induction by CAMP reported by others.i6*” FR and Intracellular Homeostasis
At maximum stimulation by a lipolytic hormone, the rate of CAMP synthesis from ATP is very high. Although the exact rate is not measurable because of the action of phosphodiesterase activity, the accumulated CAMP content could be estimated. The CAMP level 3 min after maximum stimulation could be as high as 50 nmoles/g of tissue. This means that the utilization of ATP energy was at a great rate. The initial spike of CAMP following hormone stimulation may be interpreted as a means of obtaining a rapid activation of the physiological response. One of the actions of CAMP-mediated FR may be to conserve ATP energy by inhibiting further adenylate cyclase action. It is a negative feedback controlling system. At a later time, as FR continuously increases to a relatively high level, FR starts to inhibit PDE activity to prevent CAMP levels from dropping too fast. This action of FR on inhibition of phosphodiesterase may serve as a regulatory effort to maintain or buffer the CAMP level to prevent it from rapid decrease. The stimulatory effect on protein kinase is not yet entirely understood. This action of FR may serve as a positive forward effect to potentiate CAMP action. Figure 6 represents a working hypothesis for the biological role of FR. It is based on the existing hypothesis of Sutherland et al. The consideration of FR in this diagram is self explanatory. The role of FR on guanylate cyclase and cGMP levels in a target cell is also of interest to us. The effect of FR on elevation of cGMP levels in isolated adipocytes has been demonstrated (Asakawa, T. et al. Fed. Proc. 1975). ACKNOWLEDGMENT The authors wish to thank Dr. Claudia Sutherland (Mrs. E.W.S.) for her continuing interest and support of this work before and after the death of E.W.S. We also wish to thank Messrs. Richard Snyder, Michael Hucks, and Juan Ruiz, and Mrs. Ilene Scheinbaum for their excellent
264
HO, RUSSELL,
AND ASAKAWA
technical assistance.. A special thanks should be given to Ms. Barbara Mozayeny, who is last graduate student, for her interest and enthusiasm in scientific research. We thank Dr. William J. Whelan, the American Heart Association, the NIH Heart and Institute, and the National Science Foundation for their continuous support. Finally, we Earl’s’ friends and former associates for their warm encouragements. We thank everyone tioned above: they are essential for the survival of this team and for keeping the group ative and productive.
Earl’s Lung thank menoper-
REFERENCES 1. Robison GA, Butcher RW, Sutherland EW: Cyclic AMP. New York, Academic Press, 1971, pp 17-46 2. Butcher RW, Ho RJ, Meng HC, Sutherland EW: Adenosine 3’,5’-monophosphate in biological materials. II. The measurement of adenosine 3’,5’-monophosphate in tissues and the role of cyclic nucleotides in the lipolytic response of fat to epinephrine. J Biol Chem 240:4515-4523, 1965 3. Ho RJ, Sutherland EW: Formation and release of a hormone antagonist by rat adipocytes. J Biol Chem 2466822-6827, 1971 4. Ho RJ, Sutherland EW: Cyclic AMP mediated feedback regulator in target cells, in Advances in Cyclic Nucleotide Research, vol. 5. New York, Raven Press, (in press) 5. Ho RJ, Bomboy JD, Wasner HK, Sutherland EW: Elevation of CAMP levels and subsequent formation of antihormone activity in adipocytes of rat and hamster. Fed Proc 32: 535,1973 6. Ho RJ, Sutherland EW: Inhibition of adenylate cyclase by a factor isolated from human plasma. Fed Proc 33:480, 1974 7. Schimmel RJ: Responses of adipose tissue to sequential lipolytic stimuli. Endocrinology 94: 1372- 1380, 1974 8. Ho RJ, Sutherland EW: The action of regulator on adenylate cyclase. (in preparation) 9. Ho RJ, Soderling T, Sutherland EW: Stimulatory and inhibitory actions of the feedback regulator from adipocytes. (in preparation) 10. Fain JN, Pointer RH, Ward WF: Effects of adenosine nucleotides on adenylate cyclase, phosphodiesterase, cyclic adenosine accumulation, and lipolysis. J Biol Chem 247:6866-6872, 1972 11. Ebert R, Schwabe U: Studies on the
antilipolytic effect of adenosine and related compounds in isolated fat cells. Naunyn Schmiedebergs Arch Pharmakol 278:247-259, 1973 12. Weinryb I, Michel IM, Hess SM: Adenylate cyclase from Guinea pig lung: Further characterization and inhibitory effect of substrate analogs and cyclic nucleotides. Arch Biochem Biophys 154:2&I-249, 1973 13. Butcher RW, Baird CE: Effect of prostaglandins on adenosine 3’,5’-monophosphate levels in fat and other tissues. J Biol Chem 243:1713-1717, 1968 14. Shaw JZ, Ramwell PW: Release of prostaglandin from rat epididymal fat pad on nervous and hormone stimulation. J Biol Chem 243:1498-1503, 1968 15. Zinman B, Hollenberg CH: Effect of insulin and lipolytic agents on rat adipocyte low K, cyclic adenosine 3’,5’-monophosphate phosphodiesterase. J Biol Chem 249~2182-2187, 1974 16. Manganiello V, Vaughan M: Prostaglandin Et effects on CAMP concentration and phosphodiesterase activity in fibroblasts. Proc Nat1 Acad Sci USA 69269-273, 1972 17. D’Armiento M, Johnson GS, Pastan I: Regulation of CAMP phosphodiesterase activity in fibroblasts by intracellular concentrations of CAMP. Proc Nat1 Acad Sci USA 69:459462, 1972 18. Ho RJ: Effect of feedback regulator on CAMP phosphodiesterase activity. Physiologist 17:248, 1974 19. Ho RJ, Bomboy JD, Wasner HK, Sutherland EW: Preparation and characterization of a hormone antagonist from adipocytes. Methods in Enzymology, vol. 37. New York, Academic Press (in press)
Russell, and Takeo Asakawa*
I
T WAS February of 1974. Dr. Sutherland had not been well for weeks, he was considering taking a sick leave, yet science was always turning him on, and we still had frequent scientific discussions either at the Keys or at his Coral Gables home or by telephone. I (RJH) spoke to him on February 25, 1974; our conversation focused on a few manuscripts on feedback regulation of cyclic nucleotides. It was the last conversation between us and was one of many brief influential talks by Earl in his last days. He was hospitalized 2 days later and died on March 9, 1974. From the day of the relocation of his research team to his death, we had been at the University of Miami exactly 6 months and 9 days. Within this short half-year, under his dynamic leadership, our research had progressed at a rapid pace. It was my good fortune to have such an excellent teacher and friend in my life. He had acquired recognition for his discovery of CAMP and its role in hormone action. He was even happier just before his death with the results which suggested the possible mechanism of lowering the stimulated adenylate cyclase activity: a feedback regulation process. We are writing this brief discussion in memory of Dr. Sutherland’s great and consistent interest in cyclic nucleotide research. It is a review as well as a preview of some of the results observed in this laboratory. Cellular CAMP Levels
One of the characteristics of CAMP-mediated hormone action is the elevation of CAMP levels in the target cells, which preceeds the hormone response.’ It is well established that CAMP mediates the action of lipolytic hormones in adipocytes. The quantitative difference between the hormone-stimulated rise of CAMP levels in incubated adipose tissue and isolated adipocytes has been recognized; however, this difference has not yet been explained. In isolated incubated rat epididymal fat pads a twofold stimulation of CAMP levels was observed when the tissue was incubated in the presence of 1 mM caffeine and 23 PM epinephrine.2 In the rat adipocytes, however, a 50-lOO-fold increase in this cyclic nucleotide was found in the presence of 0.6 PM epinephrine and 1 mM caffeine at 3 min (Fig. 1). This 3-min reaction time is the peak time of CAMP levels in adipocytes observed in vitro. After this peak, the CAMP level decreases precipitously in spite of the continuous presence of caffeine and *T. Asakawa
is a Career Investigator
Fellow of the American
Heart Association.
From the E. W. Sutherland Research Laboratories. Department of Biochemistry. University of Miami School of Medicine. Miami. Fla. Received for publication October I I, 1974. Reprint requests should be addressed to Ren-Jye Ho, E. W. Sutherland Research Laboratories, Biochemistry, UMED. P.O. Box 520875, Miami, Fla. 33152. o 1975 by Grune & Strarlon. Inc. Mefabolism, Vol. 24, No. 3 (March), 1975
257
258
HO, RUSSELL,AND ASAKAWA
EPINEPHRINE CAFFEINE
30 z \ z g
20
I c 3
10
0
0
z
0
9
18
27
INCUBATION
36
45
TIME (MINI
54
63
Fig. 1. CAMP levels following single and repeated exposure to caffeine and epinephrine. Adipocytesof epididymal ddipose tissue of rat were incuba+ in a KrebsRinger bicarbonate-albumin medium with epinephrine (0.6 pM) and caffeine (1 mm) for O-63 min at 37-C. Arrows indicate times of additional stimulations by epinephrine and caffeine. CAMP levels am expressed as nmoles/g of adipocyte lipid. (Ho and Sutherland, unpublished observations.)
the elevation and declination of epinephrine.3 In adipose tissue, however, CAMP levels following hormone stimulation were not rapid. This is probably due to the necessity for diffusion of the hormone from the periphery of the tissue to the deep-seated cells. Therefore, the onset of hormone action corresponded to the hormone diffusion rate, and, presumably, the biphasic time course for the whole tissue is an average of a series of many biphasic curves with different onset times and peak times. The CAMP peak and after-peak drop were absent, a characteristic of the incubated tissue. In the perfused adipose tissue this diffusion phenomenon does not take place due to the rapid distribution of perfusion medium through the vascular beds. During the period of the after-peak decrease, the cells were unresponsive to a second or third addition of both caffeine and epinephrine (Fig. 1). This unresponsiveness of the adipocytes was not the result of (1) a metabolic inhibitor derived from hormone degradation or (2) an increase in phosphodiesterase activity, since there was no response to multiple additions of caffeine (and epinephrine). Such unresponsiveness could last for 63 min of incubation. The increase and decrease in CAMP levels in the incubation system is no doubt due to a shift in the rates of synthesis and degradation of CAMP. The net decrease in CAMP levels during the after-peak decrease takes place intracellularly. The differential decrease to added ‘H-CAMP and CAMP in the incubation medium indicated that the degradation of CAMP takes place in a compartment other than the extracellular medium.3 If an increase in phosphodiesterase activity could not account for the net decrease in CAMP levels, then either the synthesis of CAMP was not at a constant rate during hormone action, or other unknown mechanisms to hydrolyze the CAMP were activated. Evidence supports the adenylate cyclase theory. The responsiveness of adipocytes to hormones was recovered when the incubation medium was replaced by fresh buffer. When insensitive cells were washed five times, each with 50 ml of fresh incubation medium, the response was completely recovered.3
FEEDBACK
REGULATION
OF CYCLIC NUCLEOTIDES
259
The restoration of hormone sensitivity of adipocytes by exchanging the incubation medium suggested the existence of an inhibitor in the incubation medium. Addition of medium from incubations of cells with hormone to incubations containing fresh cells diminished the hormone-stimulated rise of CAMP. A factor was isolated from the incubation medium of adipocytes of rats following treatment with epinephrine and caffeine. 3 This factor essentially retained the inhibitory activity. The unresponsiveness of adipocytes was not only initiated by lipolytic hormones but was also initiated by phosphodiesterase inhibitors such as caffeine. The formation and release of feedback regulator (FR) was stimulated either by adenylate cyclase activators,3 phosphodiesterase inhibitors,4 or CAMP.’ The formation of this factor appeared to be a CAMPmediated phenomenon. The formation of FR stimulated by CAMP is strong evidence for the role of CAMP in FR formation. Precautions should be taken in order to demonstrate this phenomenon without assay difficulties. When CAMP or dibutyryl CAMP are used as stimulators for the formation of FR, the partially purified FR will be contaminated with cyclic nucleotide, and the degree of contamination could upset the determination for CAMP in the assay system. It is therefore advisable to use 14C-adenine-labeled adipocytes as a tool to counter this problem. 14C-CAMP formed in the assay system can be easily determined by measuring the radioactivity in the isolated CAMP samples. This system is convenient and also sensitive, and it is especially useful for this purpose.’ Release of the FR and Its Relationship to CAMP Levels The formation and subsequent release of FR from adipocytes following hormone stimulation is time dependent. The elevation of CAMP levels preceeds the release of FR, and the release of FR is best recognized at the period of postpeak decrease of CAMP, during which time the unresponsiveness of adipocytes to further stimulation of hormone was observed.3 When adipocytes from hamster, rabbit, or human are used, a longer duration of the biphasic curve was observed following a specific hormone stimulation. In these cases, the formation and release of FR was also observed at later time periods, correlating with the later peak CAMP levels.5 In all cases the intracellular FR content appeared earlier than its release into the incubation medium. FR was formed or activated during hormone action, presumably in response to an increased level of CAMP. The discovery of FR in the circulating blood6 suggested that FR may be formed in and released from tissues other than adipocytes. The site of degradation or inactivation of FR is not yet clear. FR may be inactivated in the same tissue in which it was formed. Unpublished observations of degradation or inactivation of FR by adipose tissue cells are consistent with the requirements of FR as a biological regulator. The formation and release of FR by incubated adipose tissue was also different from that of adipocytes (Ho and Sutherland, unpublished observation). The degree of ease with which FR is released from the more deeply seated cells to the incubation medium may result in an important difference in this respect. The recent report of Schimmel’ should be reexamined along these lines.
HO, RUSSELL,
260
L
I
T
_L
FR -
-
COWT Inhibition of Adenylate
EPI
ACTH
AND ASAKAWA
6LucAcow
Fig. 2. Effect of epinephrincstimulated feedback regulator on the CAMP levels in adipocytes elc epinophrine, voted by ACTH, or glucagon.3
Cyclase as a Negative Feedback E#ect of FR
FR is formed during hormone activity and as a result of an increased CAMP level. The formation of FR caused the unresponsiveness of adipocytes to further hormone stimulation. The relationship is due to a decreased maximum stimulation of adenylate cyclase activity, i.e., adenylate cyclase is in an inhibited state due to the action of endogenous FR. The biphasic time course of CAMP levels during the action of lipolytic hormone is therefore understandable. Using partially purified FR, an inhibition of hormone-stimulated rise of CAMP and of hormone-sensitive adenylate cyclase of plasma membrane of rat adipocytes was demonstrated. (Figs, 2 and 3). It was found that the inhibitory action of FR was not hormone specific; it seems to be at the site beyond the hormone-specific receptors. Also, the inhibitory action is reversible. It lowered the V,,, of adenylate cyclase without effecting the apparent K, for ATP or K, for epinephrine.8 The inhibitory action of FR was partially overcome by in(Fig. 4) and seems to require the transferable creased Mg2+ concentrations phosphate group of ATP at the y--position, since the action of FR was greater when ATP rather than APP(NH)P was used as a substrate. Furthermore, FR stimulated the self-phosphorylation of plasma membrane of rat adipocytes.’ The link between phosphorylation of plasma membrane and inhibition of adenylate cyclase remains to be studied. Stimulation of Protein Kinase and Phosphorylase Eflect of FR
b Kinase as a Positive Forward
FR stimulated rabbit or bovine skeletal CAMP-dependent protein kinase activity. The phosphorylation of substrate histone was increased either with or without the addition of CAMP (Fig. 5). It increased the V,, without effecting the apparent K, for CAMP. It is therefore believed that the FR effect is independent of CAMP. FR increased phosphorylation of histone when the catalytic subunit was used.9 In addition to protein kinase, FR also stimulated the phosphorylation of
FEEDBACK
REGULATION
OF CYCLIC NUCLEOTIDES
Fig. 3. Effect of feedback regulator on the stimulation of phosphorylase b kinose and inhibition of adenylate cyclase. The phorphorylase kinase of rabbit skeletal muscle was incubated in a system containing 30 mM potassium phosphate buffer, pH 6.8, 7 mM Mg’+, 2 mM ATP, approximately 1 x 10’ cpm ATP-y-‘*P, and varying amounts of partially purified FR.” The volume was 70 pl. The reaction was started with the kinase and incubated at 30-C for 15 min. The rwction was stopped, and phosphorylated protein was measured. The results are expressed as fold stimulation. The activity assayed in the absence of FR was onefold. Each data point was a mean of three values. Adenylate cyclase assay curve (solid line) is also shown for the purpose of comparison. The adenylate cyclase of adipocyte plasma membrane was measured with varied amounts of FR. The incubation system consisted of
0
I FEEDBACK
3
2
UNIT
REGULATOR.
2 mM ATP, 4 mM Mgx+, 6 mM theophylline, 25 JL~ epinephrine, 50 mM glycyl glycine, pH 7.4, and 28 pg membrane protein, with or without FR. The final volume was 500 pl. The incubation was carried out at 30-C for 15 min. The rwction was stopPed by the addition of perchloric acid to a Anal concentration of 0.3 M. Results are expressed as percent inhibition which was calculated as follows: [(rate of CAMP formation, without FR - mte of CAMP formation, with FR) x (rate of CAMP formation, without FR)-‘1 x 100. The unit of FR was daRned as that amount of FR which caused a 50% decrease in hormone-stimulated adenylate cyclase activity in this assay system. Each data point is a mean of four incubations.’
1 0.5
v
0.4
Fi
4. Effect of concentrations on the inhibitory action of feedback regulator on adenylate cyclase activity. The incubation system was the same as that described in Fig. 3 except that ,,a+ concentmtions werr, varied. Each value was a mean of four incubations. Figure 4 is a lineweaver-Burk plot, l/S = mM_‘. (Ho and Sutherland, artitle in preparation.)
4
i
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/
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0
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FEEDBACK REGULATOR,
3 UNIT
‘+
CAMP
Fig. 5. Effect of feedback regulator on CAMP-dependent protein kinase activity. CAMPdependent protein kinase from rabbit skeletal muscle was assayed with varying amounts of partially purified FR” in the resence and absence of B IO- M CAMP. Results were plotted as percent stimulation versus the unit of FR added. Percent stimulation was calculated as followr: [(J”‘P incorpomted, with FR - ‘P incur 8?without FR) x ( P mted, incorporated, without FR)-’ x 100. Solid curve, -CAMP; dash curve, +cAMP, 10e6M. Each point is the mean of two observed values. (Ho, Sodarling, and Sutherland, article in preparation.)
partially purified phosphorylase b kinase (Fig. 3).’ We use these properties of FR as an assay method for estimation of FR content in fractions eluted from chromatographic columns. Using a combination of several properties of FR, the separation of FR from other adenylate cyclase inhibitors, such as adenosinerOv” and long-chain acyl CAMP,” or CAMP-lowering agents, such as PGE,,‘3*‘4 is becoming less tedious. We have found that FR does not have maximal absorbtion at 260 nM, and PGEr does not inhibit adipocytes plasma membrane adenylate cyclase; also, adenosine does not stimulate protein kinase. A personal communication by Dr. John Fain advises that adenosine has little effect on protein kinase activity of fat-cell homogenates or on adenylate cyclase at concentrations below 5 PM.
Inhibition of PDE Activity by FR as a “Bufering” E$ect on CAMP Levels It was thought that a stimulation of CAMP PDE activity by FR might be one of the causes of the postpeak decrease of CAMP levels in adipocytes. This thought was worthwhile to test, especially following the report by Zinman et al.,15 Manganiello et al.,16 and D’Armiento et al.,” that CAMP-elevating hormones or agents or CAMP alone can cause an increase in PDE activity. In the report by Zinman et al.,u’ epinephrine could cause an increase in PDE in adipocytes by 157&20%. This effect of FR on PDE was therefore tested and reported. ” PDE from adipocyte plasma membrane, as well as PDE from 10,OOOg supernatant of rat epididymal adipose tissue, was inhibited by FR. FR appeared to increase the apparent K, for CAMP and decrease the V,,,. It inhibited supernatant PDE more than PDE from plasma membrane. The concentration of FR required for 50% inhibition of membrane-bound PDE was approximately five to ten times higher than that for membrane-bound adenylate cyclase activity. The inhibition of CAMP synthesis by FR is in concert with its inhibition on PDE activity. This is consistent with the thought that a reinforcement of the hormone action by FR is through stabilization of cellular CAMP
FEEDBACK REGULATION OF CYCLIC NUCLEOTIDES
263
PHYSIOLOGICAL RESPONSE - - -*
INACTIVE
Fig. 6. A working hypothesis concoming FR. (Ho and Sutherland, unpublished mults.)
FR
levels. This effect of FR is apparently different from PDE induction by CAMP reported by others.i6*” FR and Intracellular Homeostasis
At maximum stimulation by a lipolytic hormone, the rate of CAMP synthesis from ATP is very high. Although the exact rate is not measurable because of the action of phosphodiesterase activity, the accumulated CAMP content could be estimated. The CAMP level 3 min after maximum stimulation could be as high as 50 nmoles/g of tissue. This means that the utilization of ATP energy was at a great rate. The initial spike of CAMP following hormone stimulation may be interpreted as a means of obtaining a rapid activation of the physiological response. One of the actions of CAMP-mediated FR may be to conserve ATP energy by inhibiting further adenylate cyclase action. It is a negative feedback controlling system. At a later time, as FR continuously increases to a relatively high level, FR starts to inhibit PDE activity to prevent CAMP levels from dropping too fast. This action of FR on inhibition of phosphodiesterase may serve as a regulatory effort to maintain or buffer the CAMP level to prevent it from rapid decrease. The stimulatory effect on protein kinase is not yet entirely understood. This action of FR may serve as a positive forward effect to potentiate CAMP action. Figure 6 represents a working hypothesis for the biological role of FR. It is based on the existing hypothesis of Sutherland et al. The consideration of FR in this diagram is self explanatory. The role of FR on guanylate cyclase and cGMP levels in a target cell is also of interest to us. The effect of FR on elevation of cGMP levels in isolated adipocytes has been demonstrated (Asakawa, T. et al. Fed. Proc. 1975). ACKNOWLEDGMENT The authors wish to thank Dr. Claudia Sutherland (Mrs. E.W.S.) for her continuing interest and support of this work before and after the death of E.W.S. We also wish to thank Messrs. Richard Snyder, Michael Hucks, and Juan Ruiz, and Mrs. Ilene Scheinbaum for their excellent
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AND ASAKAWA
technical assistance.. A special thanks should be given to Ms. Barbara Mozayeny, who is last graduate student, for her interest and enthusiasm in scientific research. We thank Dr. William J. Whelan, the American Heart Association, the NIH Heart and Institute, and the National Science Foundation for their continuous support. Finally, we Earl’s’ friends and former associates for their warm encouragements. We thank everyone tioned above: they are essential for the survival of this team and for keeping the group ative and productive.
Earl’s Lung thank menoper-
REFERENCES 1. Robison GA, Butcher RW, Sutherland EW: Cyclic AMP. New York, Academic Press, 1971, pp 17-46 2. Butcher RW, Ho RJ, Meng HC, Sutherland EW: Adenosine 3’,5’-monophosphate in biological materials. II. The measurement of adenosine 3’,5’-monophosphate in tissues and the role of cyclic nucleotides in the lipolytic response of fat to epinephrine. J Biol Chem 240:4515-4523, 1965 3. Ho RJ, Sutherland EW: Formation and release of a hormone antagonist by rat adipocytes. J Biol Chem 2466822-6827, 1971 4. Ho RJ, Sutherland EW: Cyclic AMP mediated feedback regulator in target cells, in Advances in Cyclic Nucleotide Research, vol. 5. New York, Raven Press, (in press) 5. Ho RJ, Bomboy JD, Wasner HK, Sutherland EW: Elevation of CAMP levels and subsequent formation of antihormone activity in adipocytes of rat and hamster. Fed Proc 32: 535,1973 6. Ho RJ, Sutherland EW: Inhibition of adenylate cyclase by a factor isolated from human plasma. Fed Proc 33:480, 1974 7. Schimmel RJ: Responses of adipose tissue to sequential lipolytic stimuli. Endocrinology 94: 1372- 1380, 1974 8. Ho RJ, Sutherland EW: The action of regulator on adenylate cyclase. (in preparation) 9. Ho RJ, Soderling T, Sutherland EW: Stimulatory and inhibitory actions of the feedback regulator from adipocytes. (in preparation) 10. Fain JN, Pointer RH, Ward WF: Effects of adenosine nucleotides on adenylate cyclase, phosphodiesterase, cyclic adenosine accumulation, and lipolysis. J Biol Chem 247:6866-6872, 1972 11. Ebert R, Schwabe U: Studies on the
antilipolytic effect of adenosine and related compounds in isolated fat cells. Naunyn Schmiedebergs Arch Pharmakol 278:247-259, 1973 12. Weinryb I, Michel IM, Hess SM: Adenylate cyclase from Guinea pig lung: Further characterization and inhibitory effect of substrate analogs and cyclic nucleotides. Arch Biochem Biophys 154:2&I-249, 1973 13. Butcher RW, Baird CE: Effect of prostaglandins on adenosine 3’,5’-monophosphate levels in fat and other tissues. J Biol Chem 243:1713-1717, 1968 14. Shaw JZ, Ramwell PW: Release of prostaglandin from rat epididymal fat pad on nervous and hormone stimulation. J Biol Chem 243:1498-1503, 1968 15. Zinman B, Hollenberg CH: Effect of insulin and lipolytic agents on rat adipocyte low K, cyclic adenosine 3’,5’-monophosphate phosphodiesterase. J Biol Chem 249~2182-2187, 1974 16. Manganiello V, Vaughan M: Prostaglandin Et effects on CAMP concentration and phosphodiesterase activity in fibroblasts. Proc Nat1 Acad Sci USA 69269-273, 1972 17. D’Armiento M, Johnson GS, Pastan I: Regulation of CAMP phosphodiesterase activity in fibroblasts by intracellular concentrations of CAMP. Proc Nat1 Acad Sci USA 69:459462, 1972 18. Ho RJ: Effect of feedback regulator on CAMP phosphodiesterase activity. Physiologist 17:248, 1974 19. Ho RJ, Bomboy JD, Wasner HK, Sutherland EW: Preparation and characterization of a hormone antagonist from adipocytes. Methods in Enzymology, vol. 37. New York, Academic Press (in press)
