- Email: [email protected]
This and That: on color and catecholamines
TiPS - February 1989 [Vol. 101
ho from diskette. Move the cursor to cell Bl and enter ‘ANALYSIS OF VARIANCE TEMPLATE’. Move the cursor to cell A3 and enter ‘Replace headings a, b, c, etc. with your own headings’. In cell A4, enter ‘Replace values in the block Bll to K20 with your own values’. In cell A5 enter the message ‘In rows 21-25 there will be a number of cells that contain ERR’. Move to cell A6 to finish the sentence, ‘erase these cells with the /RE command’. q q 0 Human nature dictates that some readers will ask why they should
reinvent
the
wheel,
even
include their names, addresses, telephone numbers and electronic mail addresses so that users can consult them directly.
though what we have described is not difficult. Some people will not have the time or the temperament to develop templates for their laboratories, so a pooling of effort may be in order. One of us (KN) would be willing to act as a point of collection and dispatch of LOTUS templates as long as the costs were covered. For about $5 to cover diskettes and handiing, copies of a collection of templates can be made and mailed to anyone on request. Send copies of templates that have been developed as well as requests for solutions to prob. lems. The templates should contain a brief description and instructions. Authors should also
KANJI NAKATSU
AND JAMES A. OWEN*
Depnrfme~lf of Phnrmacology nnd Toxicology ntzd *Depnrhmenf of Psychiatry, Queen’s University, Kingston. OMario, Cunndn K7L 3N6. Electronic Mail:
PHARDEPT@QLJCDN. Or.bitnet
netnorth
References 1 Wardlaw, A. C. (1985) Practical Statistics for Experi)rletltnl Biologists, John Wiley & sons 2 Tallerida, R. J. and Murray, R. B. (1986) Mamcnl of PharnmcologicCalculntiom wifh Conrpufer Progrows, Springer-Verlag 3 Zivin, J. A. and Bartko, J. J. (1976) Life Sci. 18, 15-26
hat: on color and catecholamines feathers. In skin, whether the dark low-sulfur eumelanins or the iighter high-sulfur phaeomelanins are formed appears to be deter-
A VISIT to Italy a few months ago, I ran into my old friend, Professor Nero Porpora, who had recently retired. He was sitting in a caf15, staring intentlv at a oassing girl. Veil& her ghoulde& wab a melanins are high sulfur, yellowcascade of that exquisite red ish to reddish brown pigments hair so characteristic of norrather similar to the trichothem Italy, and so elegantly chromes, which are reddish.’ captured by Titian in his por‘What controls which pigment is trait of La Maddelena. formed?’ I asked.
ON
I knew Nero was susceptible to what used to be the tenderer sex, so as I joined him I asked mischievously; ‘Penny for your thoughts. What are you thinking about? He looked up with a smile of recognition. ‘Phaeomelanins and trichochromes,’ he said tersely. I was not to be put off. Nero was famous for his Delphic utterances which on further probing proved to be the key to a flood of insight. I drew up a chair. ‘You were looking pointedly at that girl,’ I said pointedly. ‘The redhead.‘ ‘C&o’, he enlarged. ‘Her hair is most attractive. But I was thinking that the substance responsible for that wonderful color is also the pigment in chicken feathers.’ He pulled over a napkin and began to draw (Fig. 1). ‘You see,’ he continued, ‘tyrosine is more than a precursor for the catecholamines. Melanocytes convert tyrosine to three kinds of pigment by reaction with cysteine and glutathione. The color you get depends on the sulfur content. Capisci? The eumelanins are insoluble, low-sulfur, brown or black pigments. The phaeo-
mined by the sulfhydryl content of the melanocyte. Melanocytes in culture produce black pigments in the absence of glutathione and cysteine, but the light phaeomelanins if these substances are added to the tissue medium’. In humans, black skin contains less glutathione than white2. In suntanned skin, there are falls in the levels of glutathione and glutathione reductase. So you see,’ he ended, ‘minor details of sulfhydryl regulation in the melanocyte provide the biochemical basis for the color prejudices which darken
‘Ah!’ he said, reaching for a fresh napkin, ‘DOPA via its semiquinone can form a number of cysteinyl adducts’ (Fig. 2). ‘The commonest is at the 5-position, although 2-, 6-, 2,5- and other adducts are also formed. Further oxidation of these adducts yields the pigments. The trichochromes are found in red hair and chicken
Tyrosine
DOPA
~“me./
\
Phaeomelanins (Reddish-brown)
(Brown-black) Trichochromes
(Yellow, Red, Violet) Fig.
1. Rainbow
biochemistry:
some
pigments
deriving
from tyrosine.
TiPS - February 1989 [Vol. IO]
61
DOPA Fig. 2. Chicken feathers and trichochromes: cysteinyl-DOPA intermediates in the biosynthesis of phaeomelanins and trichochromes.
so much of the world.’ He put down his pencil and leaned back. ‘Yes,’ he said absently.
That woman had beautiful hair, but. . .’ He fell silent while he took an appreciative sip of coffee.
‘But,’. he ‘in my bat k _ continued, _ yard 1 have some beautiful chickens.’
ONE OF THE most famed and highly regarded pigments of all time is not a tyrosine but a tryptophan metabolite, 6,6’-dibromoindigotin. The dye, Tyrian purple, is produced from molluscs such as the levantine~Mur&. This dye dates to Old TestaIn the human habit of equating ment times, the Book of Exodus rareness with value without regard talking of the tabernacle curto utility, the wearing of clothing tains of ‘blue and purple and dyed with the purple was removed scarlet’. for the Roman emperor. L&eThe dye was so named because majeste’ in this regard was a capital one of the centers of production offense. Not that the right to flaunt was Tyre, or Sur, in modern the pwpura hyacintha, as the
Tiberius died of natural causes, Tiberius attaining that happy state by hiding for 11 years on the island of Capri, where no one could get at him. Those wearing the imperial purple reigned for a cumulative 50 years.
Lebanon. The loquacious Pliny the Elder, in one of his rare happy phrases, likened Tyrian purple to, ‘the color of clotted blood, dark by reflected and brilliant by transmitted light’. No purple compound is actually present in the dye-producing glands of the mollusc. When exposed to fight, the white fluid in the glands is converted through a sequence of colors, green to blue to red to deep purple. Whether the ability to make the ‘multitudinous seas incarnidine’ affords any advantage to its producer is unknown. Many of the more than 300 species of Murex are toxic, and the gland appears to have a prime defensive function. It also contains the powerful cholinergic agent, murexine. Confusingly, the calcium indicator dye, murexide, or ammonium purpurate, is a synthetic chemical. It has nothing in common with murexine but its name, and nothing in common with Tyrian purple but its color. The production of the dye was at one time a considerable indus in the eastern Mediterranean 9 . Similar industries arose in other parts of the world. In Japan, the mollusc Rapana bezoar, allied to Purpura, was the basis of a trade that arose independently of knowledge of the Mediterranean dye.
purest shade was called, provided much protection. Of the twelve Caesars, ten suffered violent deaths. Julius Caesar, Augustus, Caligula, Claudius, Salba, Vitellius and Domitian were murdered. Nero and Otho committed suicide. Titus is believed to have been poisoned by his brother, the above-mentioned Domitian. Only corrupt Vespasian and the
0
cl
cl
In medieval and Renaissance paintings, the Virgin Mary is typically shown in blue. In what sounds suspiciously like an afterthe-fact rationalization, this piece of iconography is usually attributed to the high price and rarity of blue pigment. Could it not be a cultural memory of the esteem in which the imperial purple was held? I welcome substantiated enlightenment from an informed reader.
ALTHOUGH THE DISTINCT identification in mammals of the related catecholamines, norepinephrine and epinephrine, was only made in 1946, the pharmacology of these substances is one of the pillars supporting our discipline in this century. Yet despite the array of journephrine levels’. Bombesin, connals that have fattened themversely, stimulates adrenal episelves on the results of catecholnephrine release without affecting amine research, questions are norepinephrine release5. Somatonever-ending. Fundamental statininhibits stress-induced aspects of the integration of epinephrine secretion but not adrenomedullary and symsympathetic activitys. pathetic responses, for example, The actions of the two catecholstill remain to be elucidated. amines at adrenergic terminals, however, overlap. They are A clear distinction is made in textbooks between the sympathetic release of norepinephrine as a transmitter and the adrenal release of epinephrine as a hormone. Their release can be mediated independently. Calcitonin generelated peptide, given centrally, triples circulating norepinephrine levels, as measured in the right without affecting epiatrium,
equipotent as approximately cardiac Pi-adrenoceptor agonists, but epinephrine is a more potent agonist at Bz- and ar-adrenoceptors. Adrenomedullary activation can dampen sympathetic responses because epinephrine, by its agonistic action on presynaptic norepiinhibits a-receptors, nephrine release from sympathetic
TiPS - February 1989 [Vol. 101
hl terminals. In what other ways do these two chemically related phenethylamines interact in mediating the ‘tight or flight’ response, and is the separation into transmitter and hormone as clear and simple as the textbooks would have us be: >ve? In humans, basal levels of epinephrine and norepinephrine in arterial plasma are around 0.4nM and 1.1 nM, respectively’. These levels rise severalfold during stress or excitement. From where do catecholamines plasma these derive, what do they do, and where do they go? It is easier to consider the pharmacokinetics of epinephrine because all epinephrine outside the central nervous system derives from the adrenals. Phenylethanolamine Nmethyltransferase (EC 2.1.2.28), the enzyme converting norepinephrine to epinephrine, is found only in the adrenal glands’, and, following adrenalectomy, circulating epinephrine levels fall to zeros. release, Following adrenal epinephrine is poorly removed by the lungs, and passes largely unscathed to targets downstream”. It is inactivated by removal by the liver and sympathetic nerve terminals. Epinephrine disappears from sympathetic nerve terminals after indicating adrenalectomy, its derivation from the adrenals’“. A complication is the re-release of epinephrine from terminals. Thus, although ultimately ail epinephrine in the circulation derives from the adrenals, some of it derives secondarily from sympathetic terminals. If epinephrine is a hormone, then the circulating levels should be in the pharmacological range. Is this the case? Constant infusion of epinephrine into humans produces increased heart rate at plasma levels of 0.27-0.5411~ increased systolic blood pressure at 0.4-0.7 nM, decreased diastolic pressure and elevated plasma glucose and lactate at 0%l.lnM, and decreased plasma insulin levels at plasma epinephrine levels in excess of 2.2nM (Ref. 11). Thus, these responses are produced within the physiologically achieved range of plasma epinephrine. At 4.4 nM epinephrine, heart rate had increased from 63 beats min-’ to 84 beats mm-‘, systolic blood pressure from lllmm Hg to 133mm Hg, and
blood glucose from 48mM to 142 mM. Circulating norepinephrine is released only from the adrenals and from sympathetic nerve terminals. Although norepinephrine is present in the brain, the bloodbrain barrier prevents release into the circulation’z,‘3. Adrenal release, in humans at least, appears to be insignificant compared with sympathetic release. Only about 2% of circulating norepinephrine comes from the adrenals”. The canonical belief is that this catechokrmine functions as a neurotransmitter only, and that norepinephrine in the circulation does not achieve the concentrations necessary for physiological effect; i.e. it does not act as a hormone. Powerful removal mechanisms for norepinephrine exist in vascular beds to of norepi‘spillover’ prevent nephrine. In particular, the endothelial transport of norepinephrine in the lungs serves to prevent norepinephrine from entering the arterial circulation’. Does this satisfying separation of hormonal and neurotransmitter action stand up to close inspection? Reuptake of sympathetically released norepinephrine is efficient, only about 20% escaping into the circu1a::or-r in humans’-. This amounts to some 3-10nmol min-’ entering the circulation. As expected, the lung is most efficient in removing circulating norepinephrine. About 41% is extracted by this organ. This, of course, is not because the lung has a uniquely efficient removal system, but because the lung, unique among the organs, receives the complete cardiac output. Where the apples tumble from the cart, however, is in the realization :hat all organs also release norepinephrine, the lung, probably as a consequence of its high sympathetic innervation in humans, being the most prolific, It contributes some 33% of the total circulating norepinephrine (as calculated from arterial sampling). In considering the lungs to be a sponge for norepinephrine we have fallen victims to a fallacy of radiotracer experiments. We perfuse labelled norepinephrine into the isolated lung, and the radioactivity that emerges is largely in the form of mandelic aldehyde and its further metabolites. Ergo, the lungs remove and metabolize norepinephrine. However, net re-
moval or net release is the difference between an uptake rate and a release rate. In labelling and following only one pool of norepinephrine in a multicompartment system, we are measuring only one of two relevant rates. The information obtained is insufficient to make any statement about the izet effect of the lungs on circulating norepinephrine levels. Now there is another problem. An extensive literature exists in \frhich inferences of sympathetic activity are made by measuring plasma levels of norepinephrine in venous blood. But if most organs are releasing norepinephrine from sympathetic terminals and removing it in vascular endothelial cells, and if these processes are independently regulated, plasma levels in the venous circulation draining from an organ are not going to correlate well with sympathetic activityr5. Even more misleadmeasurements, venous ingly, typically from the antecubital vein in the forearm, have been used as an index of general sympathetic status. However, about half of the norepinephrine in the antecubital vein derives locally”. Continuous infusion experiments with norepinephrine in humans show that plasma levels of 10.6nM or more must be achieved before hemodynamic and metabolic effects are seen”. This is, therefore, the concentration that must be attained in the synaptic cleft for effective neurotransmitter action. At 12.7nM, blood pressure had increased from a control 107/ 61 mm Hg to 131/80mm Hg, and heart rate had fallen from 64 beats niin-’ to 55 beatsmin-‘. These levels are about eight times the basal levels for humans, but can be achieved during violent exercise or stresses such as major surgery. Thus, it can be concluded that under certain conditions, norepinephrine does, indeed, function as a hormone. The interrelationship of the two catecholamines, and the processes releasing them, is still elusive, despite 40 years of study. Are their cardiac actions, for example, duplicative, synergistic or independent in a physiological sense? Circulating epinephrine levels have been reported to be elevated following aortic coarctation in dogs”. Is this a cause or an effect of the hypertrophy produced by this interven-
TiPS - February 2989 lVol. 101 tion? Coarcted dogs in which the adrenal medulla had been previously denervated show, of course, no increase in plasma epinephrine levels. They also do not develop cardiac hypertrophy”, leading the authors to suggest that epinephrine was a trophic factor for the heart. It is clear many problems remain to be investigated in the pharmacology of these related catecholamines. In our present state of incomplete knowledge, it appears that there is a degree of redundancy in stress responses. This makes good evolutionary sense, in that redundant systems are stable systems. It is reassuring to know you have a handbrake to use in the unlikely event your brake fluid leaks away. But this appearance of redundancy may only mask ignorance. A closer understanding of adrenomedullary and sympathetic interactions may indicate that each has clear but integrated responsibilities. B. MAX
References 1 Cleffmann, G. (1964) Exp. CelL Res. 35,
590 600 2 Haluri~, K. M. and Ohkawara, A. (1967) in . Advnnces irz Biology of Skin (Montagna, W. and Ho, F., eds), pp. 241-251, Pergamon Press 3 Baker, J. T. (1974) Et~denuour33, 11-17 4 Fisher, L. A. et nl. (1983) Nature 3C.3, 534-536 5 Brown, M. R. and Fisher, L. A. (2384) 1. Am. Med. Assoc. 251, 1310-1315 6 Best, J. D. and Halter, J. B. (1982: 1. C/in. Emfocrinol. Met&. 55, 263-268 7 Axelrod, J. (1962) /. Biol. Clzetn. 237, 1657-1660 8 Gerich, J. ef al. (1979) Am. 1. Pkysiol. 236, E380-E385 9 Gillis, C. N. and Roth, J. A. (1976) Biochent. Phnrmncol. 25, 2547-2553 10 Sudo, A. (1987) Life Sci. 41, 2477-2484 11 Clutter, W. E., Bier, 0. M., Shah, S. D. and Cryer, P. E. (1980) J. Clip. Inrrest.66, 94-101 12 Esler, M. et al. (1984) 1. Autos. New. Syst. 11, 125-144 13 Zeigler, M. G., Lake, C. R., Wood, J. H., Brook, B. R. and Ebert, H. (1977) 1. Netrrocke?n.28. 677-679 14 &own. M. J., Allison, D. J., Jenner, D. A., Lewis, P. J. and Dollery, C. T. (1981) Lmcet i, 174-177 15 Esler; M., Leonard, I’., O’Dea, K., Jackman, G., Jennings, G. and Korner, P. (1982) /. Cmfiounsc. Plrnmn~of. 4,5152 16 Hjemdahl, P. (1987) Life Sci. 41,841-844 17 Silverberg, A. 5.. Shah, S. D., Haymond, M. W. and Cryer, I’. E. (1978) Am. J. Physiol. 234, E252-E256 18 Womble, I. ‘Y, Haddox, M. K. and Russell, 6. I!. (1978) Life Sci. 23, 1951-1958 19 Womble, J. R., Larson, D. F., Copeland, J. G., Brown, B. R., Haddox, M. K. and Russell, D. H. (1980) Life Sci. 27, 2417-2420
63
Is affinity an adequate basis for receptor chssificaticm?
Will the true receptor affinity please step _ forward In a recent letter, Mackay (TiPS, May 1988)’ commented on our observation2r3 that there seems to be a continuous variation in apparent agonist affinity of norepinephrine for the cul-adrenoceptor. We surmised that this might be due, at least in part, to local cellular influence(s). Mackay suggested that if the ternary complex model of drug action is applicable, then the important cellular influ. ence on affinity might be the local concentration of effector molecules. According to the simplified ternary complex model, the apparent affinity constant &(app) of an agonist A for its receptor is: &(aPP)
=
KIA
+
&A
&A
XT
(1)
where KIA is the receptor constant, KZA is the affinity constant of the RA complex for effector molecule X and XT is the local tissue concentration of the effector molecule. This relationship has recently been further elaborated by Morgan et al4 who pointed out that, with further assumptions, [RAXI could be considered the operational determinant of tissue response. Effector molecule concentration, however, is not the only possible local cellular parameter to influence receptor affinity. For example, it is well known that certain intracellular factors such as ions and possibly the phospholipid content of the membrane microenvironment can regulate affinity5, yet these p;i;:neters do not form part of the proposed ternary complex. In fact K~A, the true receptor affinity, simple as this might be in concept, probably cannot be accurately determined at the moment because the receptor can only be adequately assessed under experimental circumstances that might influence this measurement, for example in a phospholipid or detergent environment. It is known that the receptor is associated with molecuies, e.g. phospholipids, that probably influence the three-dimensional
shape of the agonist recognition site. Thus, Eqn 1 may lack an important element, specifically the environmental influence on the receptor, the receptor-effector complex, or both. Agonist &(app) for the 01~adrenoceptor varies not only between the same tissue in different locations in the same species, but between the same tissue in different species, in the same tissue during maturation, between different individuals of the same species, as the result of disease and with the technique used to prepare tissues for affinity determination. In addition, there is increasing evidence for variation of the affinity of antagonists for the cul-adrenoceptors’. In the face of such diversity of one of the essential characteristics that determine receptor-mediated response, it might well be asked how a satisfactory comprehensive classification can be receptor achieved. It might be that an approach completely different from that presently employed will be necessary. A receptor might have to be described by an affinity profile determined under prescribed circumstances-an affinity profile to key ligands. The true affinity of a receptor molecule is an important chemical feature; however, the affinity of the receptor in situ, the K*(app) is the important functional element. JOHN AND
S.
MARTIN
A.
BEVAN
SHREEVE
Depnrfmentof Pharmncology, University Vermont, Bzrlilrgforz,VT 05405, USA.
References 1 Mackay, D. (1988) Trends 2 3 4 5
Ph~rmacol.
of
Sci.
9, 156-157 Bevan, J. A., Bevan, R. D., Kite, K. and Oriowo, M. A. (1988) Trends PharmacoL Sci. 9, 87-89 Bevan, J. A., Oriowo, M. A. and Bevan, R. D. (1986) Science 234, 196-197 Morgan, P. H., Lutz. M. W. and Kenakin. T. P. (19881 Tretzds Phnmmcol. Sri. 9, 551-352 ’ Bevan, J A., Bevan, R. D. and Shreeve, S. M. FASEB I. (in press)
ho from diskette. Move the cursor to cell Bl and enter ‘ANALYSIS OF VARIANCE TEMPLATE’. Move the cursor to cell A3 and enter ‘Replace headings a, b, c, etc. with your own headings’. In cell A4, enter ‘Replace values in the block Bll to K20 with your own values’. In cell A5 enter the message ‘In rows 21-25 there will be a number of cells that contain ERR’. Move to cell A6 to finish the sentence, ‘erase these cells with the /RE command’. q q 0 Human nature dictates that some readers will ask why they should
reinvent
the
wheel,
even
include their names, addresses, telephone numbers and electronic mail addresses so that users can consult them directly.
though what we have described is not difficult. Some people will not have the time or the temperament to develop templates for their laboratories, so a pooling of effort may be in order. One of us (KN) would be willing to act as a point of collection and dispatch of LOTUS templates as long as the costs were covered. For about $5 to cover diskettes and handiing, copies of a collection of templates can be made and mailed to anyone on request. Send copies of templates that have been developed as well as requests for solutions to prob. lems. The templates should contain a brief description and instructions. Authors should also
KANJI NAKATSU
AND JAMES A. OWEN*
Depnrfme~lf of Phnrmacology nnd Toxicology ntzd *Depnrhmenf of Psychiatry, Queen’s University, Kingston. OMario, Cunndn K7L 3N6. Electronic Mail:
PHARDEPT@QLJCDN. Or.bitnet
netnorth
References 1 Wardlaw, A. C. (1985) Practical Statistics for Experi)rletltnl Biologists, John Wiley & sons 2 Tallerida, R. J. and Murray, R. B. (1986) Mamcnl of PharnmcologicCalculntiom wifh Conrpufer Progrows, Springer-Verlag 3 Zivin, J. A. and Bartko, J. J. (1976) Life Sci. 18, 15-26
hat: on color and catecholamines feathers. In skin, whether the dark low-sulfur eumelanins or the iighter high-sulfur phaeomelanins are formed appears to be deter-
A VISIT to Italy a few months ago, I ran into my old friend, Professor Nero Porpora, who had recently retired. He was sitting in a caf15, staring intentlv at a oassing girl. Veil& her ghoulde& wab a melanins are high sulfur, yellowcascade of that exquisite red ish to reddish brown pigments hair so characteristic of norrather similar to the trichothem Italy, and so elegantly chromes, which are reddish.’ captured by Titian in his por‘What controls which pigment is trait of La Maddelena. formed?’ I asked.
ON
I knew Nero was susceptible to what used to be the tenderer sex, so as I joined him I asked mischievously; ‘Penny for your thoughts. What are you thinking about? He looked up with a smile of recognition. ‘Phaeomelanins and trichochromes,’ he said tersely. I was not to be put off. Nero was famous for his Delphic utterances which on further probing proved to be the key to a flood of insight. I drew up a chair. ‘You were looking pointedly at that girl,’ I said pointedly. ‘The redhead.‘ ‘C&o’, he enlarged. ‘Her hair is most attractive. But I was thinking that the substance responsible for that wonderful color is also the pigment in chicken feathers.’ He pulled over a napkin and began to draw (Fig. 1). ‘You see,’ he continued, ‘tyrosine is more than a precursor for the catecholamines. Melanocytes convert tyrosine to three kinds of pigment by reaction with cysteine and glutathione. The color you get depends on the sulfur content. Capisci? The eumelanins are insoluble, low-sulfur, brown or black pigments. The phaeo-
mined by the sulfhydryl content of the melanocyte. Melanocytes in culture produce black pigments in the absence of glutathione and cysteine, but the light phaeomelanins if these substances are added to the tissue medium’. In humans, black skin contains less glutathione than white2. In suntanned skin, there are falls in the levels of glutathione and glutathione reductase. So you see,’ he ended, ‘minor details of sulfhydryl regulation in the melanocyte provide the biochemical basis for the color prejudices which darken
‘Ah!’ he said, reaching for a fresh napkin, ‘DOPA via its semiquinone can form a number of cysteinyl adducts’ (Fig. 2). ‘The commonest is at the 5-position, although 2-, 6-, 2,5- and other adducts are also formed. Further oxidation of these adducts yields the pigments. The trichochromes are found in red hair and chicken
Tyrosine
DOPA
~“me./
\
Phaeomelanins (Reddish-brown)
(Brown-black) Trichochromes
(Yellow, Red, Violet) Fig.
1. Rainbow
biochemistry:
some
pigments
deriving
from tyrosine.
TiPS - February 1989 [Vol. IO]
61
DOPA Fig. 2. Chicken feathers and trichochromes: cysteinyl-DOPA intermediates in the biosynthesis of phaeomelanins and trichochromes.
so much of the world.’ He put down his pencil and leaned back. ‘Yes,’ he said absently.
That woman had beautiful hair, but. . .’ He fell silent while he took an appreciative sip of coffee.
‘But,’. he ‘in my bat k _ continued, _ yard 1 have some beautiful chickens.’
ONE OF THE most famed and highly regarded pigments of all time is not a tyrosine but a tryptophan metabolite, 6,6’-dibromoindigotin. The dye, Tyrian purple, is produced from molluscs such as the levantine~Mur&. This dye dates to Old TestaIn the human habit of equating ment times, the Book of Exodus rareness with value without regard talking of the tabernacle curto utility, the wearing of clothing tains of ‘blue and purple and dyed with the purple was removed scarlet’. for the Roman emperor. L&eThe dye was so named because majeste’ in this regard was a capital one of the centers of production offense. Not that the right to flaunt was Tyre, or Sur, in modern the pwpura hyacintha, as the
Tiberius died of natural causes, Tiberius attaining that happy state by hiding for 11 years on the island of Capri, where no one could get at him. Those wearing the imperial purple reigned for a cumulative 50 years.
Lebanon. The loquacious Pliny the Elder, in one of his rare happy phrases, likened Tyrian purple to, ‘the color of clotted blood, dark by reflected and brilliant by transmitted light’. No purple compound is actually present in the dye-producing glands of the mollusc. When exposed to fight, the white fluid in the glands is converted through a sequence of colors, green to blue to red to deep purple. Whether the ability to make the ‘multitudinous seas incarnidine’ affords any advantage to its producer is unknown. Many of the more than 300 species of Murex are toxic, and the gland appears to have a prime defensive function. It also contains the powerful cholinergic agent, murexine. Confusingly, the calcium indicator dye, murexide, or ammonium purpurate, is a synthetic chemical. It has nothing in common with murexine but its name, and nothing in common with Tyrian purple but its color. The production of the dye was at one time a considerable indus in the eastern Mediterranean 9 . Similar industries arose in other parts of the world. In Japan, the mollusc Rapana bezoar, allied to Purpura, was the basis of a trade that arose independently of knowledge of the Mediterranean dye.
purest shade was called, provided much protection. Of the twelve Caesars, ten suffered violent deaths. Julius Caesar, Augustus, Caligula, Claudius, Salba, Vitellius and Domitian were murdered. Nero and Otho committed suicide. Titus is believed to have been poisoned by his brother, the above-mentioned Domitian. Only corrupt Vespasian and the
0
cl
cl
In medieval and Renaissance paintings, the Virgin Mary is typically shown in blue. In what sounds suspiciously like an afterthe-fact rationalization, this piece of iconography is usually attributed to the high price and rarity of blue pigment. Could it not be a cultural memory of the esteem in which the imperial purple was held? I welcome substantiated enlightenment from an informed reader.
ALTHOUGH THE DISTINCT identification in mammals of the related catecholamines, norepinephrine and epinephrine, was only made in 1946, the pharmacology of these substances is one of the pillars supporting our discipline in this century. Yet despite the array of journephrine levels’. Bombesin, connals that have fattened themversely, stimulates adrenal episelves on the results of catecholnephrine release without affecting amine research, questions are norepinephrine release5. Somatonever-ending. Fundamental statininhibits stress-induced aspects of the integration of epinephrine secretion but not adrenomedullary and symsympathetic activitys. pathetic responses, for example, The actions of the two catecholstill remain to be elucidated. amines at adrenergic terminals, however, overlap. They are A clear distinction is made in textbooks between the sympathetic release of norepinephrine as a transmitter and the adrenal release of epinephrine as a hormone. Their release can be mediated independently. Calcitonin generelated peptide, given centrally, triples circulating norepinephrine levels, as measured in the right without affecting epiatrium,
equipotent as approximately cardiac Pi-adrenoceptor agonists, but epinephrine is a more potent agonist at Bz- and ar-adrenoceptors. Adrenomedullary activation can dampen sympathetic responses because epinephrine, by its agonistic action on presynaptic norepiinhibits a-receptors, nephrine release from sympathetic
TiPS - February 1989 [Vol. 101
hl terminals. In what other ways do these two chemically related phenethylamines interact in mediating the ‘tight or flight’ response, and is the separation into transmitter and hormone as clear and simple as the textbooks would have us be: >ve? In humans, basal levels of epinephrine and norepinephrine in arterial plasma are around 0.4nM and 1.1 nM, respectively’. These levels rise severalfold during stress or excitement. From where do catecholamines plasma these derive, what do they do, and where do they go? It is easier to consider the pharmacokinetics of epinephrine because all epinephrine outside the central nervous system derives from the adrenals. Phenylethanolamine Nmethyltransferase (EC 2.1.2.28), the enzyme converting norepinephrine to epinephrine, is found only in the adrenal glands’, and, following adrenalectomy, circulating epinephrine levels fall to zeros. release, Following adrenal epinephrine is poorly removed by the lungs, and passes largely unscathed to targets downstream”. It is inactivated by removal by the liver and sympathetic nerve terminals. Epinephrine disappears from sympathetic nerve terminals after indicating adrenalectomy, its derivation from the adrenals’“. A complication is the re-release of epinephrine from terminals. Thus, although ultimately ail epinephrine in the circulation derives from the adrenals, some of it derives secondarily from sympathetic terminals. If epinephrine is a hormone, then the circulating levels should be in the pharmacological range. Is this the case? Constant infusion of epinephrine into humans produces increased heart rate at plasma levels of 0.27-0.5411~ increased systolic blood pressure at 0.4-0.7 nM, decreased diastolic pressure and elevated plasma glucose and lactate at 0%l.lnM, and decreased plasma insulin levels at plasma epinephrine levels in excess of 2.2nM (Ref. 11). Thus, these responses are produced within the physiologically achieved range of plasma epinephrine. At 4.4 nM epinephrine, heart rate had increased from 63 beats min-’ to 84 beats mm-‘, systolic blood pressure from lllmm Hg to 133mm Hg, and
blood glucose from 48mM to 142 mM. Circulating norepinephrine is released only from the adrenals and from sympathetic nerve terminals. Although norepinephrine is present in the brain, the bloodbrain barrier prevents release into the circulation’z,‘3. Adrenal release, in humans at least, appears to be insignificant compared with sympathetic release. Only about 2% of circulating norepinephrine comes from the adrenals”. The canonical belief is that this catechokrmine functions as a neurotransmitter only, and that norepinephrine in the circulation does not achieve the concentrations necessary for physiological effect; i.e. it does not act as a hormone. Powerful removal mechanisms for norepinephrine exist in vascular beds to of norepi‘spillover’ prevent nephrine. In particular, the endothelial transport of norepinephrine in the lungs serves to prevent norepinephrine from entering the arterial circulation’. Does this satisfying separation of hormonal and neurotransmitter action stand up to close inspection? Reuptake of sympathetically released norepinephrine is efficient, only about 20% escaping into the circu1a::or-r in humans’-. This amounts to some 3-10nmol min-’ entering the circulation. As expected, the lung is most efficient in removing circulating norepinephrine. About 41% is extracted by this organ. This, of course, is not because the lung has a uniquely efficient removal system, but because the lung, unique among the organs, receives the complete cardiac output. Where the apples tumble from the cart, however, is in the realization :hat all organs also release norepinephrine, the lung, probably as a consequence of its high sympathetic innervation in humans, being the most prolific, It contributes some 33% of the total circulating norepinephrine (as calculated from arterial sampling). In considering the lungs to be a sponge for norepinephrine we have fallen victims to a fallacy of radiotracer experiments. We perfuse labelled norepinephrine into the isolated lung, and the radioactivity that emerges is largely in the form of mandelic aldehyde and its further metabolites. Ergo, the lungs remove and metabolize norepinephrine. However, net re-
moval or net release is the difference between an uptake rate and a release rate. In labelling and following only one pool of norepinephrine in a multicompartment system, we are measuring only one of two relevant rates. The information obtained is insufficient to make any statement about the izet effect of the lungs on circulating norepinephrine levels. Now there is another problem. An extensive literature exists in \frhich inferences of sympathetic activity are made by measuring plasma levels of norepinephrine in venous blood. But if most organs are releasing norepinephrine from sympathetic terminals and removing it in vascular endothelial cells, and if these processes are independently regulated, plasma levels in the venous circulation draining from an organ are not going to correlate well with sympathetic activityr5. Even more misleadmeasurements, venous ingly, typically from the antecubital vein in the forearm, have been used as an index of general sympathetic status. However, about half of the norepinephrine in the antecubital vein derives locally”. Continuous infusion experiments with norepinephrine in humans show that plasma levels of 10.6nM or more must be achieved before hemodynamic and metabolic effects are seen”. This is, therefore, the concentration that must be attained in the synaptic cleft for effective neurotransmitter action. At 12.7nM, blood pressure had increased from a control 107/ 61 mm Hg to 131/80mm Hg, and heart rate had fallen from 64 beats niin-’ to 55 beatsmin-‘. These levels are about eight times the basal levels for humans, but can be achieved during violent exercise or stresses such as major surgery. Thus, it can be concluded that under certain conditions, norepinephrine does, indeed, function as a hormone. The interrelationship of the two catecholamines, and the processes releasing them, is still elusive, despite 40 years of study. Are their cardiac actions, for example, duplicative, synergistic or independent in a physiological sense? Circulating epinephrine levels have been reported to be elevated following aortic coarctation in dogs”. Is this a cause or an effect of the hypertrophy produced by this interven-
TiPS - February 2989 lVol. 101 tion? Coarcted dogs in which the adrenal medulla had been previously denervated show, of course, no increase in plasma epinephrine levels. They also do not develop cardiac hypertrophy”, leading the authors to suggest that epinephrine was a trophic factor for the heart. It is clear many problems remain to be investigated in the pharmacology of these related catecholamines. In our present state of incomplete knowledge, it appears that there is a degree of redundancy in stress responses. This makes good evolutionary sense, in that redundant systems are stable systems. It is reassuring to know you have a handbrake to use in the unlikely event your brake fluid leaks away. But this appearance of redundancy may only mask ignorance. A closer understanding of adrenomedullary and sympathetic interactions may indicate that each has clear but integrated responsibilities. B. MAX
References 1 Cleffmann, G. (1964) Exp. CelL Res. 35,
590 600 2 Haluri~, K. M. and Ohkawara, A. (1967) in . Advnnces irz Biology of Skin (Montagna, W. and Ho, F., eds), pp. 241-251, Pergamon Press 3 Baker, J. T. (1974) Et~denuour33, 11-17 4 Fisher, L. A. et nl. (1983) Nature 3C.3, 534-536 5 Brown, M. R. and Fisher, L. A. (2384) 1. Am. Med. Assoc. 251, 1310-1315 6 Best, J. D. and Halter, J. B. (1982: 1. C/in. Emfocrinol. Met&. 55, 263-268 7 Axelrod, J. (1962) /. Biol. Clzetn. 237, 1657-1660 8 Gerich, J. ef al. (1979) Am. 1. Pkysiol. 236, E380-E385 9 Gillis, C. N. and Roth, J. A. (1976) Biochent. Phnrmncol. 25, 2547-2553 10 Sudo, A. (1987) Life Sci. 41, 2477-2484 11 Clutter, W. E., Bier, 0. M., Shah, S. D. and Cryer, P. E. (1980) J. Clip. Inrrest.66, 94-101 12 Esler, M. et al. (1984) 1. Autos. New. Syst. 11, 125-144 13 Zeigler, M. G., Lake, C. R., Wood, J. H., Brook, B. R. and Ebert, H. (1977) 1. Netrrocke?n.28. 677-679 14 &own. M. J., Allison, D. J., Jenner, D. A., Lewis, P. J. and Dollery, C. T. (1981) Lmcet i, 174-177 15 Esler; M., Leonard, I’., O’Dea, K., Jackman, G., Jennings, G. and Korner, P. (1982) /. Cmfiounsc. Plrnmn~of. 4,5152 16 Hjemdahl, P. (1987) Life Sci. 41,841-844 17 Silverberg, A. 5.. Shah, S. D., Haymond, M. W. and Cryer, I’. E. (1978) Am. J. Physiol. 234, E252-E256 18 Womble, I. ‘Y, Haddox, M. K. and Russell, 6. I!. (1978) Life Sci. 23, 1951-1958 19 Womble, J. R., Larson, D. F., Copeland, J. G., Brown, B. R., Haddox, M. K. and Russell, D. H. (1980) Life Sci. 27, 2417-2420
63
Is affinity an adequate basis for receptor chssificaticm?
Will the true receptor affinity please step _ forward In a recent letter, Mackay (TiPS, May 1988)’ commented on our observation2r3 that there seems to be a continuous variation in apparent agonist affinity of norepinephrine for the cul-adrenoceptor. We surmised that this might be due, at least in part, to local cellular influence(s). Mackay suggested that if the ternary complex model of drug action is applicable, then the important cellular influ. ence on affinity might be the local concentration of effector molecules. According to the simplified ternary complex model, the apparent affinity constant &(app) of an agonist A for its receptor is: &(aPP)
=
KIA
+
&A
&A
XT
(1)
where KIA is the receptor constant, KZA is the affinity constant of the RA complex for effector molecule X and XT is the local tissue concentration of the effector molecule. This relationship has recently been further elaborated by Morgan et al4 who pointed out that, with further assumptions, [RAXI could be considered the operational determinant of tissue response. Effector molecule concentration, however, is not the only possible local cellular parameter to influence receptor affinity. For example, it is well known that certain intracellular factors such as ions and possibly the phospholipid content of the membrane microenvironment can regulate affinity5, yet these p;i;:neters do not form part of the proposed ternary complex. In fact K~A, the true receptor affinity, simple as this might be in concept, probably cannot be accurately determined at the moment because the receptor can only be adequately assessed under experimental circumstances that might influence this measurement, for example in a phospholipid or detergent environment. It is known that the receptor is associated with molecuies, e.g. phospholipids, that probably influence the three-dimensional
shape of the agonist recognition site. Thus, Eqn 1 may lack an important element, specifically the environmental influence on the receptor, the receptor-effector complex, or both. Agonist &(app) for the 01~adrenoceptor varies not only between the same tissue in different locations in the same species, but between the same tissue in different species, in the same tissue during maturation, between different individuals of the same species, as the result of disease and with the technique used to prepare tissues for affinity determination. In addition, there is increasing evidence for variation of the affinity of antagonists for the cul-adrenoceptors’. In the face of such diversity of one of the essential characteristics that determine receptor-mediated response, it might well be asked how a satisfactory comprehensive classification can be receptor achieved. It might be that an approach completely different from that presently employed will be necessary. A receptor might have to be described by an affinity profile determined under prescribed circumstances-an affinity profile to key ligands. The true affinity of a receptor molecule is an important chemical feature; however, the affinity of the receptor in situ, the K*(app) is the important functional element. JOHN AND
S.
MARTIN
A.
BEVAN
SHREEVE
Depnrfmentof Pharmncology, University Vermont, Bzrlilrgforz,VT 05405, USA.
References 1 Mackay, D. (1988) Trends 2 3 4 5
Ph~rmacol.
of
Sci.
9, 156-157 Bevan, J. A., Bevan, R. D., Kite, K. and Oriowo, M. A. (1988) Trends PharmacoL Sci. 9, 87-89 Bevan, J. A., Oriowo, M. A. and Bevan, R. D. (1986) Science 234, 196-197 Morgan, P. H., Lutz. M. W. and Kenakin. T. P. (19881 Tretzds Phnmmcol. Sri. 9, 551-352 ’ Bevan, J A., Bevan, R. D. and Shreeve, S. M. FASEB I. (in press)