This and that: ruminations on stress, pitfalls and pressure

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therapeutic agents to alleviate amnesic disorders will have to wait until more is k n o w n about h o w these systems interact with one another, and w h a t systems are p r i m a r i l y affected (if this were the case) in the m a n y different forms of amnesia seen in m a n or even in laboratory animals 9. IVAN

IZQUIERDO

Laboratorio de Neuroquimica, Departamento de Bioquimica, Instituto de Biociencias,

UFRGS (centro), 90000 Porto Alegre, RS, Brazil.

References 1 GaUagher, M. (1984) in Neurobiology of Learning and Memory (Lynch, G., McGaugh, J.L. and Weinberger, N., eds), pp. 368-373, Guilford, New York 2 Izquierdo, I., Perry, M. L. S., Dias, R. D., Souza, D. O., Elisabetsky, E., Carrasco, M. A., Orsingher, O. A. and Netto, C. A. (1981) in Endogenous Peptides and Learning and Memory Processes(Martinez, J. L. Jr, Jensen, R., Messing, R. B., Rigter, H. and McGaugh, J. L., eds), pp. 269-290, Academic Press, New York

3 McGaugh,J. L. (1983)Ann. Rev. Psychol. 34, 297-323 4 McGaugh, J. L. and Gold, P. E. (1985) in Psychoendocrinology (Levine, S. and Brush, F. R., eds), (in press), Academic Press, New York 5 Baratti, C. M., Introini, I.B. and Huygens, L. (1984) Behav. Neurol. Biol. 40, 155-169 6 Introini, I. B. (1984)PhD Thesis, University of Buenos Aires 7 Introini, I. B. and Baratti, C. M. (1984) Behav. Neurol. Biol. 41, 152-163 8 Davies, P. (1985)Ann. N.Y. Acad. Sci., (in press) 9 Izquierdo, I. (1984) Trends Pharmacol, Sci. 5, 493-494

This and that: ruminations on stress, pitfalls and pressure THE SPONTANEOUSLY h y p e r t e n s i v e (SH) rat d e r i v e d b y Okamoto has become a widely used model in research on h y p e r t e n s i o n . T h i s is p r i m a r i l y b e c a u s e , d e s p i t e e x t e n s i v e s t u d y , w e k n o w as little of t h e c a u s a t i o n of h y p e r t e n s i o n in t h e S H rat as w e d o of e s s e n t i a l h y p e r t e n s i o n i n h u m a n s . The SH rat is normotensive at birth. After the first m o n t h of life, however, blood pressure slowly climbs, reaching or exceeding 220 rnm Hg systolic. A n intriguing but unexplored aspect of the model is that the heart to b o d y weight ratio is significantly elevated from birth, c o m p a r e d to normotensive Wistar rats. Wright and M c C u m b e e 7 have n o w reported the isolation of a dialysable substance from erythrocyte m e m b r a n e s of SH rats which raises blood pressure on injection into normotensive rats. Systolic pressure rose some 20-30 m m Hg, the increase persisting for 18 days following the last injection. A complication, however,

was that the saline-injected controls also s h o w e d a transient but significant elevation of blood pressure. The substance also increased the uptake of calcium b y aorta from normotensive rats. Vascular smooth muscle from SH rats takes up calcium more readily than smooth muscle from normotensive rats, so this action of the dialysate m a y provide a clue to the m e c h a n i s m of blood pressure elevation. Again, however, interpretation is complicated b y the finding that erythrocyte m e m b r a n e preparations from normotensive Wistars also stimulated aortic calcium uptake, albeit to a lesser degree.

T H E U R I N E of d o m e s t i c a t e d a n i m a l s h a s p r o v e d a c o r n u c o p i a of c h e m i c a l t r e a s u r e s . A n d r o s t e r o n e , t e s t o s t e r o n e , a n d e s t r o n e are a f e w of t h e m o l e c u l a r p u z z l e s c h e m i s t s h a v e f i s h e d o u t of t h i s r e n e w a b l e f l u i d for t h e i r p r o f e s s i o n a l d e l e c t a t i o n . The tradition continues. 'Diazep a m - l i k e ' c o m p o u n d s have n o w been isolated from bovine urine 1, p e r h a p s accounting for the placid nature of the beast. The authors are to be congratulated for the completeness of their investigations. Not only were structures derived for all c o m p o u n d s , b u t those proving to be novel were verified by synthesis. Three c o m p o u n d s exh i b i t i n g activity in a b e n z o d i a z e p i n e receptor b i n d i n g assay were s h o w n to be isoflavans. One, equo|, h a d been previously isolated t c o m mare's urine, but the other two were novel; 3',7-dihv-

droxyisoflavan and 4'-hydroxy-7methoxyisoflavan (Fig. 1). These c o m p o u n d s had approximate ICs0 in the 10-sM range, 3--4 orders of m a g n i t u d e less than diazepam

These findings are intriguing. They would be more so if such reports could range b e y o n d more p h e n o m e n o l o g y and give some indication of the chemical natures of the humoral agents involved. It has, after all, already been reported that p u p s from normotensive dams suckled b y SH dams develop elevated blood pressure, suggesting that a humoral agent secreted into the milk m a y be responsible. Is the agent thermostable? H o w long can it be left on the bench without activity being lost? H o w about enrichment b y chromatography, using calcium uptake as a bioassay, and UV spectroscopy? H o w about G C MS on an enriched sample? Is it fair to whet our appetites with a whiff of the cooking from the kitchen, leaving us to guess what is on the menu? Purine supreme? Catechol surprise? Peptide pie? Even heavy metal goulash? itself. A fourth c o m p o u n d enhanced the b i n d i n g of diazepam. This c o m p o u n d was a chlorocarbazole (Fig. 2). At 1raM, the carbazole almost d o u b l e d the b i n d i n g of diazepam. The authors claim this is the first isolation of a carbazole from a m a m m a l i a n source. The authors also isolated two

R2 R30 Fig. 1. Novel diazepam-like compounds in bovine urine. R~ = H; R2 = OH; R3 = H: 3',7-dihydroxyisoflavan R1 = OH; R2 = H; R3 = CH3: 4"-hydroxy-7-methoxyisoflavan ~) 1985, Elsevier Science Publishers B.V., Amsterdam

0165 - 6147/85/$02.00

TIPS - M a y 1985

194 dyes, one of which was indigo, probably the oldest coloring material used culturally, although the ancients isolated it from plants rather than ruminants. Ct

H

Fig. 2. 3-chloro-gH-~rbazole

Where do these mysterious compounds originate? The isoflavans presumably derive from the

diet. The carbazole may do so also, or it may derive from the rumen flora. Urine is a universal solvent the alkahest for which the medieval academician searched so unavailingly and contains the chemical echoes of the complex internal and external environments of the producer. Its composition delicately reflects subtle variations in the biochemical and nutritional milieu. There is, therefore, a rational basis for the attention that many cultures have paid to urine as an aid to diagnosis. In Chaucerian times, uroscopy was practiced, the clouds, colors and precipitates present being -

judged. Paracelsus introduced urinalysis, with his insistence on the need to separate the combustible, the volatile and the ash components. The seventeenth century saw urine being used to diagnose pregnancy. Today, the difference is quantitative rather then qualitative in that we look for molecules as well as observing the same qualities of cloudiness and color that our forebears did. As the anonymous 16th century author of the 'Judycyall of Uryns' phrased it: "It is expedient for every man to know the operation and quantities of his own b o d y . . , which can not be known so well as by the urine."

nomenclature. Thus, Am1 and Afl THE TYPE A, type B classification has been used to link personality could refer to male and female type to risk of coronary heart and artery disease. What are the type As showing decreased forecardiovascular responses of type A c o m p a r e d to type B w o m e n arm resistance on mental stress, w h e n b o t h g r o u p s are challenged b y a task of mental arithmetic? and Af2 the larger group of The workers w h o studied this question (in P s y c h o p h y s i o l o g ys) females not showing such a escape the charge of sex bias b y h a v i n g p u b l i s h e d an earlier p a p e r change in resistance. The approon mental arithmetic in type A and B males (in Science6). priate elastic nomenclature could The task was to subtract 13 reIf this paper is an indication of a keep many workers happy and peatedly from a four digit number. busy, which should be good for trend in subclassifying us As and The appropriate element of comstress reduction. Bs, workers in this field might petition was introduced by the consider adapting receptor offer of a bottle of champagne to the person with the highest rate of correct subtractions. The exIF YOU HAVE the m o n e y , it is easy to b u y a gleaming mass of perimental information was inanalytical e q u i p m e n t into one e n d of w h i c h a sample is injected, to sufficient to judge whether this be followed after an appropriate period of digestion b y the selected the finest at mental arithrecording of a peak at the other end. M a n y are m a d e very h a p p y metic, the thirstiest, or the most b y such toys. Analysis turns out to be easy and clean - the discriminating eye for a vintage fulfilment of a scientist's dream. (not given). by decapitation or by catheterizaType A females, it is reported, However, those symmetrical and tion. Following decapitation, the respond differently to type A easily obtained peaks can be levels of over half the amino acids males. The latter are hyper-resgrossly misleading. Although a in plasma were higher than in ponsive in that they release more response is readily recorded from samples obtained by the presumepinephrine, cortisol and norea sample injected into an amino ably less stressful route of pinephrine, pointing to activation acid analyser, an HPLC or a GLC, catheterization. Taurine levels, for of the adrenomedullary, adrenoreducing a peak to meaning is not example, were 487 nmolem1-1 necessarily readily achieved. Too cortical and peripheral sympplasma as compared to 87. Phosathetic systems, respectively. often one can forge past inphoethanolamine increased from Although the As of both sexes strumental artefacts, biases of responded to the task with insample preparation and insuffi7.4 to 31.1 nmole m1-1, and ~alanine from 1.7 to 9.3. Anserine creased heart rate, blood pressure cient experimental design without and carnosine were undetectable and forearm blood flow, males awareness that these may have a in samples obtained from showed a decrease in forearm more profound effect on the incatheterization, but were 25 and vascular resistance (due to strumental response one is in21 nmolem1-1, respectively, in epinephrine release). Only the terested in than does the concensubset of females with a family tration in the sample of the samples obtained following dehistory of hypertension showed capitation. substance being analysed. the same decreased vascular reAnother report 2 has joined the Similar findings have been resistance. The authors conclude ported for catecholamines 3. The long list of cautionary articles on that both behavior and genetics the pitfalls of amino acid analysis. ease with which analytical instrushould be taken into account in ments may be used belies the A total of 41 ninhydrin-positive considering the implications of substances in rat plasma were difficulty of obtaining accurate' quantitative data 4. determined on samples obtained the type A/type B classification.

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Metallic value judgements: magnesium is good...

h y p o t e n s i v e action of Mg has b e e n k n o w n for sixty years. More recently, the incidence of hypert e n s i o n in certain areas has been correlated with Mg intake. The f i n d i n g s of Altura et al. s provide a rational basis for both the cause a n d treatment of one class of hypertension.

ANOTHER THEORY has entered the fertile and burgeoni n g f i e l d of h y p e r t e n s i o n . T h e r e s u l t s of a t r a n s - A t l a n t i c collaboration indicate that magn e s i u m d e f i c i e n c y l e a d s to i n creased blood pressure s. This a p p e a r s to r e s u l t f r o m t h e decreased lumen size in arteri o l e s a n d v e n u l e s of M g - d e f i c i e n t rats. Rats fed a Mg-deficient diet for twelve weeks had an increase in systolic blood pressure from 111 m m H g -1 to 143 m m Hg -1. C o r r e s p o n d i n g serum concentrations of Mg were 1.0 mM and 0.3 mM. The precapillary sphincters of deficient animals had a 33% decrease in lumen size. Microvessels from these animals were also h y p e r - r e s p o n s i v e to the constrictive actions of vasopressin and catecholamines. A n i m a l s on a Mgdeficient diet that were given 4 mm Mg in the d r i n k i n g water had, after 12 weeks, serum Mg level of 0.7 mm, intermediate between the control and Mg-deficient groups. The blood pressure and circulatory pathology in these animals were also intermediate

b e t w e e n those of the control and deficient groups These f i n d i n g s could initiate a major change in therapy. A n association of Mg and hypertension h a d long b e e n suspected. The

ETHANOL, heroin, thalidomide, heavy metals: these a g e n t s s h a r e t h e p r o p e r t y of fetal t o x i c i t y . T h e p r o t e c t i o n afforded by the womb and the p l a c e n t a is i l l - d e s i g n e d to shield the unborn from the inc r e a s e d c h e m i c a l c o m p l e x i t y of the twentieth century. Pregnant smokers have amniotic cadmium levels three times higher t h a n t h o s e of p r e g n a n t n o n s m o k e r s 9. C a d m i u m l e v e l s a r e p r o p o r t i o n a l to t h e n u m b e r of cigarettes smoked, but are unr e l a t e d to t h e s t a g e of p r e g nancy. This consequence of s m o k i n g launches a t w o - p r o n g e d attack on the child-to-be. The first is the direct developmental conse-

quences of the increased exposure to a toxic h e a v y metal. The second is that the child is born with an initial h i g h e r level of a substance having a long biological half life. The b o d y b u r d e n of such subtances typically increases slowly t h r o u g h o u t life. A h i g h e r initial level ensures that a toxic threshold will be attained earlier in the person's life. Amniotic lead levels d i d not vary b e t w e e n smokers and nonsmokers, being a little over 20 ng 1-1. H o w e v e r , this s t u d y was performed in the industrialized city of Lubeck, birthplace of Thomas Mann, so it is possible that an effect of smoking was m a s k e d b y ubiquitous environmental exposure to lead.

References

144-148 4 Perry, T. L. and Hansen, S. (1969) Clin. Chim. Acta 25, 53-58 5 Lane, J. D., White, A. D. and Williams, R. B. (1984) Psychophysiology 21, 39-46 6 Williams, R. B., Lane, J. D., Kuhn, C. M., Melosh, W., White, A. D. and Schanberg, S. M. (1982) Science 218, 483-485

7 Wright, G. L. and McCumbee, W.D. (1984) Life Sci. 34, 1521-1528 8 Altura, B. M., Altura, B. T., Gebrewold, A., Ising, H. and Gunther, T. (1984) Science 223, 1315-1317 9 Siegers, C. P., Jungblut, J. R., Klink, F. and Oberheuser, F. (1983) Toxicol. Lett. 19, 327-331

1 Luk, K. C., Stern, L. and Weigele, M. (1983) J. Nat. Prod. 46, 852M361 2 Milakofsky, L., Hare, T. A., Miller, J. M. and Vogel, W. H. (1984) Life Sci. 34, 13331340 3 Popper, C. W., Chiueh, C. C. and Kopin, I.J. (1977) ]. Pharmacol. Exp. Ther. 202,

... and cadmium

is bad

A program for random numbers which are roughly norma!!y distributed In m a n y statistical tests it is a s s u m e d that the errors attached to an observation are r a n d o m and normally d i s t r i b u t e d about the mean. This applies, for instance, to the c o m p a r i s o n of two mean values with a test using Student's t - d i s t r i b u t i o n , or to the setting of confidence limits to an estimate o b t a i n e d from linear (or curvilinear) regression, such as the

estimation of protein spectrophotometrically, the concentration of h o r m o n e in a r a d i o i m m u n o assay, or to estimates of concentrations p r o d u c i n g half-maximal responses (ECso or ICso). It is therefore useful to be able to generate normally distributed r a n d o m n u m b e r s to assess, by trial, the extent to which w h a t you calculate from such experiments is

B. A~AX

likely be influenced by the errors in your observations. All m o d e r n microcomputers are able to produce r a n d o m n u m b e r s b e t w e e n 0 and 1 b u t these will not be d i s t r i b u t e d normally. Each n u m b e r can, however, be regarded as a fraction and the integrated form of the normal frequency curve can be used to calculate the c o r r e s p o n d i n g normal equivalent deviation. The series of r a n d o m n u m b e r s will thus be replaced b y a series of deviations which will be normally d i s t r i b u t e d about zero. It is impossible to write an equation for the integrated form of the normal frequency curve: the integration is intractable and m u s t

,q'C)1985,ElsevierSciencePublishersB.V.,Amsterdam 0165 6147/85/$02.00