Tryptophan pyrrolase in haem regulation. Experiments with administered haematin and the relationship between the haem saturation of tryptophan pyrrolase and the activity of 5-aminolaevulinate synthase in rat liver.

1. Administration of haematin to rats decreases 5-aminolaevulinate synthase activity in whole liver homogenates. 2. An inverse relationship between this decrease and the increase in saturation of apo-(tryptophan pyrrolase) with haem is observed during the initial phase of treatment with haematin. 3. Significant changes in both functions are caused by a 1 mg/kg dose of haematin, whereas the maximum effects are achieved by the 5 mg/kg dose. 4. Prevention by allopurinol of the conjugation of exogenously administered haematin with apo-(tryptophan pyrrolase) renders this haem available for further repression of 5 aminolaevulinate synthase. 5. The various aspects of the relationship between synthase activity and the haem saturation of tryptophan pyrrolase are discussed.     

The effects of acetate, metal cations, phenobarbitone, porphyrogens and substrates of glycine acyltransferase on the utilization of haem by rat liver apo-(tryptophan pyrrolase).

1. The utilization of haem by rat liver apo-(tryptophan pyrrolase) under basal conditions and after enhancement of the enzyme activity by various mechanisms was studied under the influence of treatments affecting various aspects of liver haem metabolism. 2. These treatments were: benzoate and p-aminobenzoate as substrates of glycine acyltransferase, acetate as an inhibitor of 5-aminolaevulinate synthase activity, enhancement of 5-aminolaevulinate dehydratase by aluminium, destruction of haem and inhibition of ferrochelatase by porphyrogens, increased haem utilization by phenobarbitone and enhancement of haem oxygenase activity by metal cations. 3. The results show that the haem saturation of the apoenzyme is sensitive to all these treatments. 4. The possible usefulness of tryptophan pyrrolase in studying the regulation of liver haem is suggested.     

The effects of chemical porphyrogens and drugs on the activity of rat liver tryptophan pyrrolase

1. Drugs such as phenobarbitone and phenylbutazone, which increase the concentration of microsomal haem and cytochrome P-450, also increase the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator, as does the haem precursor 5-aminolaevulinate. 2. At 4h after the administration of the porphyrogens 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and griseofulvin, the total pyrrolase activity is increased whereas the haem saturation of the apoenzyme is decreased. This decreased saturation is prevented by pretreatment of the animals with the inhibitor of drug-metabolizing enzymes, SKF 525-A. 3. Pretreatment of rats with the above porphyrogens inhibits the rise in holo-(tryptophan pyrrolase) activity produced by subsequent administration of cortisol, tryptophan and 5-aminolaevulinate with two single exceptions, the possible reasons for which are discussed. 4. At 24h after the administration, in starved rats, of a single daily injection of the above porphyrogens for 1 or 2 days, the holoenzyme activity is significantly increased. 5. It is suggested that the saturation of rat liver apo-(tryptophan pyrrolase) with its haem activator can be modified by treatment known to cause destruction, inhibition of synthesis, increased utilization and enhanced synthesis of liver haem. The possible involvement of the latter phenomenon in the aetiology of mental disorders in some patients with porphyria is discussed.     

Tryptophan pyrrolase in haem regulation. The mechanisms of enhancement of rat liver 5-aminolaevulinate synthase activity by starvation and of the glucose effect on induction of the enzyme by 2-allyl-2-isopropylacetamide

1. Rat liver tryptophan pyrrolase activity is enhanced by a hormonal-type mechanism during the first 2 days of starvation and by a substrate-type mechanism during the subsequent 2 days. 5-Aminolaevulinate synthase activity is also enhanced during the first 2 days of starvation, but returns thereafter to values resembling those observed in the fed rat. Treatments that prevent or reversé the enhancement of tryptophan pyrrolase activity in 24–48h-starved rats also abolish that of 5-aminolaevulinate synthase activity. Starvation of guinea pigs, which does not enhance the pyrrolase activity, also fails to alter that of the synthase. It is suggested that the decrease in 5-aminolaevulinate synthase activity in 72–96h-starved rats represents negative-feedback repression of synthesis, possibly involving tryptophan participation, whereas the enhancement observed in 24–48h-starved animals is caused by positive-feedback induction secondarily to increased utilization of the regulatory-haem pool by the newly synthesized apo-(tryptophan pyrrolase). 2. Glucose, fructose and sucrose abolish the 24h-starvation-induced increases in rat liver tryptophan pyrrolase and 5-aminolaevulinate synthase activities. Cortisol reverses the glucose effect on 5-aminolaevulinate synthase activity, presumably by enabling pyrrolase to re-utilize the regulatory-haem pool after induction of synthesis of this latter enzyme. 3. The impaired ability of 2-allyl-2-isopropylacetamide to enhance markedly 5-aminolaevulinate synthase activity in 24h-starved rats treated with glucose is associated with a failure of the porphyrogen to cause loss of tryptophan pyrrolase haem. Cortisol restores the ability of the porphyrogen to destroy tryptophan pyrrolase haem and to enhance markedly 5-aminolaevulinate synthase activity, presumably by enhancing tryptophan pyrrolase synthesis and, thereby, its re-utilization of the regulatory-haem pool. It is tentatively suggested that 2-allyl-2-isopropylacetamide destroys the above pool only after it has become bound to (or utilized by) apo-(tryptophan pyrrolase).     

Regulation of rat liver tryptophan pyrrolase by its cofactor haem: Experiments with haematin and 5-aminolaevulinate and comparison with the substrate and hormonal mechanisms.

1. The administration of haematin or 5-aminolaevulinate to rat enhances the activity of liver tryptophan pyrrolase; both endogenous and newly formed apoenzymes become strongly haem-saturated. Haem activation does not stabilize tryptophan pyrrolase. 2. Actinomycin D, puromycin or cycloheximide prevent the activation of the enzyme by 5-aminolaevulinate but not that by haematin. The latter is inhibited by haem-destroying porphyrogens. 3. The combined injection of either haematin or 5-aminolaevulinate with cortisol does not produce an additive effect, whereas potentation is observed when tryptophan is jointly given with either the cofactor or the haem precursor. 4. Further experiments on the substrate (tryptophan) mechanism of pyrrolase regulation are reported, and a comparison between this and the cofactor and hormonal mechanisms is made. 5. It is suggested that the substrate mechanism may also involve increased haem synthesis. 6. The role of tryptophan pyrrolase in the utilization of liver haem, and as a possible model for the exacerbation by drugs of human hepatic porphyrias, is discussed.     

Tryptophan pyrrolase in haem regulation. The relationship between the depletion of rat liver tryptophan pyrrolase haem and the enhancement of 5-aminolaevulinate synthase activity by 2-allyl-2-isopropylacetamide.

Rat liver tryptophan pyrrolase haem is maximally depleted at 30 min after administration of a 400 mg/kg dose of 2-allyl-2-isopropylacetamide. This depletion lasts for 24 h, by which time 5-aminoleevulinate synthase activity becomes maximally enhanced. 2. though the above maximum depletion of pyrrolase haem (at 0.5h) is also produced by a 100 mg/kg dose of the porphyrogen, this does not enhance synthase activity at 24 h. It and smaller doses, however, cause a smaller but earlier enhancement of synthase activity (maximum at 2 h) and produce a similarly short-lived deplation of pyrrolase haem. 3. The depletion of pyrrolase haem and the enhancement of synthase activity by the porphyrogen are inhibited by compound SKF 525-A and phenazine methosulphate, and are potentiated by nicotinamide but not by phenobarbitone. Phenazine methosulphate and nicotinamide also exert opposite effects on hexobarbital sleeping-time. 4. 2-Allyl-2-isopropylacetamde also the depletes pyrrolase haem in vitro. It does so in liver homogenates of control rats in the presence, and in those of phenobarbitone-treated rats in the absence of added NADPH. 5. A discussion of the present results in relation to previous work with other haemoproteins suggests that, whereas cytochrome P-450 (haem) is primarily involved in the production of the active (porphyrogenic) metabolite(s) of 2-allyl-2-isopropylacetamide, the haem pool used by tryptophan pyrrolase may play an important role in the effects of this compound on haem biosynthesis.     

Effect of endotoxin on tryptophan pyrrolase and delta-aminolaevulinate synthase: evidence for an endogenous regulatory haem fraction in rat liver.

Endotoxin was administered to rats at a dose shown previously to stimulate hepatic haem oxygenase activity and to block induction of delta-aminolaevulinate synthase, apparently by causing redistribution of haem from cytochrome P-450 to a regulatory haem pool in the liver. Within 5h of the administration of endotoxin (at a time when the effect of the compound on cytochrome P-450 is maximal) the relative saturation of tryptophan pyrrolase with intrinsic haem rose, from a basal value of 50% to 90%, indicating that 'free' haem had become available. Concurrently, the activity of delta-aminolaevulinate synthase was decreased to 25% of its basal value. Haem oxygenase reached peak activity 13h after endotoxin administration. These findings provide new evidence for the existence of an 'unassigned' hepatic haem fraction, which exchanges with cytochrome P-450 haem and regulates these three enzyme functions.     

Tryptophan catabolism by tryptophan pyrrolase in rat liver. The effect of tryptophan loads and changes in tryptophan pyrrolase activity.

We investigated how changes in tryptophan pyrrolase activity and tryptophan loads affect the breakdown of tryptophan was estimated by injecting rats with [ring-2-14-C]tryptophan and measuring respiratory 14-CO2. We concluded, contrary to previous reports, that induction of tryptophan pyrrolase definitely will increase the rate of tryptophan breakdown. Tryptophan loads also increase tryptophan breakdown even in circumstances where there is no increase in tryptophan pyrrolase activity, presumably by increasing the saturation of the enzyme. After a tryptophan load (50 mg per kg) the increase in liver tryptophan concentration lasts only 30 min. The rapid return of liver tryptophan to normal may be due partly to the high turnover rate of liver tryptophan. We estimate that tryptophan pyrrolase degrades tryptophan in vivo at a rate that is equivalent to the whole liver tryptophan concentration in 7.5 min or less.     

Effects of the haem precursor 5-aminolaevulinate on tryptophan metabolism and disposition in the rat.

5-Aminolaevulinate administration to rats inhibits cerebral 5-hydroxytryptamine synthesis by decreasing tryptophan availability to the brain secondarily to activation of hepatic tryptophan pyrrolase. The results show that tryptophan metabolism and disposition can be influenced by changes in liver haem concentration, and are discussed briefly in relation to mood disorders in the hepatic porphyrias.     

The functions and regulation of tryptophan pyrrolase.

The regulation and functions of rat liver tryptophan pyrrolase are reviewed. The enzyme is regulated by four mechanisms: hormonal induction by glucocorticoids, substrate activation and stabilization by tryptophan, cofactor activation by haem and feedback inhibition by NAD(P)H. Possible functions of the pyrrolase are the detoxication of tryptophan and the regulation of brain 5-hydroxytryptamine synthesis, liver haem metabolism, synthesis of nicotinamide-adenine dinucleotides (phosphates) and hepatic gluconeogenesis.It is suggested that the regulation and functions of tryptophan pyrrolase are physiologically interrelated, and that the enzyme may play important roles in vital body processes.     

Tryptophan pyrrolase in haem regulation. The mechanism of the permissive effect of cortisol on the enhancement of 5-aminolaevulinate synthase activity by 2-allyl-2-isopropylacetamide in the adrenalectomized-rat liver.

The decreased ability of 2-allyl-2-isopropropylacetamide to enhance liver 5-amino-laevulinate synthase activity in the adrenalectomized rat is not associated with a marked depletion of the already low amount of tryptophan pyrrolase haem. Cortisol permits the porphyrogen markedly to enhance synthase activity by rendering it capable of causing a stronger depletion of pyrrolase haem, presumably as result of hormonal induction of pyrrolase synthesis.     

Enhancement of rat brain tryptophan metabolism by chronic ethanol administration and possible involvement of decreased liver tryptophan pyrrolase activity.

1. Chronic ethanol administration enhances rat brain 5-hydroxytryptamine synthesis by increasing the availability of circulating tryptophan to the brain. This increased availability is not insulin-mediated or lipolysis-dependent. 2. Under these conditions, tryptophan accumulates in the liver and apo-(tryptophan pyrrolase) activity is completely abolished, but could be restored by administration of regenerators of liver NAD+ and/or NADP+. 3. All four regenerators used (fructose, Methylene Blue, phenazine methosulphate and sodium pyruvate) prevented the ethanol-induced increase in liver tryptophan concentration and the increased availability of tryptophan to the brain. 4. It is suggested that the enhancement of brain tryptophan metabolism by chronic ethanol administration is caused by the decreased hepatic tryptophan pyrrolase activity. The results are briefly discussed in relation to previous work with ethanol. 5. Fructose enhances the conversion of tryptophan into 5-hydroxyindol-3-ylacetic acid in brains of ethanol-treated rats, whereas Methylene Blue inhibits this conversion in both control and ethanol-treated animals.     

Tryptophan pyrrolase, the regulatory free haem and hepatic porphyrias. Early depletion of haem by clinical and experimental exacerbators of porphyria.

1. The importance of the early depletion of liver haem in the production of porphyria is discussed and further supporting evidence is presented from experiments with tryptophan pyrrolase, under conditions of exacerbation of experimental porphyria by therapeutic and other agents. 2. In addition to the early depletion of pyrrolase haem by porphyrogens, a further depletion is produced when rats are given a porphyrogen plus an analogue or one of 19 drugs known to exacerbate the human disease. 3. Non-exacerbators of human porphyrias do not cause a further early depletion of pyrrolase haem and it is suggested that this system may be used as a screening test for possible exacerbation of the disease by new and existing drugs. 4. A similar further early depletion of haem is produced by combined administration of lead acetate plus phenobarbitone, thus suggesting that the depletion is a more general phenomenon in experimental porphyria. 5. The relationship between tryptophan pyrrolase and the regulatory free haem is discussed. It is suggested that pyrrolase may play an important role in the regulation of haem biosynthesis.     

Liver tryptophan pyrrolase. A major determinant of the lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL mice.

The lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL, compared with -non-preferring CBA, mice is caused by a decrease in circulating tryptophan availability to the brain secondarily to a higher liver tryptophan pyrrolase activity associated with a higher circulating corticosterone concentration. Activity or expression of liver tryptophan pyrrolase and/or their induction by glucocorticoids may be important biological determinants of predisposition to alcohol consumption.     

Guinea-pig liver tryptophan pyrrolase. Absence of detectable apoenzyme activity and of hormonal induction by cortisol and possible regulation by tryptophan

1. When assayed in fresh homogenates, guinea-pig liver tryptophan pyrrolase exists only as holoenzyme. It does not respond to agents that activate or inhibit the rat liver enzyme in vitro. Only by aging (for 30min at 5 degrees C) does the guinea-pig enzyme develop a requirement for ascorbate. 2. The guinea-pig liver enzyme is activated by the administration of tryptophan but not cortisol, salicylate, ethanol or 5-aminolaevulinate. 3. The tryptophan enhancement of the guinea-pig liver pyrrolase activity is prevented by 0, 34 and 86% by pretreatment with actinomycin D, cycloheximide or allopurinol respectively. 4. The guinea-pig liver tryptophan pyrrolase is more sensitive to tryptophan administration than is the rat enzyme. On the other hand, the concentrations of tryptophan in sera and livers of guinea pigs are 45-52% less than those in rats. 5. It is suggested that tryptophan may regulate the activity of guinea-pig liver tryptophan pyrrolase by mobilizing a latent form of the enzyme whose primary function is the detoxication of its substrate.     

Possible involvement of the enhanced tryptophan pyrrolase activity in the corticosterone- and starvation-induced increases in concentrations of nicotinamide-adenine dinucleotides (phosphates) in rat liver.

1. Deoxycorticosterone, which does not enhance tryptophan pyrrolase activity, also fails to alter the concentrations of the NAD(P) couples in livers of fed rats, whereas corticosterone increases both pyrrolase activity and dinucleotide concentrations. 2. Starvation of rats increases serum corticosterone concentration, lipolysis, tryptophan availability to the liver, tryptophan pyrrolase activity and liver [NADP(H)]. Glucose prevents all these changes. 3. The beta-adrenoceptor-blocking agent propranolol prevents the starvation-induced lipolysis and the consequent increase in tryptophan availability to the liver, but does not influence the increase in serum corticosterone concentration, liver pyrrolase activity and [NADP(H)]. 4. Actinomycin D, which prevents the starvation-induced increases in liver pyrrolase activity and [NADP(H)], does not affect those in serum corticosterone concentration and tryptophan availability to the liver. 5. Allopurinol, which blocks the starvation-induced enhancement of pyrrolase activity, also abolishes the increases in liver [NADP(H)], but not those in serum corticosterone concentration or tryptophan availability to the liver. 6. It is suggested that liver tryptophan pyrrolase activity plays an important role in NAD+ synthesis from tryptophan in the rat.     

Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450.

After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic 'free' haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.     

Role of haem in the induction of cytochrome P-450 by phenobarbitone. Studies in chick embryos in ovo and in cultured chick embryo hepatocytes.

The role of haem synthesis during induction of hepatic cytochrome P-450 haemoproteins was studied in chick embryo in ovo and in chick embryos hepatocytes cultured under chemically defined conditions. 1. Phenobarbitone caused a prompt increase in the activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, and in the concentration of cytochrome P-450. This induction response occurred without measurable initial destruction of the haem moiety of cytochrome P-450. 2. When intracellular haem availability was enhanced by exogenous haem or 5-aminolaevulinate, phenobarbitone-medicated induction of cytochrome P-450 was not affected in spite of the well known repression of 5-aminolaevulinate synthase by haem. These data are consistent with the concept that haem does not regulate the synthesis of cytochrome P-450 haemoproteins. 3. Acetate inhibited haem biosynthesis at the level of 5-aminolaevulinate formation. When intracellular haem availability was diminished by treatment with acetate, phenobarbitone-medicated induction was decreased. 4. This inhibitory effect of acetate on cytochrome P-450 induction was reversed by exogenous haem or its precursor 5-aminolaevulinate. These data suggest that inhibition of haem biosynthesis does not decrease synthesis of apo-cytochrome P-450. Moreover, they indicate that exogenous haem can be incorporated into newly formed aop-cytochrome P-450.     

The role of liver tryptophan pyrrolase in the opposite effects of chronic administration and subsequent withdrawal of drugs of dependence on rat brain tryptophan metabolism.

1. Chronic administration of morphine, nicotine or phenobarbitone has previously been shown to inhibit rat liver tryptophan pyrrolase activity by increasing hepatic [NADPH], whereas subsequent withdrawal enhances pyrrolase activity by a hormonal-type mechanism. 2. It is now shown that this enhancement is associated with an increase in the concentration of serum corticosterone. 3. Chronic administration of the above drugs enhances, whereas subsequent withdrawal inhibits, brain 5-hydroxytryptamine synthesis. Under both conditions, tryptophan availability to the brain is altered in the appropriate direction. 4. The chronic drug-induced enhancement of brain tryptophan metabolism is reversed by phenazine methosulphate, whereas the withdrawal-induced inhibition is prevented by nicotinamide. 5. The chronic morphine-induced changes in liver [NADPH], pyrrolase activity, tryptophan availability to the brain and brain 5-hydroxytryptamine synthesis are all reversed by the opiate antagonist naloxone. 6. It is suggested that the opposite effects on brain tryptophan metabolism of chronic administration and subsequent withdrawal of the above drugs of dependence are mediated by the changes in liver tryptophan pyrrolase activity. 6. Similar conclusions based on similar findings have previously been made in relation to chronic administration and subsequent withdrawal of ethanol. These findings with all four drugs are briefly discussed in relation to previous work and the mechanism(s) of drug dependence.     

Role of tryptophan pyrrolase in endotoxin poisoning

Using substrate induction as a tool, we attempted to determine the role of tryptophan pyrrolase in the response to endotoxin in mice. Previous results have shown that the administration of the ld(50) of endotoxin lowers tryptophan pyrrolase activity. alpha-Methyltryptophan was found to maintain tryptophan pyrrolase activity above control levels in endotoxin-poisoned mice without increasing survival. 5-Hydroxytryptophan, by contrast, lowered tryptophan pyrrolase activity but did not sensitize mice to endotoxin. These results suggest that tryptophan pyrrolase per se does not play a unique role in survival of mice poisoned with endotoxin. This enzyme, however, may reflect the fate of other liver enzymes inducible by adrenocorticoids. In mice given concurrent injections of tryptophan and endotoxin, tryptophan pyrrolase activity was elevated to a level intermediate between that of normal mice and that of mice given tryptophan alone. The mice injected with tryptophan and endotoxin also had about the same mortality as mice given endotoxin alone. Mice treated with tryptophan 4 hr after endotoxin, at a time when the substrate did not fully elevate tryptophan pyrrolase activity, died convulsively and in larger numbers than those given endotoxin alone. This effect was reversed by prior treatment with cyproheptadine, an antiserotonin drug. These results indicate that the depression of tryptophan pyrrolase activity previously observed in vitro after injection of endotoxin reflects an actual decrease in the in vivo activity of the enzyme.