The control of the concentrations of cyclic nucleotides and adenosine in animal tissues

The aim of this thesis is to provide information concerning the control of the concentrations and the physiological roles of cyclic nucleotides and adenosine in animal tissues. For this purpose the activities, kinetics and some properties of adenyl cyclase, cyclic AMP and cyclic GMP phosphodiesterase, 5'-nucleotidase, adenosine kinase and adenosine deaminase have been studied in muscles, nervous tissues and livers from a number of vertebrates and invertebrates.
Chapter 1
Metabolic events within an organism are controlled and coordinated at a number of levels of complexity. Cyclic nucleotides and adenosine are messenger molecules concerned with the coordination of metabolism.
In Section A some aspects of cyclic nucleotide metabolism are considered and certain points are made which are relevant to the interpretation of the data presented in later chapters. Some factors which modulate the activities of adenyl cyclase and phosphodiesterase in vitro are described and the relevance of their effects to the in vivo situation is discussed. The significance of the basal and fluoride-stimulated activities of adenyl cyclase and the unusual kinetics of phosphodiesterase is considered.
Cyclic AMP, and possibly cyclic GMP, transmit hormonal stimuli to the interior of the cell but it is pointed out that they may have further roles. In particular, cyclic AMP may play an important role in nervous tissue. Some of the effects of cyclic AMP depend on the phosphorylation of enzymes or other proteins by a cyclic AMP-dependent protein kinase. The mediation of protein kinase means that there may be a low concentration of cyclic AMP in the cell but nevertheless a small change in the concentration of cyclic AMP may produce a large and rapid effect on the flux through a metabolic pathway. The low concentration of cyclic AMP means that a high rate of turnover of cyclic AMP is associated with only a low utilization of energy. The high rate of turnover of cyclic AMP is necessary for a rapid response of the cell to hormonal stimulation of adenyl cyclase. It has frequently been claimed that the concentration of cyclic AMP available to protein kinase or phosphodiesterase is very different from that measured in the whole cell. It is shown that it is often possible to provide different explanations for the data which have led to this claim.
In Section B adenosine metabolism and the role of adenosine are discussed in some detail. It is pointed out that it does not now appear that adenosine is a metabolite on a main pathway of intermediary metabolism. There have been few measurements of tissue concentrations of adenosine and there is no consensus of opinion concerning the primary location of adenosine intra- or extracellular. The simplest hypothesis is that adenosine equilibrates across the cell membrane. Evidence is presented to show that in vivo adenosine is produced by 5'-nucleotidase and utilized by adenosine kinase and adenosine deaminase. The regulation of the activities of these enzymes for the purpose of controlling the concentration of adenosine is discussed. A theory of the control of the concentration of adenosine by changes in the phosphorylation state of the cell is presented. This theory predicts variations in the rate of output of adenosine from the cell which are consistent with data in the literature. The output and uptake of adenosine by the cell are discussed in detail. Low concentrations of adenosine within a tissue are mostly phosphorylated within the cell; deamination of adenosine, which may be partly extracellular, becomes relatively more important when the concentration of adenosine is high.
Exogenous adenosine has a number of effects on the physiology of mammalian tissues. For example, it relaxes most smooth muscles. Adenosine produces a large increase in the content of cyclic AMP in mammalian brains and it is suggested that a number of the physiological effects of exogenous adenosine are mediated by a change in the concentration of cyclic AMP. In the final section of Chapter 1 it is argued that adenosine plays a role in the regulation of blood flow in some vertebrate tissues. It may also play a role in neurotransmission.
Chapter 2
For comparative studies it is necessary that enzyme assays are applicable to enzymes in homogenates of very different tissues. In some cases very small quantities of tissue are available. Moreover, when maximal activities are being measured, optimal conditions for the assays are required. Radiochemical assays were developed to meet these requirements; they are described in Section A of Chapter 2. In the succeeding sections the development of each assay is described. A number of preliminary results are presented, including pH curves. Finally, the graphical methods which were used to analyze the results and the significance of the data obtained are discussed.
Chapter 3
As a preliminary to the discussion of the results of Chapter 3, it is reported that the extraction of adenyl cyclase using a hypotonic medium and a ground-glass homogenizer and pestle leads to higher basal activities and a greater degree of stimulation of enzyme activity by fluoride than extraction in isotonic medium using an ordinary glass homogenizer. The former method was used for a comparative study of basal and fluoride-stimulated activities of adenyl cyclase. The activities and Km values of adenyl cyclase and cyclic AMP and cyclic GMP phosphodiesterase are reported for a number of animal tissues. In general the activities decrease in the order nervous tissues> hearts > muscles with a high oxidative capacity and livers > muscles with a low oxidative capacity. The kinetic data for phosphodiesterase are analyzed on the assumption that at least two enzymes (high and low Km) contribute to the kinetics. It is argued that the analysis supports this assumption. It is also argued.that, in vertebrate tissues, one high Km enzyme hydrolyzes both cyclic AMP and cyclic GMP.
The data are applied to a simple model of the turnover of cyclic AMP in the cell. Maximum and minimum tissue concentrations of cyclic AMP are predicted from the model. The predictions are consistent with much reported data. It is suggested that tissue concentrations of cyclic AMP do not vary greatly in vivo. The results, as interpreted by the model, indicate that the extrusion of cyclic AMP from the cell does not play an important role in the turnover of cyclic AMP in most tissues.
The roles of the high and low Km phosphodiesterases are discussed. It is proposed that the high Km enzyme prevents the accumulation of cyclic AMP in tissues where the activity of adenyl cyclase may be higher than the activity of low Km phosphodiesterase. However, it is more efficient for the cell to synthesize the low Km enzyme. Possible explanations for variations in the proportions of high and low Km enzymes between tissues are suggested. One of these explanations is consistent with the present data only if the simple model of the turnover of cyclic AMP is modified. The modification of the model permits the explanation of some further data. According to the modified model adenyl cyclase and low K
m phosphodiesterase are located at the cell membrane, whereas high Km phosphodiesterase is located in the cytosol. Nevertheless, it is claimed that, unless the local activity of adenyl cyclase is much greater that that of low Km phosphodiesterase, the compartmentation of cyclic AMP is not great.
The roles of cyclic AMP in nervous tissue and muscle are discussed. It is argued that high activities of adenyl cyclase and phosphodiesterase are found in those tissues in which the concentration of cyclic AMP must change rapidly, rather than in tissues which contain a large number of enzyme molecules whose activity is regulated by cyclic AMP-dependent protein kinase.
Chapter 4
The activities and Km values of 5'-nucleotidase, adenosine kinase and adenosine deaminase are reported for a number of animal tissues. The activities of the enzymes are higher in nervous tissues and red muscles than in white muscles. The brain of the new-born mouse contains low activities of 5'-nucleotidase and adenosine kinase. The activity of adenosine kinase is very high in vertebrate livers.
It is reported that methylxanthines and papaverine activate adenosine kinase. Na+ also activates adenosine kinase, whereas K+ and Ca2+ inhibit. The significance of these effects in relation to the control of tissue concentrations of adenosine is discussed. During basal metabolism the concentration of adenosine is low; it is proposed that when the cell is under stress the activity of 5'-nucleotidase increases and the activity of adenosine kinase decreases so that the concentration of adenosine is increased. Some of this adenosine passes out of the cell. The data are consistent with the view that one of the functions of extracellular adenosine in muscle and brain is to cause the smooth muscle of the vasculature to relax so that blood flow increases. It is suggested that the liver, and possibly the lung and blood cells remove adenosine from the circulation. Further roles for adenosine are suggested.
By comparing measured Km values with reported tissue concentrations of AMP and adenosine, it is deduced that the activities of 5'-nucleotidase, adenosine kinase and adenosine deaminase are partly regulated by their substrate concentration in vivo. The proportion of exogenous adenosine which is deaminated rather than rephosphorylated is increased when the concentration of adenosine is high because the Km value of adenosine deaminase is greater than that of adenosine kinase and the activity of adenosine kinase is inhibited by a high concentration of adenosine. Moreover, if a high concentration of adenosine is associated with the cell being under stress, the activity of adenosine kinase may be reduced by other factors. It is argued that reduced phosphorylation and increased deamination of adenosine help to maintain the tissue ATP/ADP concentration ratio constant when the cell is under stress.
Chapter 5
The control of lipolysis in the rat heart is discussed. It is pointed out that variations in the activities of triglyceride lipase, adenyl cyclase or phosphodiesterase could cause the increased lipolysis which has been measured for hearts from diabetic and starved rats. It is reported that no differences in the activities or kinetics of these three enzymes are found between normal, starved and diabetic rats. There is further speculation concerning the factors which increase lipolysis in the diabetic and starved conditions.
Chapter 6
The role of adenosine is discussed further. It is suggested that the control of the activity of adenosine kinase by Ca2+, Na+ and K+ would be consistent with a role for adenosine in neurotransmission. It is proposed that adenosine stimulates the activity of adenyl cyclase in a number of tissues but usually the effect cannot be detected because it is limited to a small proportion of the cells. The difficulties involved in obtaining effects of adenosine on the activity of adenyl cyclase in tissue homogenates are discussed and some results are presented concerning this problem.
It is pointed out that there are a number of similarities between the metabolism of cyclic AMP and adenosine. These similarities derive from the similar roles of the two molecules as messengers. The mechanisms which the cell uses to metabolize messenger molecules are therefore discussed. However, there are also some differences between the metabolism of cyclic AMP and adenosine. It is argued that the differences can be accounted for in terms of the differing roles of the two messengers; adenosine functions extracellularly as a first messenger, whereas cyclic AMP functions intra-cellularly as a second messenger.