Serotonin and Behavior

Serotonin and Behavior contains the proceedings of the 1972 symposium on the behavioral effects of changes in brain serotonin, held at Stanford University in California. The papers explore the role of serotonin in behavior as well as the key biochemical and pharmacological issues involved in behavioral studies of severe psychiatric disorders in both humans and animals. The book is organized into eight sections comprised of 65 chapters, with topics ranging from the fundamental biochemistry and pharmacology of the enzymes synthesizing serotonin, particularly, tryptophan hydroxylase and its inhibitors, to the physiology and pharmacology of serotonin. Some papers discuss the link between the telencephalic content of serotonin and pain sensitivity. Other papers focus on the effects of altering serotonin on neurons in the central nervous system. There are chapters that explain the effects of altering serotonin on animal behavior, the relationship between serotonin and sleep, the use of high doses of probenecid to estimate central serotonin turnover in affective disorders and addicts, the behavioral and metabolic effects of L-tryptophan in unipolar depressed patients taking methadone, and amygdala unit activity as a reflection of functional changes in brain serotonergic neurons. Biochemists, pharmacologists, psychiatrists, psychologists, and anyone interested in psychopharmacology will find this book extremely useful.

• Language: English
• Publisher: Academic Press
• Release date: Dec 2, 2012
• ISBN: 9780323143660

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WELCOMING STATEMENT

I want to greet you very briefly on behalf of Stanford University. We are immensely pleased to have this meeting here, for in our view, the conference deals with one of the principal frontiers in science.

One of the most remarkable developments in science during the 1960s was the elucidation of the role of catecholamines in brain. With impressive speed, this work not only provided fundamental new insights into brain organization and function, but also yielded significant improvements in the treatment of Parkinson’s disease and of depression. Understandably this dynamic pace of events regarding catecholamines drew attention away from closely related problems involving indoleamines in brain. However, by the early 1970s, it was apparent that important new developments were occurring in this area of inquiry also. Scientists in the brain-and-behavior field began to see the potential importance of new developments in research on serotonin. This research was being conducted in many different disciplines, many different countries, and many different settings including both basic and clinical investigations. Thus, there was a need to bring together this diversity of workers under conditions that would foster a full and stimulating exchange of information and ideas.

The notion of such a conference arose in the Preclinical Psychopharmacology Research Review Committee a couple of years ago, and gradually came to fruition, thanks, of course, to the support of the National Institute of Mental Health. Indeed, most of the research to be presented here was also sponsored by the NIMH. The ultimate results in treatment and prevention of disease will surely be due in large part to the farsighted efforts of the excellent staff of this Institute.

David A. Hamburg, M.D.,     Professor and Chairman, Department of Psychiatry, Stanford University School of Medicine

Session 1

Biochemistry and Pharmacology: Tryptophan Hydroxylase and Inhibitors of Tryptophan Hydroxylase

INTRODUCTION TO BIOCHEMISTRY AND PHARMACOLOGY: TRYPTOPHAN HYDROXYLASE AND INHIBITORS OF TRYPTOPHAN HYDROXYLASE

Moderator. M.A. Lipton

Introduction

This promises to be a very comprehensive symposium with topic areas ranging from the fundamental biochemistry of the enzymes synthesizing serotonin through the effects of spontaneous and experimental alterations in serotonin level upon many aspects of behavior in man and animals. The program is organized in an ascending order of complexity and perhaps a descending order of precision. It might appear from this that investigators working in this area have systematically moved from chemistry and anatomy to psychology and clinical research. But, we all know that this is far from the case. Research on serotonin has moved forward very rapidly on many fronts simultaneously. The initial problem choices of investigators are determined by their training, talent and curiosity. Clinical research has often preceded basic research and interesting clinical hypotheses as often stimulate basic investigators as the other way around.

Nonetheless, with retrospective wisdom, it is appropriate to organize a symposium as this one has been. The first session begins with a series of seven papers dealing with fundamental biochemistry, physiology and pharmacology. The speakers will discuss the biochemistry of serotonin formation, our current knowledge of the mechanisms involved in the regulation of tissue serotonin concentrations, and the chemical nature of serotonin inhibitors and our knowledge of their mode of action.

DOES THE TOTAL TURNOVER OF BRAIN 5-HT REFLECT THE FUNCTIONAL ACTIVITY OF 5-HT IN BRAIN?

D.G. Grahame-Smith

Publisher Summary

This chapter discusses whether the total turnover of brain 5-hydroxytryptamine (5-HT) reflects the functional activity of 5-HT in brain. Many studies have been conducted in which the turnover of brain 5-HT has been determined based on the overall steady state system where synthesis of 5-HT equals its metabolism to 5-hydroxyindoleacetic acid (5-HIAA). Concentration of tryptophan in the brain is the most important factor determining the rate of brain 5-HT synthesis, because the rate-limiting enzyme tryptophan hydroxylase is not saturated with its substrate tryptophan. The small and large changes in the concentration of brain tryptophan alter the rate of synthesis of 5-HT. If tryptophan alone is given to rats, the brain 5-HT concentration rises only slightly, but the brain 5-HT turnover markedly increases, the evidence being the rise in brain 5-HIAA production.

Many studies have been done in which the turnover of brain 5-HT has been determined on the basis of the overall steady state system where synthesis of 5-HT equals its metabolism to 5-HIAA, as shown below.

Implicit for the relevance of these studies to the behavioural role of 5-HT is the assumption that the 5-HIAA, which is produced, reflects the amount of 5-HT being used in functional activity within the brain. This must be the thought in the minds of most people, because there would be little point in studying the effect of alterations in behaviour upon the turnover of 5-HT, or the levels of 5-HIAA in the CSF of patients with mental disease, unless it was thought that there was some relationship between total turnover and the functional activity of 5-HT in brain.

Now if indeed the steady state situation was such that every molecule of 5-HT that was synthesized in brain was metabolized to 5-HIAA after having been functionally active, moment to moment synthesis of 5-HT would be crucially important in determining the functional level of 5-HT within the neuron. The situation would then hold that if synthesis were not turned off at an appropriate moment, than pushing a molecule of 5-HT in would cause one to spill over into functional activity. Even on biological first principles this is unlikely. Some experiments I have recently reported (Grahame-Smith, 1971) imply this is not the case.

It is apparent that the concentration of tryptophan in the brain is the most important factor determining the rate of brain 5-HT synthesis, because the rate-limiting enzyme tryptophan hydroxylase is not normally saturated with its substrate tryptophan. I (Grahame-Smith, 1971) and Fernstrom and Wurtman (1971a) have shown that both small and large changes in the concentration of brain tryptophan alter the rate of synthesis of 5-HT.

If tryptophan alone is given to rats it can be shown that the brain 5-HT concentration rises only slightly, but that the brain 5-HT turnover more markedly increases, the evidence being the rise in brain 5-HIAA production. However, with tryptophan loading alone no obvious behavioural change occurs in rats.

Contrast this situation with that which occurs if tryptophan is given to rats pretreated with a monoamine oxidase inhibitor. After pretreatment with a monoamine oxidase inhibitor, small doses of tryptophan (5 mg/kg) cause the animals to become extremely hyperactive and hyperpyrexial in a stereotyped manner, the degree of this excitation correlating with the rate of accumulation of 5-HT in the brain. The evidence is now very good that it is the accumulation of brain 5-HT or a derivative of it which causes the excitation.

These two situations differ only in regard to the inhibition of monoamine oxidase. In the first, 5-HT is being synthesized then metabolized to 5-HIAA without any obvious behavioural change and in the second, 5-HT is being synthesized at an equivalent rate, accumulating because of MAO inhibition, exceeding the binding mechanisms, and spilling over into functional activity. How is it that in the absence of MAO inhibition, the production of 5-HIAA is not associated with evidence of serotonergic functional activity if indeed all the 5-HIAA which is produced derives from a functionally active 5-HT? That is difficult to explain, and it seems more likely that when no MAO inhibitor is given, 5-HT is formed during tryptophan loading, but is metabolized intraneuronally without ever becoming functionally active. Because very small increases in brain tryptophan which minimally increase the rate of brain 5-HT, nevertheless cause hyperactivity and hyperpyrexia when MAO is inhibited, it is possible that normally 5-HT is synthesized in excess of functional requirements. If this is so, newly synthesized 5-HT probably passes into one of two compartments, either into a storage compartment or when that is full or adequate, into a compartment in which it is accessible to monoamine oxidase when it is metabolized intraneuronally. The controlled activity of monoamine oxidase, the ability of 5-HT to get to that enzyme, the control of the storage compartment, would all combine to finely control the level of a functional pool of 5-HT within the neurons. I find it very difficult to conceive that the regulation of a crucial functional pool of neuronal 5-HT can depend upon the fine regulation of tryptophan hydroxylation when this enzymatic activity is so dependent upon the brain tryptophan concentration which is obviously not finely controlled. If the foregoing postulates are correct, then measurement of total turnover rates may in no way reflect the control and turnover of the functionally active compartment. As shown below, if both functionally active and functionally inactive 5-HT are both metabolized to 5-HIAA, then the estimation of total turnover rates of 5-HT will include both these compartments of 5-HT, and depending upon the proportion of one to another will not necessarily reflect the functional activity of 5-HT dependent neurons.

It is of importance to determine whether or not this hypothesis is true in view of the current tendency in animal behavioural studies and in human psychiatric disease to accept turnover studies of 5-HT to 5-HIAA as evidence of serotonergic neuron activity.

A corollary of accepting that intraneuronally metabolism of nonfunctional brain 5-HT may be an important regulating mechanism, concerns the mechanism of action of reserpine and tetrabenazine. It has been generally assumed that reserpine causes the release of either noradrenaline or 5-HT on to post-synaptic receptor sites and that the resulting neuronal activity produces the Reserpinized state. An alternative explanation might be that reserpine causes an unloading of monoamine from storage granules onto intraneuronal monoamine oxidase by which it is metabolized. In this case, the Reserpinized state would be due to a deficiency of the monoamine within behaviourally excitant neuronal networks. The unloading of 5-HT from its granular storage sites onto intraneuronal monoamine oxidase and its subsequent intraneuronal metabolism producing an intraneuronal deficiency of 5-HT may well be the cause of the Reserpinized state (Grahame-Smith, 1971).

Although in the long term, the synthesis of 5-HT is an important background upon which the functional level of this monoamine can be regulated, compartmentation with regard to storage in granular binding sites and elsewhere and a controlled intraneuronal metabolism of synthesized in excess of needs amounts of 5-HT seem more likely mechanisms regulating the size and activity of those pools of 5-HT which are of such crucial functional importance.

METABOLISM OF p-CHLOROPHENYLALANINE AND THE MOLECULAR ASPECTS OF ITS ACTION

E.M. Gál

Publisher Summary

This chapter discusses the metabolism of p-chlorophenylalanine (p-CP) and the molecular aspects of its action. Some of the monooxygenases get inhibited by DL-p-chlorophenylalanine in vitro and in vivo. The monooxygenases that are affected by administration of DL-p-CP are phenylalanine 4-hydroxylase and cerebral tryptophan 5-hydroxylase. It was postulated that the inactivation of these monooxygenases was primarily because of the incorporation of p-CP into the enzyme protein near or at the active site. Earlier studies indicated that that hepatic phenylalanine hydroxylase was markedly inhibited, and in the brain, the inhibition of hydroxylation of tryptophan became coincident with the increase of cerebral p-CP. Although this was confirmed, a quantitative difference was found with respect to the correlation between the extent of inhibition and the amount of p-CP present in the brain.

Inhibition of some of the mono-oxygenases by DL-p-chlorophenylalanine (p-CP) in vitro and in vivo is well established (Koe and Weissman, 1966a; Jequier, Lovenberg & Sjoerdsma, 1967; Gál, Roggeveen & Millard, 1970). The mono-oxygenases affected by administration of DL-p-CP are phenylalanine 4-hydroxylase (EC 1.14, 3.1) and cerebral tryptophan 5-hydroxylase (EC 1.99, 1.4). Many explanations have been advanced for the nature of this inhibition. To this date, however, only one explanation has received experimental support (Gál and Millard, 1971). It was postulated that the inactivation of these mono-oxygenases was primarily due to the incorporation of p-CP into the enzyme protein near or at the active site. Thus, this inactivation might well be another example of a single amino acid substitution imparting critical changes to the physical and biochemical properties of the enzymes so affected.

This paper will present additional evidence in support of the above explanation as well as some corollary results obtained from metabolic studies with DL-p-CP that are supportive of the above thesis.

For detailed description of methodology the reader is referred to other publications (Gál, Roggeveen & Millard, 1970; Gál and Millard, 1971; Gál, 1972).

Metabolism of p-CP and Related Metabolites

Earlier studies (Koe and Weissman, 1966a) indicated that hepatic phenylalanine hydroxylase was markedly inhibited and in the brain the inhibition of hydroxylation of tryptophan became coincident with the increase of cerebral p-CP (Jequier, Lovenberg & Sjoerdsma, 1967). Although this is confirmed by our studies (Gál, Roggeveen & Millard, 1970) we found a quantitative difference with respect to the correlation between the extent of inhibition and the amount of p-CP present in the brain. It is revealing that the amount of inhibitor present in a tissue (expressed per g wet weight) apparently has little correlation with the inhibition found at the time of maximal concentration of the inhibitor. The distribution of [2-¹⁴C]DL-p-CP demonstrates this points (Table 1) where the liver has almost three times the p-CP per g of tissue as compared to the brain at six hours while the inhibition of phenylalanine 4-hydroxylase is about 15-20% against 50% for cerebral tryptophan 5-hydroxylase. This inhibition was measured with enzyme preparations at the first stage of purification to remove any free p-CP present in the crude supernatant solutions. This was necessary to eliminate the possibility of any competitive inhibition in vitro by p-CP or any of its metabolites. Of course, one cannot ignore the possibility that in addition to other factors (e.g., different rates of protein synthesis) there might be more active enzyme per g tissue of liver than that of the brain.

TABLE 1

Distribution of Radioactivity in Organs of the Rat Following i.p. Administration of [2-¹⁴C]DL-p-CP-Ethylester HCl

Rats (300 gm) were injected with 135 mg (0.51 mmol) of DL-[2-¹⁴C]p-CP-ethylester HCl (containing 7.6 × 10⁶ dis/min). Number of animals in brackets.

The inhibition of cerebral tryptophan hydroxylase is maximal at 48 hours while the level of p-CP is about 1/5th of that of the maximum concentration obtained at 6 hours (Table 2).

TABLE 2

Inhibition of Cerebral Tryptophan-5-Hydroxylase In Vivo by [2-C¹⁴]DL-p-CP and [2-C¹⁴]p-CPPA

Rats were injected with p-CP-ethylester HCl as in Table 1 and those given [2-C¹⁴]p-CPPA received 200 mg/kg (containing 6.6 × 10⁶ dis/min/animal). Number of animals in brackets.

It is noteworthy that at the time of maximal inhibition of the enzyme the distribution of p-CP in the different areas of the brain was very similar, averaging about 0.23 μmole/g.

D-p-CP and p-chlorophenylpyruvic acid (p-CPPA) were reported to be fully as potent as DL-p-CP (Koe and Weissman, 1966a). We first postulated that the active inhibitor might be p-CPPA or p-chlorophenylacetaldehyde the first product following its decarboxylation. This latter compound would be particularly reactive in forming Schiff bases with certain active groups of proteins. Although this hypothesis was unattractive since it failed to explain the specifity of inhibition, we nevertheless explored this aspect by administering different p-chloro-derivatives to rats which were then studied for their inhibition of mono-oxygenases. The following compounds produced absolutely no inactivation: p-chlorophenylacetic acid, p-chlorophenylacetaldehyde, p-chlorobenzaldehyde, p-chlorobenzalmalononitrile, and 3-chlorotyrosine. This latter compound was described as a major product following the action of Pseudomonas phenylalanine 4-hydroxylase on p-CP (Guroff, Kondo & Daly, 1966). The inhibition of p-CPPA was confirmed and studies on its metabolic fate were initiated with p-CPPA labeled in carbon-2.

In order to determine whether p-CP was converted to p-CPPA in the brain [¹⁴C]DL-p-CP was intravenously administered to rats which were killed 30 min later and the supernatant of the brain homogenate was analyzed for p-CPPA by isolating the 2,4-dinitrophenylhydrazone of labeled p-CPPA. It was found that 0.8% of the radioactivity present in the homogenates could be recovered in this derivative (Table 3).

TABLE 3

Extraction of p-CPPA-DNPH From Rat Brain 30 Minutes After IV Injection of DL-[2-C¹⁴]-p-CP-ethylester

Above are averages of four animals.

Animals received IV 30 mg (or 4.6 × 10⁷ dpm) p-CP-ethylester HCl.

*Corrected for recovery.

Therefore, there is clear evidence in vivo as well that p-CP serves as a substrate for transaminases. In 30 min this represented only 2.5 nmole of p-CPPA per g of brain – an amount too small to be significant as an inhibitor. In fact, administration of p-CPPA and the appearance of the same in much greater amounts in the brain failed to produce any inhibition within four hours and only 21% inhibition within six hours while at the same time equimolar amounts of DL-p-CP or even smaller amounts would lead to about 50% inhibition (Table 2). It was interesting to note this six-hour lag period before any inhibition of tryptophan hydroxylase appeared following p-CPPA administration particularly since the degree of enzymic inhibition by 24 hours was the same for both compounds. In view of these experiments and the fact that both p-CP and p-CPPA served as substrates for brain transaminases, we have pursued this problem by investigating the extent to which p-CPPA was converted to p-CP in vivo. A group of rats received 500 mg/kg of [¹⁴C]p-CPPA i.p. and were sacrificed four hours later at a time where the cerebral tryptophan 5-hydroxylase was not inhibited in spite of the almost toxic doses. Neither at 4 hours nor at 24 hours could any radioactivity be recovered in the ether extract of the acidified supernatant of the brain. The label remained in the aqueous layer. In contrast to this 1.2% of the label in the liver and about 16% of the plasma was ether-extractable. Over one-half of this radioactivity corresponded to p-CPPA while the remainder was identified as p-chlorophenylacetic acid. Of the original radioactivity from p-CPPA over 50% was recoverable in the acidified protein-free aqueous supernatant fraction. This activity was recovered as p-CP in all organs examined. The p-CP was identified by paper chromatography. Small amounts of [¹⁴C]tyrosine were also discernible from brain extracts – additional evidence for a degree of p-hydroxylation of halo-aromatic amino acids in vivo which was previously reported with p-fluorophenylalanine in vitro (Kaufman, 1961b. These experiments demonstrated very active transamination of p-CPPA to p-CP indicating that the inhibition was due to p-CP itself and not to p-CPPA or any of its other metabolites.

To explain the marked inhibition of some of the monooxygenases upon administration of D-p-CP the following working hypothesis was advanced. We have already demonstrated the reversibility of L-p-CP to p-CPPA. It was postulated that D-p-CP is rapidly converted to p-CPPA in the liver and kidneys of the animals by D-amino acid oxidase and that this compound then was enzymatically transaminated to L-p-CP – the latter being finally responsible for the inactivation of some of the hydroxylases. We have, in fact, experimentally established the inversion of D-p-CP to L-p-CP (Table 4). It is obvious that when D-p-CP is incubated with rat kidney or liver homogenates for 30 min marked conversion to p-CPPA occurs at pH 8.5. The incubation mixture was then adjusted to pH 7.5 with Tris-HCl buffer and fortified with pyridoxal phosphate and glutamic acid. The incubation was allowed to go another 15 min to enable an active transamination to take place. The demonstration of L-p-CP can be achieved as follows. The reaction was stopped with trichloroacetic acid and the supernatant obtained after centrifugation was re-adjusted to pH 7.5 with Tris and incubated with L-amino acid oxidase (crude Crotalus adamanteus venom) for an additional 20 min. The difference of p-CPPA value obtained after incubation with L-amino acid oxidase and that obtained from the TCA supernatant before transamination will correspond to L-p-CP formed from D-p-CP.

TABLE 4

Inversion of D-p-CP to L-p-CP by Rat Kidney and Liver Homogenates

5 μmol D-p-CP was added to the system.

*Determined by L-amino acid oxidase with Crotalus admanteus venom. All values were obtained by measuring the products formed against blanks from tissue samples without added D-p-CP.

It is to be noted that all values were obtained by measuring p-CPPA against blanks from tissue samples similarly incubated but without added D-p-CP. Furthermore, the D-p-CP sample was assayed with L-amino acid oxidase for any possible contamination by L-p-CP. In all instances the p-CPPA was determined by the FeCl3 method following its extraction into ether (Gál, Roggeveen & Millard, 1970) using the molar extinction of 1500 at 600 nm and by the spectrophotometric-assay in arsenate-borate buffer (Wellner, 1966) in which p-CPPA has a molar extinction coefficient of 9700 at 300 nm. Another demonstration of the inversion of D-p-CP to L-p-CP in vivo was obtained by injection of [2-¹⁴C]-D-p-CP into rats and the demonstration of incorporation of label into purified phenylalanine 4-hydroxylase. This experiment was based on our earlier studies in which we demonstrated the appearance of radioactivity as L-p-CP in purified hepatic phenylalanine hydroxylase, cerebral tryptophan 5-hydroxylase, and tyrosine 3-hydroxylase (Gál, Roggeveen & Millard, 1970). It is to be emphasized here that tyrosine 3-hydroxylase unlike the other two monooxygenases, is not inactivated irreversibly in spite of the presence of p-CP, assumedly in the enzyme protein. It is true for all three enzymes that p-CP as well as p-CPPA will competitively inhibit them in vitro (Figures 1 and 2). From these experiments the ki of p-CP of 3 × 10−4M (Jequier, Lovenberg & Sjoerdsma, 1967) for tryptophan hydroxylase is confirmed and is 1 × 10−3M for tyrosine hydroxylase. The ki of p-CPPA is 8 × 10−4M for tryptophan hydroxylase and 5 × 10−4M for tyrosine hydroxylase.

Figure 1 Kinetics of p) and p).

Figure 2 Kinetics of p) and p).

There is discernible competitive inhibition in vivo during the first 72 hours following administration of p-CP. This reversible competitive inhibition may account for reports of a 30% decrease in cerebral norepinephrine in these animals. Of course the competitive inhibition of phenylalanine 4-hydroxylase and tryptophan 5-hydroxylase, while being part of the slowing down of the enzyme activity in the early period does not explain the irreversible inactivation lasting 6 to 7 days.

The time course studies on both enzymic activity and incorporation of [¹⁴C]p-CP assayed with phenylalanine 4-hydroxylase purified to the Alumina Cγ stage also indicate (Gál and Millard, 1971) that the aspect of competitive inhibition is a minor component in vivo.

Active incorporation of p-chloro and p-fluorophenylalanine into partially purified monooxygenases has been previously demonstrated by us (Gál and Millard, 1971). These results revealed that while the incorporation of p-CP was about 54 dpm/mg enzyme protein for tryptophan 5-hydroxylase and 33 dpm/mg for tyrosine hydroxylase two days after administration of [¹⁴C]p-CP hydroxylation of tryptophan was inhibition by 85% whereas tyrosine hydroxylase retained its full activity when compared to enzyme from control animals. Incorporation of p-CP was due to an active protein synthesis utilizing p-CP to make p-CP AMP. This was demonstrated in vivo (Gál and Millard, 1971).

Our results revealed that cycloheximide or puromycin inhibited incorporation of p-CP into liver or brain protein when the inhibitors of protein synthesis were administered simultaneously with the p-haloamino acid. The inhibition was identical to that in control animals which received labelled tyrosine. However, when the animals received cycloheximide two hours prior to intraperitoneally administered p-CP the incorporation of p-CP was significantly prevented with consequent decrease in inhibition of enzyme activity (Table 5).

TABLE 5

Effect of Cycloheximide on Inhibition of Hepatic Phenylalanine-4-Hydroxylase by p-CP

Enzyme purified to AlCγ stage from combined livers of 3 animals; duplicate enzyme assays.

¹p-CP (300 mg/kg) administered i.p. 2 hrs after cycloheximide (3 mg/kg).

²Liver from single animal due to high mortality at 4 mg/kg cycloheximide.

Similar inhibition of incorporation into cerebral tryptophan hydroxylase could be achieved by intracerebral administration of cycloheximide. Intraperitoneally administered inhibitors of protein synthesis do not get in the brain in amounts sufficient to produce this effect unless given in very toxic doses which, of course, preclude long-term studies.

We must re-emphasize that it is critical that these assays be performed with at least partially purified enzymes. For instance, the amount of free p-CP recoverable from the liver over a 48 hour period can be as much as 350 μg/gm (Gál, Roggeveen & Millard, 1970); consequently, any system with the 100,000 × g supernatant from liver may contain 3-5 × 10−4M p-CP the Ki of p-CP being 3 × 10−4M (Jequier, Lovenberg & Sjoerdsma, 1967; Gál, Roggeveen & Millard, 1970).

It was reported that corticosteroid administration affects cerebral tryptophan hydroxylase activity as measured either by tryptophan hydrolase activity (Azmitia and McEwen, 1969) or by measurement of 5-HT turnover (Azmitia, Algeri & Costa, 1970). These experiments suggested that it might be possible to stimulate the de novo synthesis of tryptophan 5-hydroxylase in p-CP-treated animals by administration of ACTH.

Two groups of rats received a single intraperitoneal injection of 300 mg/kg p-CP. One group received 9 μ/kg ACTH 12 hours after p-CP and every 12 hours thereafter. Animals were sacrificed the second, third, fourth, and fifth days after p-CP and the 5-HT levels in the brains were determined with o-phthalaldehyde spectrophotofluorimetrically. A small but statistically significant increment in 5-HT resulted from administration of ACTH in the p-CP treated animals. This increment, when expressed in the ratio of experimental to control (A/B) appeared to be linear (Figure 3). Earlier, others (Guroff, 1969) as well as ourselves have demonstrated that injections of inhibitors of protein synthesis prevented the reappearance of phenylalanine 4-hydroxylase activity. This suggests that the reappearance of the enzyme activity is due to the de novo synthesis. Our experiments with ACTH approach this problem from the other direction and seem to lead us to the very same conclusion. It is, however, conceded that further experiments with p-CP and ACTH must be carried out in which tryptophan hydroxylase activity is directly assayed rather than 5-HT levels.

Figure 3 Stimulation of cerebral tryptophan 5-hydroxylase activity by ACTH (9 units/kg) in p-CP (300 mg/kg) pre-treated rats. A. p-CP and ACTH. B. p-CP. Each point represents an average of six animals.

During the course of this work, we successfully purified rat liver phenylalanine 4-hydroxylase to a degree of electrophoretic homogeneity consistent with increase of enzymatic activity. This purification was achieved by preparative gel electrophoresis of the enzyme protein obtained through the calcium gel stage of purification (Kaufman and Fisher, 1970). Details of the purification of the enzyme are described elsewhere¹ (Gál, 1972). This purification enabled us to compare the enzyme from control and p-CP treated animals. The results indicated that 100% inactivation of the enzyme would result from incorporation of one mole of p-CP into one mole of enzyme. According to the amino acid composition of the pure enzyme this would mean, for instance, replacing one phenylalanine residue out of 20 with p-CP.

Investigation of the physio-chemical difference between the pure enzyme protein of control and that of liver from p-CP-treated animals revealed no difference with respect to excitation and emission spectra, O.R.D. and C.D. spectra (Gál, 1972) nor with respect to movement by analytic electrophoresis in various buffers. However, treatment of the enzyme with mercaptoethanol and sodium dodecylsulphate for four hours followed by dialysis overnight resulted in the appearance of four major and one minor band from the control but only three bands from the "p-CP"-enzyme protein. The disappearance of band 5 (Figure 4, a, b) from the p-CP enzyme is intriguing. At present our studies are concerned with the separation of these bands by column chromatography in order to ascertain if the incorporation of p-CP is selective for one of the subunits only. Incorporation of p-CP also served to impart greater instability to the enzyme structure (Figure 4,c, d) as the electrophoretic pattern indicates for samples ran after two weeks of storage at −24°C. Finally, we had already demonstrated in vivo that formation of Phe∼t-RNA was 3:1 to that of p-CP∼t-RNA (Gál and Millard, 1971). Experiments with rat liver and brain extracts in vitro now also revealed that p-CP incorporation cannot be decreased by increasing the UUC ratio to UUU¹. This observation is dissimilar to that reported for cell-free E. coli system (Dunn and Leach, 1967).

Figure 4 Electrophoresis of pure phenylalanine hydroxylase on SDS (0.1%) containing polyacrylamide gels. Gels - a: p-CP enzyme 120 μg. b: Control enzyme, 120 μg. Electrolyte was 0.05 M phosphate-buffer at pH 7.0. Samples were run 8 hours at 25°C at 8 ma/tube. c,d: 100 μg samples of control and PCP enzyme after 3 weeks of storage at −24°C. 0.02 M Tris-glycine pH 8.75, length of run 23 hrs. at 6°C.

Current studies in our laboratories are aimed at confirming the non-random labeling of phenylalanine-4-hydroxylase by p-CP. We are now attempting to locate by fingerprinting the position of p-CP in the peptides from different subunits of the pure enzyme following its tryptic digestion.

The results reported here give further credence to the idea that the p-halophenylalanine bring about irreversible inactivation of phenylalanine-4-hydroxylase and tryptophan-5-hydroxylase by affecting the primary structure of these enzyme proteins though incorporation of p-CP near or at their active center. Hopefully the results of peptide sequencing will yield the final proof of molecular mechanism of p-CP produced inhibition proposed by us.

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¹Gál, in preparation.

¹Gál, in preparation.

ASSAYS AND PROPERTIES OF TRYPTOPHAN 5-HYDROXYLASE

J. Renson

Publisher Summary

This chapter provides an overview of assays and the properties of tryptophan 5-hydroxylase. The search for an enzymatic system responsible for the formation of 5-hydroxytryptophan (5-HTP)—the direct precursor of 5-hydroxytryptamine (5-HT)—was first directed toward a microorganism called Chromobaoterium violaeeum. It was because chemical analysis of violacein—a purple pigment produced by this organism—had revealed the existence of a 5-hydroxyindole moiety. Hydroxylation of tryptophan in a cell-free system was reported in mammalian intestinal mucosa. The assay of brain tryptophan hydroxylase requires a radioisotopic method. Several rapid and sensitive assays for brain tryptophan hydroxylase are now available that have partially unraveled several intriguing properties of tryptophan 5-hydroxylase. The complexity of the regulation of hepatic phenylalanine hydroxylase—another enzyme using tetrahydrobiopterin as an electron donor source—illustrates and provides a model to pursue the purification of tryptophan hydroxylase to elucidate its regulatory properties.

A new phenolic substance, detected as early as 1933 by various histochemical stains in the enterochromaffin cells of gastrointestinal mucosa (hence its name, enteramine), was isolated and studied for many years by Erspamer who edited the most comprehensive review about indole-alkylamines (Erspamer, 1966).

In the United States, a group led by Page (1968) was studying a vasoconstrictive substance found in serum and called serotonin. Their work culminated in the elucidating work of Rapport (1948) who demonstrated the exact chemical structure of serotonin to be the creatinine sulfate complex of 5-hydroxytryptamine (5-HT).

By 1951, total chemical synthesis by several laboratories demonstrated that enteramine and serotonin were identical. The indolic nature of this new biogenic amine immediately led to the hypothesis that it was derived from tryptophan, an essential amino acid.

The first experimental demonstration that tryptophan was the dietary precursor of 5-HT came from the studies of Udenfriend, Titus, Weissbach and Peterson (1956) who demonstrated that administration of radioactive tryptophan to the toad Bufo marinus and the rabbit, led to the formation of radioactive 5-HT.

The search for an enzymatic system responsible for the formation of 5-hydroxytryptophan (5-HTP), the direct precursor of 5-HT, was first directed toward a microorganism called Chromobacterium violaceum because chemical analysis of violacein, a purple pigment produced by this organism, had revealed the existence of a 5-hydroxyindole moiety. Mitoma, Weissbach and Udenfriend (1956) were able to isolate 5-hydroxytryptophan from the growth medium. Resting cells of C. violaceum, when incubated in the presence of L-tryptophan, could also produce sufficient amounts of 5-HTP to be detected by the nitrosonaphthol colorimetric assay of Udenfriend and his co-workers (Udenfriend, Weissbach & Clark, 1955). Further attempts to obtain a cell-free system capable of hydroxylating tryptophan in this organism have been unsuccessful thus far.

Hydroxylation of tryptophan in a cell-free system was reported for the first time by Cooper and Melcer (1961) in mammalian intestinal mucosa. Like the non-enzymatic hydroxylating system described by Udenfriend, Clark, Axelrod and Brodie (1954), Cooper’s preparation required ascorbic acid and cupric ions, but no molecular oxygen. This biological system was investigated further by Renson, Weissbach and Udenfriend (1962) and was found to be of no physiological importance, because it was due to a non-enzymatic hydroxylation of tryptophan coupled to enzymic decarboxylation of L-5-HTP and L-tryptophan. The system described by Cooper was clearly an artifact.

Freedland, Wadzinski and Waisman (1961) reported the first unequivocal demonstration of enzymatic formation of 5-HT in a cell-free system using rat liver homogenate in the presence of a large concentration of L-tryptophan (1.6 × 10−2M). This tryptophan hydroxylase activity was shown to be due to phenylalanine hydroxylase (EC 1.14.3.1), the enzyme controlling the catabolism of phenylalanine via tyrosine formation (Renson, Goodwin, Weissbach & Udenfriend, 1961). It was further demonstrated that this tryptophan activity of liver phenylalanine hydroxylase had no physiological significance for serotonin biosynthesis in the brain (Renson, Weissbach & Udenfriend, 1962).

A major breakthrough in the study of aromatic amino acid hydroxylases of phenylalanine, tyrosine, and tryptophan was accomplished by Kaufman (1958) in the course of his work on phenylalanine hydroxylase purification. He discovered that a boiled rat liver extract contained an unknown cofactor necessary for the hydroxylation of phenylalanine. In a series of elegant studies, Kaufman (1963) established that this natural compound necessary for phenylalanine hydroxylation was dihydro-biopterin (BH2) or dihydro-2-amino-4-hydroxy-6-[1,2-dihydroxypropyl-(L-erythro)]-pteridine. A new enzyme necessary to reduce this cofactor to its tetrahydro form (BH4) was purified and shown to utilize NADPH as a reducing cofactor (Kaufman, 1961a). Several synthetic unconjugated pteridines were found to be convenient substitutes for the natural and more unstable tetrahydrobiopterin. The most commonly used is 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine (DMPH4). Subsequent in vitro studies, however, have shown that substitution of the natural cofactor by these synthetic compounds alters the kinetic parameters of all hydroxylases studied so far (Friedman, Kappelman & Kaufman, 1972).

Mast cells in rodents normally contain, in addition to histamine and heparin, a significant amount of serotonin (Sjoerdsma, Waalkes & Weissbach, 1957). A murine mastocytoma, which could be transplanted and grown in an ascitic form in mice, was used by Lovenberg, Levine and Sjoerdsma (1965) to obtain a cell-free system capable of hydroxylating both tryptophan and phenylalanine in the presence of DMPH4 and ferrous ion. Further purification of this tryptophan hydroxylating system permitted the conclusion that this enzyme was different from liver phenylalanine hydroxylase and brain tryptophan hydroxylase (Sato, Jequier, Lovenberg & Sjoerdsma, 1967).

The first evidence of tryptophan hydroxylase activity in the brain was made by Gál, Poczik and Marshall (1963). Intracerebral administration of labelled tryptophan in the rat and pigeon was followed by local formation of radioactive 5-HT. Within the next three years, using radioactive tryptophan and the new pteridine cofactor substances discovered earlier by Kaufman, specific hydroxylases for tryptophan were reported in dog and rabbit brain (Grahame-Smith, 1964, 1967), and in carcinoid tissue, rat brain and mammalian pineal (Lovenberg, Jequier & Sjoerdsma, 1967).

Both tryptophan hydroxylating systems from C. violaceum and from murine mast cells were employed to develop a novel assay with 5-³H-tryptophan and to discover an intramolecular migration of tritium during enzymatic tryptophan hydroxylation (Renson, Daly, Weissbach, Witkop & Udenfriend, 1966).

Assays of Tryptophan Hydroxylase

The assay of brain tryptophan hydroxylase (EC 1.14.3.3) generally requires a radioisotopic method. The first demonstration in mammalian brain of this enzymatic activity in a cell-free system was achieved by Grahame-Smith (1964, 1967). At that time, an elaborate and time-consuming procedure involving a series of purification and isolation steps by column and paper chromatography was necessary. Such methodology was not suitable for subsequent studies of this enzyme. Other methods, more rapid and sensitive, had to be found before further progress could be achieved. Indeed, an interesting property of several aromatic hydroxylases was discovered while trying to devise a new assay procedure using 5-³H-tryptophan as a substrate (Renson, Daly, Weissbach & Udenfriend, 1966).

1 Methods Using Ring Labelled ¹⁴C-Tryptophan or ³H-(U)-Tryptophan

A common feature of these methods is the requirement that the substrate must be isolated from 5-HTP or 5-HT. Håkanson and Hoffman (1967) for instance, incubated intact rat pineals in the presence of a decarboxylase inhibitor and were able to separate tryptophan from 5-HTP satisfactorily by thin-layer chromatography. The most useful assay in this category was proposed by Lovenberg, Jequier and Sjoerdsma (1967). The originality and usefulness of this assay is based on the low affinity of tryptophan for aromatic L-amino acid decarboxylase (Km ± 1.4 × 10−2M) compared to the high affinity of the same enzyme for 5-HTP (Km ± 5.4 × 10−6M). After tryptophan hydroxylation, a second enzymatic step using a purified decarboxylase preparation is necessary. Following the hydroxylation step, a known amount of cold 5-HTP is added. This assay requires measurement of the specific activity of 5-HT enzymatically formed and isolated by column chromatography. Although this method gives invaluable information, it is time consuming; it involves two separate enzymic incubations – the assay of 5-HT by fluorescence, and the counting of radioactive 5-HT isolated by column