Pharmacological effects of ionophore A-23187 on isolated guinea-pig atria and isolated cat nictitating membrane

Gen Pharmac Vot 13 pp 111 to 116 1982 Printed in Great Britain All rights reserved

0306-3623/82/020111-0650300/0 Copyright © 1982 Pergamon Press Ltd

P H A R M A C O L O G I C A L E F F E C T S O F I O N O P H O R E A-23187 O N I S O L A T E D G U I N E A - P I G ATRIA A N D I S O L A T E D CAT N I C T I T A T I N G M E M B R A N E VIVIANA TARIZZO and MODESTO C RUBIO* Inshtuto de Investigaciones Farmacologlcas, CONICET, Junin 956, 5°Piso, 1113 Buenos Aires, Argentina (Recewed 27 July 1981) The present paper is concerned with some pharmacological actions of lonophore A-23187 on both isolated guinea-pig atria and cat's nictitating membrane 2 The drug showed an agomst effect on both tissues 3 There is an indirect effect by releasing catecholamlnes which is antagonized by reserpine and beta receptor blockers m atria and alfa receptor blockers in nictitating membrane 4 The ionophore exhibited a direct positwe chronotroplc effect on the atria This effect is blocked by Ca 2+ flux antagonist such as verapamil and pentobarbital and not by reserpine 5 Ionophore produced no direct effect on the activity of the isolated tyrosme hydroxylase but showed an inhibitory action m intact tissues, mediated by released of endogenous catecholammes, since this effect is antagomzed by reserpine 6 Monoamme oxldase activity was unaffected by ionophore Abstract--1

INTRODUCTION

trahons) NaCI, 1180, KCI, 47, CaCI2, 26, MgCI2, 1 2, NaH2PO4, 1 0, NaHCOs, 250, glucose, 11 1, ethylendlamlne tetraacetlc acid (EDTA), 0 004, ascorbic acid, 0 11 The dissected atria were set up In a 10 ml organ bath at 37°C and with continuous bubbling with 95% 02 and 5% CO2 mixture The upper thread of the atria was connected to a force displacement transducer (Grass FT 03) and the spontaneous beats of the atria were recorded on a Grass polygraph The preparations were kept m the bath until their spontaneous rate did not change by more than 5 beats/mm during a 10 mln period of observation, this took about 1 hr Dose-response curves were determined by stepwlse increases of the total lonophore concentration The response of the pacemaker usually reached a steady level after 3-4 min The concentration of the ionophore or ( - ) noradrenahne was then Increased by a factor of about 3 without changing the bathing soluhon The lonophore A 23187 stock solution was prepared in pure dlmethylsulfoxlde, which did not show any effect on control experiments, and then diluted with water For some experiments the animals received I p injectlons of 0 3 mg/kg or 3 mg/kg of reserpine 24 hr prior to the experiments When used, propranolol was added to the organ bath 20rain before the begmlng of dose-response curves and kept in the organ bath throughout In experiments in which used sodium pentobarbltone (124pg/ml) or verapamil (02#g/ml) were added to the organ bath 15min after the ionophore 10-Tg/ml was added

It is known that some antlblot~cs act as ionophores by facdltatmg the selective transport of ions through biological membranes and artLficml membranes Although the chemical structure of ionophore may be different, m all cases their action occurs through an increase m 1on solublhty at the membrane non polar medm by surrounding the 1on and thus removing the hldrtc layer (Pressman, 1973) The lonophore A 23187 showed a very high affinity to form hposoluble complexes with Ca 2 + (Caswell & Pressman, 1972, Scarpa et al, 1972, Schadt & Haeusler, 1974) The catecholamme releasing effect of this drug from adrenal medulla (Cochrane et al, 1976) and guineapig vas deferens (Thoa et al, 1974), has been described Consequently, it was considered of interest to study whether the responses eltctted by the lonophore m adrenergtcally innervated tissues are due solely to the release of noradrenahne or ff It has a post synaptlc effect F o r this purpose the effects of A-23187 on hssues m which the receptors are of the beta type, guinea-pig atrm, and m t~ssues with p r e d o m | n a n c e of alpha receptors, cat's mct~tatlng membrane, were analysed METHODS

Isolated gumea-pl O atma Guinea-pigs of 300-500 g of either sex were killed by a blow on the head The hearts were quickly removed and placed in a Petri dish with Krebs soluUon at room temperature for the dissection of the atria The composition of the Krebs solution was the following (mflhmolar concen* Member of Carrera del Investigador CIentifico, Consejo Nacional de Investigaclones Clentlficas y Tecmcas Argentina

Isolated cat's mCtltatmg membrane Cats of 2-4kg of either sex were anesthetized with sodium pentobarbltone 35 mg/kg (l p ) The eyeball was excised and the nictatmg membrane with all the adjoining tissue was removed from the orbit The tissue was placed in a Petn dish with cold Krebs solution The medial muscle was dissected under a binocular dissecting microscope The cartilage on which the fibers of the medial muscle are inserted was fixed to the bottom of a 10 mi organ bath The upper end of the muscle was connected to a force displacement transducer Grass FTO3

111

112

VIVIANATARIZZOand MODESTOC RUalO A

and recorded with a Grass polygraph The temperature was maintained at 37°C and the organ bath was bubbled with a 95%/002 5% CO2. mixture A period of l hr was allowed to elapse before starting the experiments During this period the Krebs solution was replaced every 10 mln The resting tension of the muscle was repeatedly readjusted to 2 5 g and it reached steady condltmns after 40-50mm The resting tension was not readjusted once the experiment had begun Dose response curves to lonophore or ( - ) noradrenahne were determined by adding the drug cumulatively In such a way that the concentration in the bath was increased by a factor of about 3. whenever a steady response to the previous concentration was achieved When used phentolamine (l/zg/ml) was added 15 rain before the addition of the agonlst Biochemical experiments In the experiments In which the endogenous stores of noradrenahne of the nictitating membrane were labelled, the tissues were incubated for 30ram at 37°C with 5 ml of Krebs solution containing 5 ,uCl/ml of DL-7-aH-noradrena hne (New England Nuclear Corporation, Boston, Mass, specific activity, 6 9 Ci/mmol) and then was washed for 1 min in each of eight beakers contalmng 50ml of fresh Krebs solution After washing, the nictitating membrane was suspended in the isolated organ bath as indicated in the secUon on isolated cat nictitating membrane The washings continued until the spontaneous outflow of radioactivity had leveled off This took about 100-120mln after the end of the incubation with 3H-noradrenalIne The total radioactivity in the spontaneous outflow of trltlated compounds from nictitating membrane into the bathmg fired was momtored by counting 0 5 ml samples of the Krebs solution which had been in contact with the tissues for 5 min Samples were counted in 5 ml scintillation fluid of the following composition 5 5 g PPO, 0 1 g POPOP, 600 ml tolueno and 300 ml Triton x 100 Assay of tyrosine hydroxylase in homogenates The cat nictitating membranes were homogenized in the cold room with an all glass homogenizer in a 150 mM KCI solution in a 1/10 (w/v) ratio and tyroslne hydroxylase activity determined as described by Waymire et al (1971) Assay of tyrosine hydroxylase activity in intact tissue The assay was carried out m a glass tube In a final volume of 04 ml The Krebs solution employed in the incubation medium was the one described m the first section of methods 1-1*C-L-Tyrosme 001#mol (520pCi/gmol), D M P H , 0 1 pmol and ascorblc acid 5 0 #mol were added to 1 ml of Krebs solution Blanks were obtained in the presence of 3-1odotyroslne 2 0 mM A plastic well was suspended from the rubber injection stopper and contained folded paper imbibed with 0 2 ml NCS (Nuclear Chicago solublhzer) to trap the 1'CO2 formed In the decarboxylatlon step of the assay The reaction was stopped by injection of 0 4 m l 10% trichloroacetic acid Subsequently, the incubation continued for 2hr at 37°C and with continuous shaking to recover the i'*CO2 Then the plastic wells were removed, wiped with absorbent tissue and placed In 10 ml of scintillation fluid Assay of monoamine ox~dase actwity In homogenates The atria were homogenelzed in a 150 mM KC! solution in a 1/10 (w/v) ratio and then 25 #l of this homogenate was used as the source of enzyme in each experiment The method employed for the determmauon of monoamine Oxldase activity was the one described by McCaman et al (1965)

100 80 ~o 4o

10-10 i0-9 i0-8 i0-7 10% 10-5 (-)Norodrenohne(g/ml)

r

s

1001-80 60 40 20

10-11 10-10 10-9 10-8 10-7 10-6 10-5 Ionophore(9/roll Fig 1 Effect of propranolol on the positive chronotroplc response to ( - ) noradrenal,ne and lonophore A-23187 Isolated guinea-pig atria Krebs solution 37°C. ordinates-frequency increase in beats per minute, abscissae--(a) ( - ) noradrenahne concentration (g/ml). (B) ionophore concentration (g/ml) ---O--- control. --O--- propranolol 30 ng/ml Shown are mean values + SEM Statistical calculations and dru#s Statistical calculations were performed according to conventlonal procedures (Snedecor & Cochrane, 1967) The following drugs were used lonophore A 23187 was a gift of Ely Lilly U S A Propranolol chloride (Lazar S A ), Phentolamlne chlorhydrate (Clba Gelgy S A I C ), 6.7-dimethyl tetrahydropterldlne (DMPH., Aldrich S A U S A ). histamine (Calblochem. U S A ). dlphenhidramIne (Benadryl. Parke-Davls). sodium pentobarbitone (Embutal. Abbot. S A ). metiamlde (Smith Klein and French) and verapamll (Isoptmo. Knoll S A ) RESULTS Isolated Oulnea-p~g atria In Isolated spontaneously beating atria, the ionophore A-23187 ehcited a positive chronotroplc effect (Fig 1 B) The dose response curve was biphaslc The first phase started at rather low concentration (10 T M g / m l ) a n d reached its maximum at 1 0 - 9 g / m l while the second one extended from 1 0 - g g / m l to 10 -6 g/ml exhibiting the highest effect of lonophore A-23187 The positive chronotroplc lonophore A-23187 effect was compared with the ( - ) - n o r a d r e n a h n e effect (Fig

1 A) The ionophore A-23187 showed more potency than the neurotransmttter since its pD2 was higher (Table 1) The maximal response was lower than that of ( - ) - n o r a d r e n a h n e (Table 1) In order to elucidate the mechanism mvolved m the posltwe chronotroptc biphasic effect of lonophore A-23187, we tried different drugs on lonophore actions Proprano.lol, 30ng/ml shifted to the right the ( - ) - n o r a d r e n a h n e dose-response curve by 1 log unit, (Fig 1 A)

113

Pharmacological effects of ionophore A-23187 Table 1 pO 2 and the maximal response to the ( - ) noradrenahne and lonophore A-23187 on the isolated guineapig atria Drug (-)noradrenahne A-23187

N

pD2

Maximal response (beats/mln)

16 8

6 52 + 0 07 924+010

130 + 5 85+6

B

,1o

30

B

10

N--number of experiments pD2--negatlve logarithm of the medium effective molar concentration + SEM The same concentration of propranolol abolished only the responses to the low concentration of lonophore (Fig 1 B) A similar effects was observed when noradrenahne endogenous store was depleted by reserpine (Fig 2) After 24 hr reJection of 0 3 mg/kg reserpine, a partial decrease m the first component of response to lonophore A-23187 was observed (Fig 2 A) while from a 3 mg/kg dose such a component was almost absent, the second one remaining unaffected (Fig 2 B) Since lonophore A-23187 is able to release histamine from mast cell (Foreman, Mongar, 1973) and considering that histamine produces a positive chronotropic effect on isolated guinea-pig atria, it was thought possible that the effect observed was partially mediated by the release of endogenous histamine from mast cells Histamine 0 3 #g/ml showed a positive chronotropic effect (Fig 3) This effect remained almost unchanged by diphenhydramme, a H1 receptor antagomst, while tt was completely blocked by metiamide, a H2 receptor antagonist Since the lonophore response did not change by addition of H i and H2 receptor antagonists, the m-

HS

HIS 70

riM_

II

-30

Fig 3 Effect of antihistamines on the ionophore response Isolated guinea-pig Krebs solution, 37°C, ordinates--frequency increase in beats per minute, Histamme (His) 03#g/ml, diphenhydramine (B) 5 pg/ml, ionophore (I) 3 x 10 -6 g/ml, metlamlde (M) 20/ag/ml volvement of histamine was discarded m the posmve chronotrop~c response to lonophore Since the second component did not appear to be related either to B-receptors and H1 or Hz receptors, it was thought to be concerned with the Ca 2 + permeablllty directly m cardiac muscle In order to elucidate this posstb]hty, two Ca 2÷membrane-transport antagonists were tried The action of 2 10 -7 g/ml verapamll (Haeusler, 1972, Bayer et al, 1975) on Ionophore A-23187 positive chronotropic effect in reserpmezed atria was shown (Fig 4 A) Ionophore A-23187 (10-7g/ml) m control conditlons produced a positive chronotropic effect which dechned only about 20% within 45 min However,

10C

A

Veropomll I 2 10-7g/ml

_~-~

60

A

\

,o

60

20

40

o

"'~....a...a" i

i

i

i

i

i

i'"F--~

20 --~

0

A-2 3187 10-1g/mr

~, u

u~

Pen~oborbltol

80

8C

60

6C

40 20

4C 2( 0i

B

100 ~

I

I

]

I0-~ i0-~0 I0-9 i0-8 g/ml

I

I

I

i0-7 i0-6 10-s

Fig 2 Effect of reserpine on the posmve chronotroplc response to lonophore Isolated guinea-pig atria Krebs solution, 37°C, ordinates--frequency increase in beats per minute, abscissae--]onophore concentration (g/ml), (A) animals pretreated with reserpine 03mg/kg l p 24hr before the experiment, (B) animals pretreated with reserpine 3 mg/kg l p 24 hr before the experiment, The mean values _ SEM are shown

5 A-23187 10-7g/ml

10 15 20 25 30 35 40 45 Tlrne (m~n)

Fig 4 Effect of two Ca 2+ membrane transport antagonists verapamll and pentobarbltal on the lonophore response Isolated guinea-pig atria, reserpintzed animals (3 mg/kg 24 hr) Krebs solution 37°C, ordinates--frequency Increase m beats per minute, abscissae--time in minutes The ionophore 10- 7 g/ml was added at zero time - - A - control group, --O--- the arrow indicates the time at which the verapmll 2 10 - 7 g/ml (A) and sodium pentobarbitone 1 24 x 10-4g/ml were added Shown are mean values + SEM

114

VIVIANA TARIZZOand MODESTOC RUBIO 30G

Table 2 pD2 and maximal response to the ( - ) noradrenahne and lonophore A-23187 m cat's mctltatmg membrane

¢-

E

20C

t/1

N

pD2

Maximal response (g)

(-)noradrenahne A-23187

10 6

503 _ 005 572+010

160 ___02 55+04

N--number of experiments pD2--negatwe logarithm of the medmm effective molar concentration _ SEM

IOG

LL 1"

2*

3*

4*

5"

Fig 5 Tachyphylaxls of posmve chronotroplc effect of the ionophore A-23187 Isolated guinea-pig atria Krebs solutmn 37°C The isolated guinea-pig atrm were stimulated Consecutively 5 times with ionopfiore 1 #g/m] and w]aen the maximal response at th~s concentration was reached, the atrm were successively washed w~th Krebs solutmn ! basal frequency, [] frequency after the addition of mnophore A-23187 (1 #g/ml)

when verapamfle was added into the organ bath, a strong decrease m the posmve chronotrop]c effect of lonophore was observed, moreover such a n effect almost &sappeared 20 m m later A similar effect was observed w]th s o d m m pentobarbltone, another C a 2+ flow antagomst (Fig 4 B) (Blaustem & Christie, 1975) It was considered of interest to find out ff the direct pos]tlve chronotroplc response to lonophore A-23187 exhibits a tachyphylaxls p h e n o m e n o n W h e n reaching maximal response, they were washed w]th cold Krebs solution, this procedure bemg repeated 5 times (Fig 5) The ]onophore ehcited a 85 b e a t s / m m increase m frequency at first stlmulatmn, th~s response was decreased by successive stimulations, (to a b o u t 50% of

,r

Drug

f f

/

m m a l value at the second one a n d 15% at the followmg ones) This clearly suggests a fast tachyphylactlc effect O n the other h a n d the basal frequency was not statistically affected in these successive st~mulatmns

Isolated cat mctztatmg membrane C o n t r a c t d effect produced by b o t h lonophore A-23187 a n d ( - ) - n o r a d r e n a h n e on medial smooth muscle can be seen m F]g 6 Although the lonophore exhibited a potency slm]lar to ( - ) - n o r a d r e n a h n e (Table 2), the maximal response reached was only 34% of the neurotransm~tter one Th~s effect presented, unhke isolated guinea-pig atria, only one c o m p o n e n t and rose at 3 x 10 -8 g/ml Phentolamme, 1/~g/ml produces a shift to the right m the ( - ) - n o r a d r e n a h n e dose-response curve (Fig 6A) a n d almost a b o h s h e d the response to lonophore In the cat mctltatmg m e m b r a n e (Fig 6 B) In order to test releasing effect of m n o p h o r e A-23187, endogenous neurotransm~tter stores were labelled with a H - n o r a d r e n a h n e Release of t n t m m by 3 x 10- 6 g/ml lonophore__was measured after 100-120 m m l n c u b a t m n with aH-noradrenahne, when the e q m h b r m m was reached (Fig 7) I o n o p h o r e showed a 3-fold increase m t n t m m release which was maintained for 2 0 m m after subsequent washmgs

Actton of tonophore on tyrosme hydroxylase and monoamme oxtdase In order to elucidate ff m n o p h o r e A-23187 modify either the bmsynthesls or m e t a b o h s m of catechol-

/

10-8 10-7 10-6 10-5 10-z, 10-3 (-) Norodrenohne (g/ml)

10-9

(~ 15F

B

,°f 3O

'°f,

I

I

10-9 10-8 10-7 10-6 10-6 10-4 Ionophore(g/m I)

Fig 6 Effect of phentolamme on the contractfl response to (-)-noradrenahne and ionophore A-23187 Isolated cat's mctltatmg membrane Krebs solutmn 37°C, ordinates-development of tensmn m g, absossae---(A) ( - ) noradrenahne concentratmn (g/ml), (B) mnophore A-23187 concentratmn (g/ml), + control, - - O - - phentolamine 1/ag/ml, shown are mean values _ SEM

!

!

A-231B7 Fig 7 Release of tritium ehclted by lonophore A-23187 Isolated mctltatmg membranes were incubated with Krebs solution containing ( _ ) aH-noradrenahne 5pCl/ml Each bars represents the released nC1 per 100mg of tissue m 5 mm samples, collection of samples started 120 mm after the end of the incubation with aH-noradrenahne The period of exposure to 3 10 -6 g/ml of Ionophore is shown by the horizontal bars The four samples obtained after the exposure to A-23187 correspond to the period of washings

Pharmacological effects of lonophore A-23187

115

Table 3 Effect of lonophore A-23187 on tyrosme hydroxylase of cat's mCtltatmg membrane A Homogenates

Tissue Homogenate Homogenate Homogenate Homogenate B Intact tissue Tissue Intact Control Intact Reserpine 3 mg/kg pretreated animals

A-23187 -01 1 10 A-23187

TH (J) 3 20 3 15 2 98 3 32

_ + + +

% Inhibition 0 35 0 18 0 20 042

TH (b)

1

13 008 _ 3 215 6699+833

1

26 150 + 2391 24 8 4 2 _ 2 2 7 5

-16 _ 12 69 + 32 004 + 3 1 % Inhibition 485+95" 5_43

(a)Tyrosme hydroxylase activity expressed in nmoles of dopa formed per gram tissue and per hour Tyroslne concentration 10/~M DMPH4 concentration 100/~M (b) Hydroxylatlon of tyrosme m intact tissue expressed in dpm of dopa formed per gram tissue In 20 rain *P <005

amines, it was assayed on the activity of tyrosme hydroxylase, the h m l t m g enzyme in the catecholammes biosynthesis (Lewtt et al, 1965) as well as on the activity of m o n o a m m e oxldase, a n enzyme revolved In the metabolism of catecholammes I o n o p h o r e h a d no direct effect on tyrosme hydroxylase m cat mctltatmg m e m b r a n e homogenates (Table 3) Determinations were made at under-saturant concentrations of substrate (0 01 m M ) a n d cofactor (0 1 m M ) However the enzymatic activity was lnhlbtted by 50% m intact tissues (Table 3) Since the drug had n o direct effect o n the enzyme it was t h o u g h t possible that the observed effect was mediated by endogenous n o r a d r e n a h n e release, consldermg that n o r a d r e n a h n e a n d some of its metabohtes competitively inhibit the enzyme (Rubto & Langer, 1973) This hypothesis was demonstrated by the fact that the inhibitory effect of lonophore disappeared in reserpmlzed animals when the endogenous n o r a d r e n a h n e store had been depleted (Table 3) At 10 - l ° a n d 10-5 g/ml, the drug had no effect on m o n o a m m e oxldase activity m isolated guinea-pig astrla homogenates ( M A O activity m /~mol/g h control 1 63 _+ 0 17, 1 0 - 1 ° g / m l lonophore 1 56 4- 0 15, 10 -5 g/ml lonophore 1 59 _+ 0 18) DISCUSSION In the present paper the pharmacological actions of lonophore A-23187 on b o t h guinea-pig atria a n d cat nictitating m e m b r a n e were studied In these adrenerglcally lnervated tissues the drug showed agomst effect with distinctive features In atria a blphaslc positive chronotroplc effect was observed Its first c o m p o n e n t appeared at low concentrations (about 10- xi g/ml) and is due to the release of endogenous catecholammes since it was antagonized by a blockade of beta adrenoreceptors or by pretreating the animals with reserpine

This releasing effect was also exhibited in mctltatmg m e m b r a n e whose contractll response is antagonIzed by phentolamlne, also a release of tritium from tissues labelled with the aH-neurotransmltter have been demonstrated However in the nictitating m e m b r a n e this effect was lower than that of the atria, since it was observed only at a concentration higher than 10-7 g/ml Although the sensitivity of fl responses to ( - ) - n o r adrenaline is higher than that of ~-responses (see pD2 in atria and nictitating m e m b r a n e for ( - ) - n o r a d r e n a line) it is clear that there are differences in the sensitivity between the releasing mechanism In b o t h tissues The second c o m p o n e n t of the response to lonophore in atria Is a direct effect on cardiac muscle which is not observed m the nictitating m e m b r a n e s m o o t h muscle The sensitivity of car&ac muscle to extracellular C a 2÷ concentration is well k n o w n , a clear positive chronotroplc effect as well as modifications in action potentials related to Ca 2 + increase in such a compartment have been detected (Westfall & Flemmg, 1968, Toda, 1972) The effect observed in atria is not related to the releasing effect of histamine exhibited by this drug since it is not antagonized by H l and H2 receptor blockers when the positive chronotroplc effect of histamine is clearly impaired The direct effect on cardmc muscle Is clearly related to the entrance of Ca 2÷ from the extracellular medmm, since it is antagomzed by two Ca 2+ flow blockers Verapamll (Haeusler, 1972, Bayer et al, 1975) and pentobarbltal (Blausteln & Christie, 1975) T h o u g h this effect on cardiac muscle is long lasting 0t can be observed at least for 45 mm), the exposure of guinea-pig atrm to successive stlmuh with the ~onophore produces a clear tachyphylactlc effect It is noticeable that this effect it is not related to the well

116

VIVIANATARIZZO and MODESTOC RUBIO

known action produced by amines of indirect action (Bhagat et al, 1965) which would be related to depletion of catecholamlnes stores In the present paper, where response to lonophore is studied in reserptnlzed animals, a direct cardiac muscle response is being involved This tachyphylaxis is thus a tolerance mechanism related to the events occurring between the Ca 2 ÷ entrance and the cardiac frequency increase, and could be related to an inhibition of ATP synthesis by Ionophore as has been described in liver (Frledmann et al, 1979) The lonophore A-23187 has been reported to produce a mixed (exocltOtlC and not exocltOtlC) release of NA in guinea-pig vas deferens (Thoa et al, 1974) F r o m our experiments we would agree with these authors Since the drug did not affect monoamlne OXldase activity, ff the mechanism were completely non exocitotic, noradrenahne released from granules would be almost fully metabolized by the enzyme to metabohtes inactive on ct- or fl-receptors (Adler-Graschinsky et al, 1971, Langer & Rublo, 1973) O n the other hand supporting a not-exocltOtlC release the lonophore showed an inhibitory action on tyroslne hydroxylase actlvtty in intact tissues, without any effect on the isolated enzyme This inhibitory effect is related to the release of noradrenahne to cytoplasm since either NA ttself or its deammated metabohte 3-4 dihydroxyphenllglycol are able to inhibit the enzyme (Rublo, 1976) Acknowledgements--We are grateful to Dr Francisco J E Stefano for critical reading of the manuscript, to Lldla Caballero for her excellent technical assistance and Isabel Mercado for her expert secretarial assistance

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