Earl W. Sutherland

Effects of derivatives of adenosine 3′,5′-phosphate on liver slices and intact animals

Abstract Cyclic AMP and its N6-2′-O-dibutyryl derivative were found to be relatively non-toxic in mice. The chief effect noted when mice were injected with 500 mg/kg of either compound was drowsiness. Cardiovascular responses resulting from intravenous injection of 13 μmoles/kg of cyclic AMP or derivatives in anesthetized dogs were small and variable. Acyl derivatives of cyclic AMP with substitution on the N6 or 2′-O position or both were more active than the parent compound in producing hyperglycemia in intact dogs. These derivatives were also more effective in causing phosphorylase activation in liver slices. An initial rise of blood lactate was observed when cyclic AMP or derivatives were injected into intact dogs. This initial rise was followed by a prolonged decrease in the blood lactate concentration.

Effects of glucagon on adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate in human plasma and urine

Glucagon, infused intravenously into fasting, well-hydrated, normal men in doses of 25-200 ng/kg per min, induced up to 30-fold increases in both plasma and urinary cyclic AMP. Cyclic GMP levels were unaffected by glucagon. Simultaneous cyclic AMP and inulin clearance studies demonstrated that the glucagon-induced increase in urinary cyclic AMP was entirely due to glomerular filtration of the elevated plasma levels of the nucleotide. The cyclic AMP response to glucagon was not mediated by parathyroid hormone or epinephrine, and trypsintreated glucagon was completely inactive. The perfused rat liver released cyclic AMP into the perfusate in response to glucagon, indicating that the liver is a possible source of the cyclic AMP entering the extracellular fluids in response to glucagon in vivo.

The effect of epinephrine and other agents on adenyl cyclase in the cell membrane of avian erythrocytes

Abstract 1. 1. Cell membranes were prepared from turkey erythrocytes by fragmentation into small particles or larger pieces. Extensively fragmented bits of membrane did not respond to addition of epinphrine while larger pieces did respond to epinephrine. 2. 2. Aging of cell membrane preparations in the cold led to a loss of responsiveness to epinephrine. The responsiveness could be restored by prior incubation with a sulfhydryl reagent such as mercaptoethanol. 3. 3. ATP and magnesium ions applied to the exterior of intact erythrocytes did not cause synthesis of adenosine 3′,5′-phosphate. Several proteolytic enzymes were unable to inactivate adenyl cyclase when applied to intact cells but did inactivate the adenyl cyclase of hemolyzed preparations. 4. 4. The ATPase activity of the cells was apparent when ATP was added to intact erythrocytes. Some of the ATPase activity was lost when proteolytic enzymes were added to intact cells but most ATPase activity was retained unless the cells were first hemolyzed before addition of the enzymez.

The Role of Cyclic AMP in the Control of Carbohydrate Metabolism

Cyclic AMP plays an important role in the regulation of metabolism generally. Emphasis in the present review has been placed on carbohydrate metabolism, but lipid metabolism has also been discussed to some extent. The chief role of cyclic AMP in several tissues seems to be to facilitate or promote the mobilization of glucose and fatty acid reserves. In the liver, glucagon and the catecholamines cause an increase in the intracellular level of cyclic AMPby stimulating adenyl cyclase. This increase in the level of .cyclic AMP leads to a net increase in hepatic glucose production by at least three mechanisms: stimulation of phosphorylase activation, suppression of glycogen synthetase activity, and stimulation of gluconeogenesis. The catecholamines also stimulate adenyl cyclase in muscle and adipose tissue. Among the principal effects of cyclic AMP in these tissues are glycogenolysis in muscle and lipolysis in adipose tissue. Another role of cyclic AMP is to enhance or promote the release of insulin from pancreatic beta cells. Insulin then travels to the liver and adipose tissue to suppress the accumulation of cyclic AMP, and may also antagonize the action of cyclic AMP in muscle. Cyclic AMP is thus seen to mediate the actions of several catabolic hormones as well as promote the release of an anabolic hormone which acts in part by opposing cyclic AMP. Since cyclic AMP is involved in the release as well as several of the actions of insulin, the possible role of cyclic AMP in diabetes has been discussed. Human diabetes mellitus is recognized as the result of a basic genetic defect, the nature of which is undefined. One line of evidence implicates basement membrane thickening as an early event in the patho genesis of diabetes. Further study of the formation and breakdown of the basement membrane may therefore lead to a better understanding of the genetic defect. Whether or not cyclic AMP plays a regulatory role in basement membrane synthesis is presently unknown. Another defect recognizable in prediabetics is faulty insulin release in response to glucose infusion. This could be secondary to basement membrane thickening, but there is also evidence that the cyclic AMP mechanism may be defective. At another level, the role of cyclic AMP is more obvious: insulin deficiency leaves unopposed the actions of hormones which stimulate the production of cyclic AMP, thereby contributing to the glucose plethora and ketosis so often seen in the later stages of the disease.

THE EFFECTS OF THE CATECHOLAMINES, ADRENERGIC BLOCKING AGENTS, PROSTAGLANDIN E1, AND INSULIN ON CYCLIC AMP LEVELS IN THE RAT EPIDIDYMAL FAT PAD IN VITRO*

The Role of Cyclic A M P in the Lipolytic Response of Adipose Tissue. Cyclic A M P t appears to mediate a t least in part the lipolytic actions of the catecholamines and several other lipolytic hormones in rat epididymal fat pads in ~ i t r o . ’ ~ The data which led to this conclusion may be summarized as follows: a. Exogenous cyclic AMP, or the derivative N6-2’ O-dibutyryl cyclic AMP, stimulated lipolysis when added to incubating fat pads or isolated fat cells.’-3 b. Lipolytic rates and cyclic A M P levels were compared in fat pads incubated in a variety of conditions.”’ Incubation or perfusion of fat pads with epinephrine produced increases in cyclic A M P which occurred a t least as rapidly as the increased rate of free fatty acid release. These changes in cyclic A M P were elicited by concentrations of epinephrine in the same range as those required for the lipolytic action. Caffeine (an inhibitor of the cyclic nucleotide phosphodiesterase) acted synergistically with epinephrine in stimulating cyclic A M P accumulation and lipolysis, and dichloroisoproterenol (DCI) antagonized the effects of epinephrine on both. c. Cell-free preparations of rat epididymal fat contained an epinephrinesensitive adenyl cyclase ~ y s t e m . ~ These data, taken together, indicated that the adenyl cyclase system was the adrenergic receptor for lipolysis in fat. (For a detailed discussion of the relationship of the adenyl cyclase system to adrenergic receptors, see Reference 5 . ) The effects of hormones on lipolysis are so numerous and interrelated that it seemed desirable to investigate in depth the actions of hormones and blocking agents on the adenyl cyclase system. In the recently reviewed.studies characterizing the lipolytic receptor of fat, the effects of the adipokinetic hormones and blocking agents on lipolysis (FFA or glycerol release) have been Unfortunately, the nature and the number of steps between increased intracellular cyclic A M P and increased lipolysis are not completely

Preparation of derivatives of adenosine 3′,5′-phosphate

Abstract The preparation of the N1-oxide, of N6- and 2′-O-monoacyl derivatives and of N6-2′-O-diacyl derivatives of adenosine 3′,5′-phosphate, is described. The syntheses of the acyl derivatives are based on the difference in the speeds of acylation of the NH2 and the OH groups and on the difference in the susceptibility to the action of alkalis of the two acyl groups.

Analysis of adenosine 3′,5′-monophosphate with luciferase luminescence☆

Abstract A sensitive, relatively simple, assay with wide range linearity has been developed for adenosine 3′,5′-monophosphate (cyclic AMP). It is based on the conversion of cyclic AMP to adenosine triphosphate (ATP), which is then measured by its luminescent reaction with luciferase. A linear standard curve for cyclic AMP was demonstrated using samples of 100 μl containing from 7.2 × 10−9 to 7.2 × 10−6M cyclic AMP. The assay was used to measure the rise in human urine cyclic AMP levels produced by glucagon infusion. Urine samples needed only to be filtered and buffered prior to assay and values agreed with those obtained using another assay method.

Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm.

Abstract Sperm from several invertebrates contained guanylate cyclase activity several-hundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn 2+ was up to several hundred-fold greater than with Mg 2+ and was increased 3–10-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mn 2+ -dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than 10-fold more active with Mn 2+ than with Mg 2+ . Invertebrate sperm contained phosphodiesterase activities against 1.0 μ m cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by Triton. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1–1 nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive to stimulation by cyclic AMP than by cyclic GMP. 5

Effect of epinephrine on cyclic adenosine 3′,5′-phosphate and hexose phosphates in intestinal smooth muscle

Abstract Physiologically active concentrations of epinephrine produce an increase in the concentrations of cyclic adenosine 3′,5′-phosphate in isolated intestinal smooth muscle preparations from the taenia coli of the guinea-pig. This effect is not associated with a change in the concentrations of hexose phosphate esters in this tissue.

CYCLIC AMP AND THE FUNCTION OF EUKARYOTIC CELLS: AN INTRODUCTION

The chemical structure of adenosine 3’,5’-monophosphate, otherwise known as cyclic AMP, is shown in FIGURE 1. It was discovered as the mediator of the hepatic glycogenolytic effect of epinephrine and glucagon,’ and is now recognized as a versatile regulatory agent mediating a host of hormonal effects.2 Research in this area is expanding at such a rate that it is very difficult, if not impossible, for a single group to keep track of it all. Our hope for this present monograph was that it would update and supplement an earlier one,2 which we hope will continue to be useful as a source of background material. Both of these hopes will probably be realized to some extent, but the incredible pace of research in this area cannot be overemphasized. We are writing these words in October, 1970, with the conference less than a month away, and we fully expect that they will have a faintly old-fashioned ring to them by the time they are in print. Perhaps by extrapolating from what is said here and in the rest of this monograph, the serious reader will gain at least some idea of where the subject is likely to lead in the future. The structure of adenyl cyclase is still poorly understood. Information about this enzyme was summarized previously,”.’ and an important recent development is discussed in this monograph by Lefkowitz et Still more recently, Rodbell and his colleagues 6 * have made some useful contributions. There is no longer any question that the receptors for some hormones are very closely related to the adenyl cyclase system as a whole, but most of the details remain to be worked out. We still do not know how the hormone-receptor interaction leads to a change in the catalytic activity of the enzyme. To whatever extent our earlier model (FIGURE 2) represents an aspect of reality, it would appear that the catalytic and regulatory subunits do not necessarily develop at the same time. A more operationally correct way of summarizing the available data R , would be to say that the catalytic activity of adenyl cyclase and its ability to be stimulated by hormones do not necessarily develop at the same time. There is now evidence that these two components of the system can be separated,*O and further work along these lines will be watched with interest. The suggestion that GTP or GDP may play an important role in the effects of hormones on adenyl cyclase has also been of interest, but whether future research will substantiate or invalidate this hypothesis remains to be seen. The detrimental effects of cell breakage on adenyl cyclase, and especially on its sensitivity to hormonal stimulation, were emphasized in an earlier review.J However, we can now see that this may be even more variable than it was then thought to be. Platelet adenyl cyclase, for example, seems to be just as sensitive to stimulation by prostaglandins in broken cell preparations as it is in intact cells, as noted by Krishna and his colleagues.ll Brain cyclase stands at the other end of the scale, with most other cells and tissues falling some-