Current Topics in Experimental Endocrinology: Volume 2

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Preface

V.H.T. James and L. Martini

The second volume of this book follows the same policy as the first; the Editorial Board has again selected a number of active investigators and has encouraged them to review their own fields of interest selectively and critically. Readers thus continue to be kept informed of major developments in the ever-expanding field of endocrinology, and at the same time stimulated by each individual writer’s viewpoint. Our contributors have had the unenviable task of writing when they probably would prefer to be researching, and to them, and to the publisher’s editorial staff, we extend our thanks. We hope that they are not displeased with the result.

Recent Progress in Cyclic Nucleotide Research

S.J. Strada and G.A. Robison,     Program in Pharmacology, University of Texas Medical School at Houston, Houston, Texas

Publisher Summary

Adenosine 3’, 5’-monophosphate (cyclic AMP) is formed from ATP through the action of adenylyl cyclase, which seems to be an integral component of the cell membrane in most cells. Hormones appear to stimulate the enzyme by interacting with specific receptors on the external surface of the membrane. Calcium is involved in several ways; it is required for cyclase activation by at least one hormone (ACTH), and there is evidence that some hormones can influence calcium transport independently of their effect on cyclise. Fluoride stimulates adenylyl cyclise activity in broken cell preparations of most eukaryotic cells, but so far, this has not led to any important insights into the mechanism of hormonal stimulation. Cyclic AMP is metabolized to 5’-AMP under the catalytic influence of one or more phosphodiesterases. Phosphohpids, ions, and one or more endogenous proteins are involved in regulating phosphodiesterase activity, and cyclic AMP itself appears capable of inducing the formation of at least one isozyme. Most of the physiologically important effects of cyclic AMP in higher forms are the result of protein kinase activation. Cyclic AMP-dependent protein kinases are composed of catalytic and regulatory subunits.

I Introduction

II Adenylyl Cyclase

A Relation to Hormone Receptors

B Possible Importance of Calcium

C Phospholipids

D The Fluoride Response

E The Effect of Guanyl Nucleotides

F Effects of Prostaglandins

G The Effect of Cholera Toxin

H Inhibitors of Adenylyl Cyclase

III Cyclic Nucleotide Phosphodiesterases

A Multiple Molecular Forms

B Endogenous Modulators of Cyclic Nucleotide Phosphodiesterases

IV Mechanism of Action of Cyclic AMP

A Protein Kinase Activation

B Possible Importance of Microtubules

C Other Possible Mechanisms

V Cyclic GMP

VI Summary

References

I Introduction

Adenosine 3′,5′-monophosphate (cyclic AMP) was discovered in 1956 in the course of endocrinological research (see Sutherland and Rall, 1960, for an early review). Most of the research on this substance for the next 10 years or so was concerned directly or indirectly with its role as a regulator of differentiated eukaryotic cell function. It was shown first to mediate the hepatic glycogenolytic effect of glucagon and epinephrine, and was eventually recognized as a second messenger mediating many of the effects of a variety of other hormones, including ACTH (adrenocorticotropic hormones), TSH (thyroid-stimulating hormone), vasopressin, luteinizing hormone, MSH (melanocyte-stimulating hormone), and parathyroid hormone. This aspect of the subject has been discussed in a number of recent monographs and review articles (e.g., Robison et al., 1971 a,b; Hardman et al., 1971; Cheung, 1972; Gill, 1972; Major and Kilpatrick, 1972; Greengard et al., 1972a; Sutherland, 1972).

One of the reasons for the slow initial progress in understanding the role of cyclic AMP was that methodology was difficult, but this is no longer the most important limiting factor (Greengard et al., 1972b; Chasin, 1972). Cyclic AMP has now transcended its endocrinological beginning and has been shown to function in almost all animal species, including bacteria and other unicellular organisms. In Escherichia coli and other gram-negative bacteria, cyclic AMP appears to be required for the synthesis of a number of inducible enzymes, and the ability of glucose to suppress cyclic AMP formation appears to account satisfactorily for catabolite repression (Pastan and Perlman, 1972). Cyclic AMP has also been implicated in lysogeny (Hong et al., 1971) and bacterial transformation (Wise et al., 1973). In certain species of cellular slime molds, cyclic AMP appears to be responsible for initiating the aggregation of slime mold amebae, leading to the formation of a multicellular organism (Bonner, 1971).

Evidence has now begun to accumulate to suggest that cyclic AMP may also play an important role during the growth and development of higher organisms. Although data are presently insufficient to define this role precisely, it would appear that in some types of cells reduced levels of cyclic AMP are needed to permit rapid cell division, whereas higher levels are associated with differentiation (Weiss and Strada, 1973). Changes in cyclic AMP during the cell cycle are now being explored (Willingham et al., 1972; Burger et al., 1972), and an important complementary role for cyclic GMP (guanosine 3′,5′-monophosphate) has been suggested (Hadden et al., 1972). It now seems possible that reduced levels of cyclic AMP or perhaps increased levels of cyclic GMP are involved in a number of proliferative disorders, including psoriasis (Voorhees et al., 1972) and certain forms of cancer (Otten et al., 1972). Cyclic nucleotides may also play an important series of roles during the immune response (see, for example, Orange et al., 1971; Parker, 1972; Hadden et al., 1972; Bourne et al., 1973), although it may be some time before these roles become clarified.

It is no longer possible to discuss intelligently all aspects of cyclic nucleotide research in a single review article. Our purpose in this review will be to summarize what is known about the formation, metabolism, and action of cyclic AMP, with major emphasis on eukaryotic cells. In all cells studied, cyclic AMP is formed from ATP through the catalytic influence of adenylyl cyclase, and is metabolized to 5′-AMP under the influence of one or more phosphodiesterases (Fig.1). The intracellular level of cyclic AMP is therefore determined by the rates of these reactions, as well as by the rate at which it is released into the extracellular space. Most of the effects of cyclic AMP are poorly understood, but the glycogenolytic and lipolytic effects have been shown to involve the activation of a protein kinase. This may be the mechanism of many and perhaps most of the physiologically important effects of cyclic AMP in differentiated eukaryotic cells.

Fig. 1 Reactions involved in the formation and metabolism of cyclic AMP.

II Adenylyl Cyclase

A Relation to Hormone Receptors

The particulate nature of hepatic adenylyl cyclase was established by early experiments of Sutherland and Rall and their colleagues (Sutherland and Rall, 1960; Sutherland et al., 1962). Adenylyl cyclase in most eukary-otic cells appears to occur predominantly in the plasma membrane (Davoren and Sutherland, 1963), although significant activity may also occur in other membranous components in some cells, such as the sarcoplasmic reticulum in muscle cells (Levey, 1971a). The ability of glucagon to stimulate hepatic adenylyl cyclase and of ACTH to stimulate the adrenal enzyme led to the idea that the receptors for some hormones might be closely related to adenylyl cyclase (Sutherland and Rall, 1960).

Based on these and other observations, a model was developed according to which the protein component of the adenylyl cyclase system was envisioned as a two-component system embedded in the lipid matrix of the cell membrane (Robison et al., 1967). A regulatory subunit possessing receptors for one or more hormones was postulated to be in contact with the extracellular space, with a catalytic subunit in contact with cytoplasmic ATP. An effective interaction between hormone and receptor on the external surface of the membrane could thus lead to a conformational perturbation leading to a change in catalytic activity on the inner surface of the membrane. A variant of this model postulated a third component, a transducer interposed between an external discriminator and an internal amplifier (Rodbell, 1972). Such models are perhaps best viewed in the light of the fluid mosaic theory of membrane structure (Singer and Nicolson, 1972), according to which many membrane proteins are inserted into the lipid bilayer in essentially this way. Some of these proteins, such as glycophorin from erythrocyte membranes, have actually been shown to be amphipathic, with hydrophilic regions at either end separated by an intervening hydrophobic portion. Unfortunately much less is known about the physicochemical structure of adenylyl cyclase.

Our earlier model was based on the knowledge that the conversion of ATP to cyclic AMP was ordinarily an intracellular event (Øye and Sutherland, 1966) combined with the presumption that large polypeptide hormones such as glucagon and ACTH probably interacted with receptors on the external surface of their target cells. This presumption is now supported by experiments showing that hormones covalently linked to glass or agarose beads, too large to possibly enter cells, nevertheless retain their ability to stimulate the intracellular accumulation of cyclic AMP (Schimmer et al., 1968; Venter et al., 1972; C. B. Johnson et al., 1972). Copurification of receptors (as determined by specific hormone binding) with adenylyl cyclase activity from several tissues has lent additional support (e.g., Lefkowitz et al., 1971; Rodbell, 1972) for the validity of the model, at least in broad outline.

The recognized inadequacies of the earlier models have been further emphasized, however, by a number of recent observations which are as yet poorly understood. These observations are discussed briefly in the following paragraphs.

B Possible Importance of Calcium

Adenylyl cyclase under most conditions is inhibited by calcium, but stimulation by at least one hormone, ACTH, requires calcium (Lefkowitz et al., 1971). Since most other hormones do not have this requirement, a reasonable hypothesis seemed to be that calcium would be required in order for ACTH to bind to its receptors. This was shown to be incorrect, however, when it was demonstrated that calcium did nothing but inhibit the binding of ACTH (Lefkowitz et al., 1971). Thus the reason why ACTH requires Ca++ in order to stimulate cyclase, while other hormones do not have this requirement, continues to be obscure.

Rubin and his colleagues (Carchman et al., 1971; Rubin et al., 1972) have suggested that the interaction of ACTH with its receptors in the adrenal cortex leads to an effect on calcium translocation in addition to stimulation of adenylyl cyclase. They showed that perfusion of isolated cat adrenal glands with calcium-free Locke’s solution led to a three- to six-fold increase in tissue cyclic AMP but did not augment steroid output. Addition of ACTH under these conditions produced only a small additional increment in cyclic AMP but a pronounced increase in steroid release. It had previously been shown that the steroidogenic effect of ACTH was associated with a redistribution of tissue calcium from a more readily exchangeable fraction to a less readily exchangeable fraction. It would thus appear that the effect on calcium may be required not only for cyclase stimulation but also for the steroidogenic effect of cyclic AMP to be expressed.

Some recently reported observations by Moyle et al (1973) can be understood in terms of this hypothesis. Moyle and his colleagues compared the effects of ACTH and its o-nitrophenyl sulfenyl derivative (NPS-ACTH) on both cyclic AMP accumulation and steroidogenesis in rat adrenal cells. They found that the effect of NPS-ACTH was proportionately much greater on steriodogenesis than it was on cyclic AMP, i.e., that a given increment in cyclic AMP in response to NPS-ACTH was more effective in stimulating steroidogenesis than the same increment in response to ACTH. This would be understandable if the structural requirements for the postulated effect on calcium translocation differ slightly from those required for cyclase stimulation. It seems possible, for example, that one part of the ACTH molecule might be primarily responsible for cyclase stimulation and another part primarily responsible for the effect on calcium. The impression created by the results of Moyle et al. is that NPS-ACTH might be relatively less effective than the parent compound in stimulating cyclase but relatively more effective in causing the redistribution of calcium, although this remains to be demonstrated experimentally.

The phenomenon of the same hormone-receptor interaction leading to two effects, both of which are required in order for the final response to be expressed, might not be restricted to large polypeptide hormones such as ACTH. Evidence has been presented, for example, that some of the effects of serotonin (Berridge and Prince, 1972) and epinephrine (Øye and Langslet, 1972) may also involve changes in calcium translocation in addition to cyclase stimulation.

At the risk of wandering too far into the realm of speculation, it is possible to imagine that some membrane proteins could serve as conduits for calcium while simultaneously functioning as the regulatory subunits of an adenylyl cyclase system, in addition to providing sites with which hormones could interact. Clearly more experimentation is needed before the relation of calcium to the hormonal stimulation of adenylyl cyclase can be understood.

C Phospholipids

The importance of phospholipids in the structure and function of mem-branes in general has been recognized for a long time. More recently their possible importance for the control of adenylyl cyclase has been explored, with results which are interesting but which are also to some extent contradictory.

Among the more interesting of these results are those reported by Levey and his colleagues. Levey (1971a) used the nonionic detergent Lubrol-PX to solubilize cat heart adenylyl cyclase, which in particulate preparations responds to stimulation by catecholamines, glucagon, or histamine. After solubilization, however, either in the presence of the detergent or freed of detergent by DEAE-cellulose chromatography, activity was unresponsive to any of the hormones, although it could still be stimulated by fluoride (see Section D). The addition of phosphatidylserine but not phosphatidylinositol was then found to restore and even enhance the sensitivity to glucagon (Levey, 1971a) and histamine (Levey and Klein, 1972). By contrast, phosphatidylinositol but not phosphatidylserine was found to restore sensitivity to norepinephrine (Levey, 1971b). These results are not easily interpreted, in the light of present knowledge, but might suggest that the receptors for norepinephrine are located on regulatory subunits which are separate and distinct from the ones possessing glucagon and histamine repectors. The phospholipids used in these experiments were chromatographically pure preparations from bovine brain.

Using a similar solubilized preparation, Lefkowitz and his colleagues (1972, 1973) were able to partially purify a protein which binds catecholamines with at least some of the characteristics of an adrenergic β-receptor. Since phosphatidylinositol did not alter the rate or extent of norepinephrine binding to this protein, Lefkowitz and Levey (1972) concluded that the phospholipid was most likely affecting the system at a site between the receptor and the catalytic subunit of adenylyl cyclase, possibly part of the transducer postulated by Rodbell and his colleagues. This conclusion may not be warranted, partly because the binding experiments were performed under conditions different from those of the adenylyl cyclase measurements and partly because it is far from clear that the norepinephrine was being bound to an adrenergic β-receptor. The lack of stereospecificity and the low affinity for β-adrenergic blocking agents would argue against this possibility. The work of Levey and Lefkowitz and their colleagues nevertheless represents an interesting early step toward gaining a better understanding of the role of phospholipids in the hormonal activation of adenylyl cyclase.

Different results were obtained in studies employing rat liver membranes. Rethy and his colleagues (1972) found that treatment with a mixture of petroleum ether and butanol for 2 minutes at 4° C reduced basal adenylyl cyclase activity and abolished the response to fluoride as well as to either glucagon or epinephrine. The addition of phosphatidylserine partially restored the response to all of these agents whereas phosphatidylinositol was ineffective.

Incubation with phospholipase A also led to decreased activity and loss of hormonal and fluoride sensitivity which could be partially restored by phosphatidylserine. Phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine were all found to be ineffective. Since phosphatidylserine was effective regardless of whether it was obtained from rat liver or bovine brain, Rethy et al. (1972) concluded that the nature of the fatty acid moieties was probably unimportant.

These results differ somewhat from those reported by Birnbaumer et al. (1971), who found that phospholipase A caused a selective loss of the glucagon response while actually enhancing the stimulatory effect of the fluoride ion. The reason for this discrepancy remains to be resolved. Like Rethy and his colleagues, Birnbaumer et al. found phosphatidylserine to be more effective than other phospholipids in restoring hormonal sensitivity.

An interesting recent observation by Tomasi et al. (1973), albeit of uncertain relation to the foregoing work with phospholipids, was that most of the adenylyl cyclase activity in homogenates of Yoshida hepatoma cells was found not in association with the plasma membrane fraction but rather in soluble fractions. There appeared to be a gradual transfer of activity from membrane to soluble fractions as a function of tumor growth.

It is obviously impossible at present to define the role of phospholipids in the regulation of adenylyl cyclase activity. Some interesting experiments have been done, but much additional work is needed.

D The Fluoride Response

Fluoride stimulates adenylyl cyclase activity in broken cell preparations but apparently not in intact cells. This effect has been seen in preparations of most mature eukaryotic cells and tissues studied. It was thought at one time that activity in the presence of fluoride was invariably greater than in the presence of hormones and other agents, such that it represented something of a maximum, but this is not the case. For example, the effects of prostaglandin E1 (PGE1) on platelet cyclase (Krishna et al., 1972), of glucagon on hepatic cyclase (Birnbaumer et al., 1971), and of treatment with Triton X-100 on rat brain cyclase (Perkins and Moore, 1971) are all greater than the effect of fluoride. Fluoride increases the activity of adenylyl cyclase from some bacteria, such as Streptococcus salivarius (Khandelwal and Hamilton, 1971), but not from others, such as Brevibacterium liquefaciens (Hirata and Hayaishi, 1967).

The significance of the fluoride effect is that by studying it we might be led to a better understanding of how the membrane adenylyl cyclase system functions, although to date this expectation has not been fulfilled. The lack of effect on certain bacterial systems suggests that it will not teach us much about the catalytic mechanism per se. The lack of effect in intact eukaryotic cells, despite the fact that fluoride penetrates most of these cells readily, might suggest that fragmentation of the membrane leads to an inhibitory alteration which can in some manner be reversed by fluoride.

Birnbaumer et al. (1969) plotted increased activity of rat fat cell adenylyl cyclase as a function of increasing fluoride concentration and found the curves so generated to be sigmoidal. Reducing the temperature from 37° to 30°C led to a marked increase in the apparent Km of activation. By contrast, the curves produced by increasing ACTH concentrations were hyperbolic, and reducing the temperature had only a slight effect on the apparent Km of activation. The presence of Mg++ or Mn++ appears to be necessary for the effect of fluoride, which increases with increasing time of exposure (Perkins and Moore, 1971) and which is not easily reversed by washing in fluoride-free medium (Schramm and Nairn, 1970; Perkins and Moore, 1971; Severson et al., 1972). Drummond and his colleagues (1971) studied adenylyl cyclase from skeletal muscle and concluded that the effect of fluoride was to increase Vmax with no significant effect on the affinity for ATP. Perkins and Moore, using particulate preparations of both cardiac muscle and brain, also observed an apparent increase in the affinity for Mg++, possibly at an allosteric site.

A potentially important observation by Pastan et al. (1970); (see also Lefkowitz et al., 1971) was that fluoride appeared to be necessary for the solubilization of an ACTH-sensitive cyclase from adrenocortical tissue.

E The Effect of Guanyl Nucleotides

An apparent requirement for GDP or a related nucleoside phosphate was first reported by Rodbell et al. (1971) for the stimulation of hepatic adenylyl cyclase by glucagon. A similar requirement has now been demonstrated in a variety of other systems, including pancreatic β-cell cyclase in response to glucagon (Goldfine et al., 1972), platelet cyclase in response to PGE1 (Krishna et al., 1972), frog bladder cyclase in response to oxytocin (Bockaert et al., 1972), and thyroid cyclase in response to either TSH or PGE1 (Wolff and Cook, 1973; Burke, 1973). It is beginning to appear, therefore, that this requirement may be universal for the stimulation of eukaryotic adenylyl cyclase by hormones or prostaglandins. By contrast, bacterial adenylyl cyclases appear only to be inhibited by guanyl nucleotides (Ide, 1971; Khandelwal and Hamilton,