Camp. Biochem. Physiol.Vol. IOlC, No. 2, pp. 197-201, 1992 Printed in Great Britain

0306-4492/92 $5.00 + 0.00 0 1992 Pergamon Press plc

EFFECTS OF CATECHOLAMINES ON THE ISOLATED AORTA OF THE SNAKE BOTHROPS JARARACA N. YAMANOUYE,

L. C. SALOM~O*

and Z.

P. PICARELLI~

Serviqo de Farmacologia, Instituto Butantan, Av Vital Brazil, 1500, CP 65, 05504, Slo Paulo, and *Departamento Fisiologia Geral, USP, Slo Paulo, SP, Brazil (Received

30 May

SP, Brazil

1991)

Abstract-l. Effects of catecholamines in snakes have been examined using an aorta preparation isolated from Bothrops jururaca. Adrenaline, noradrenaline and isoprenaline produced dose-dependent contractions on this preparation. The relative potency was adrenaline > noradrenaline > isoprenaline. 2. Phentolamine displaced, to the right, the concentration-response curve of the three catecholamines tested, showing the presence of alpha-adrenoceptors in this preparation. 3. Isoprenaline has never produced a relaxation, even when the aorta was first contracted by BaCl, and pretreated with phentolamine, indicating that beta-adrenoceptors are absent in this preparation. 4. In this Bothrops juraraca preparation, exclusively neuronal uptake was found, thus demonstrating

that

its

existence was preserved during evolution.

INTRODUCTION

agonists and antagonists, with the excitatory response developing more rapidly than the inhibitory. Bolton and Bowman (1969) have also detected both alpha- and beta-adrenoceptors in the domestic fowl aorta. In the present work, the effects of some catecholamines, the receptors involved and the participation of neuronal and extraneuronal mechanisms in the ending of their actions have been studied on the isolated aorta of Bothrops juraraca, as part of an analysis of the evolution of the adrenergic system, in the vascular system of the lower vertebrates.

Cardiovascular function has undergone pronounced changes during vertebrate evolution, a progressive increase occurring in the adrenergic component compared to the cholinergic one (Burnstock, 1969). Since Ahlquist (1948) classified adrenoceptors into alphaand beta-receptors, many studies of them have been performed on the cardiovascular system of mammals. However, there are few data about these receptors in lower vertebrates. In general, results have just shown the qualitative effects of some of the main catecholamines. It has been reported that adrenaline and noradrenaline produced contractions of large arteries in fishes, amphibians, reptiles and domestic fowl (Burnstock and Kirby, 1968; Kirby and Burnstock, 1969a; Bolton and Bowman, 1969; Forster, 1981; Stephens, 1984; Yung and Chiu, 1985) and caused an increase in arterial blood pressure in opossum, toad, lizard, snake, turtle, alligator and fowl (Akers and Peiss, 1963; Kirby and Burnstock, 1969b; Bolton and Bowman, 1969; Baysal and Vural, 1975; Breno et al., 1984; Picarelli, 1987). Hypertensive and contractile effects in the toad and lizard were blocked by alphaand beta-adrenoceptor antagonists unspecifically (Burnstock and Kirby, 1968; Kirby and Burnstock, 1969a, 1969b). Disagreement about isoprenaline effects is observed: it has been shown that isoprenaline had no effect on the arterial strip preparations of toad and teleost fish; however, it caused contraction on the lizard aorta preparation and it never produced relaxation even when the muscle tone had been previously raised (Burnstock and Kirby, 1968; Kirby and Burnstock, 1969a). On the other hand, Forster (1981) using ventral aorta of eels, showed the presence of either contractions, due to alpha-adrenoreceptors, or relaxation, due to beta-adrenoreceptors in response to catecholamine tTo whom

correspondence

should

MATERIALS

AND METHODS

Animals Bothrops juraruca snakes of both sexes (100-200 g) were used throughout this study. They were captured in the wild, were received and classified by SeqLo de Herpetologia, Instituto Butantan, and, as described by Breno et al. (1990), after an observation period, were kept in a room under controlled environmental conditions (photoperiod of 12 hr light/l2 hr dark, temperature 26°C and relative humidity 65.3 + 0.9%). In vitro preparations The snakes were killed by decapitation the day before the experiment and their bodies were kept at 4°C. At the moment of the experiment, aortas were removed and dissected from the connective tissue and cut into 1 cm length rings. These rings were suspended in an organ chamber and connected to a transducer (Amp&e) for measurement of isometric tension, which was recorded on a ECB-RB 202 recorder. The organ chamber was filled with Krebs solution for snakes with the following composition mM: NaCl 144.17, KC1 4.95, CaCl, 2.75, MgSO, 1.21, NaH,PO, 1.20, NaHCO, 29.60, glucose 5.50 and osmolarity 285 mOsm/l, bubbled with 95% 0, and 5% CO,. The composition of the Krebs solution for snakes was established by determination of the electrolyte concentration (Table 1) and osmolarity of Bothrops jararaca plasma. Osmolarity was found to be 281.78 (272.81-290.75) mOsm/l. The experiments were performed at 37°C. Each ring was placed under 1.0 g tension

be addressed. 197

198

N. YAMANOLJYEet al.

and allowed to equilibrate for 60 min. Cumulative concentration-response curves for adrenaline, noradrenaline and isoprenaline were constructed. Only one concentration-response curve was made on each preparation. In two series of experiments, cocaine (10m4 or 3 x lo-4 M) or P-estradiol (10m4 or IO-‘M) were added to the organ chamber, 30 min before the agonist, in order to detect the presence of neuronal or extraneuronal uptake, respectively, in this preparation. The abilities of the adrenergic antagonists phentolamine (10m7 M) and propranolol (10-j or lO-4 M) to shift the concentration-response curve were tested. The preparations were preincubated for 30 min with the antagonists. These experiments were performed in the presence of cocaine (10m4 M), with the purpose of eliminating the neuronal uptake present in Bothrops jararaca aorta.

For the relaxation tests, aortas were pre-contracted with BaCl, (3 x lo-‘M) in the presence of phentolamine (3 x lo-’ M). Concentrations causing an increase in tension of 50% of the maximal response (ECso) to each agonist were calculated from each individual concentration-response curve and presented as the mean negative logarithms (pD,). In experiments using adrenoceptor antagonists the ECU,,

obtained for each agonist in the absence and in the presence of antagonists was determined and compared statistically. Measurement of electrolyte concentration and osmolarity of Bothrops jararaca plasma Na+ and K+ concentrations were determined by flame emission with a Digimed NK-2002 Flame photometer; Ca*+ and Mg r+ by flame absorption with a Zeiss PM-Q11 Atomic Absorption Spectrophotometer; and Cl- by the modified ultramicro Schales and Schales method with a Beckman microtitrator Model 153, where 10~1 of plasma were titrated with mercuric nitrate solution in the presence of diphenylcarbazone as indicator. Osmolarity was determined with a Fiske G-66 osmometer.

hydrochloride (Ciba, (Baker, Brazil). Statiscal

Switzerland)

and

barium

analysis

were expressed either as means + SEM or mean values and 95% confidence intervals. Statistical differences between two means were determined by Student’s r-test for unpaired observations. Where more than two means were compared, analysis of variance was used. Results

RESULTS Histological

structure

of Bothrops

jararaca

Electrolyte Bothrops

determination

1 shows jararaca

the electrolyte concentration of plasma and of the Krebs solution

which is commonly used as a nutritive liquid for working with isolated aorta of mammals (Furchgott and Zawadzki, 1980). Na+, KC, Ca2+ and Cl- concentrations differed significantly in both liquids. By using a physiological solution with electrolyte concentrations similar to those of Bothrops jararaca plasma (Krebs solution for snakes), the contractions produced on Bothrops jararaca aorta were greater than those produced in the usual Krebs solution.

A segment of aorta was fixed in Bouin and embedded in paraffin. In order to verify the aorta structure, 7-pm sections were cut and stained; hematoxylin-eosin, Masson’s trichrome or Weigert’s resorcine methods, respectively, were used for layer identification of collagenic and smooth muscle fibers, or elastic fibers.

Effect

of catecholamines

Adrenaline, noradrenaline and isoprenaline produced dose-dependent contractions of the snake isolated aorta (Fig. 2). The relative order of potency, expressed in therms of pD, values, was adrenaline, noradrenaline and isoprenaline (Table 2). No significant differences in the maximal contractions produced by these catecholamines were detected. As

Drugs L-epinephrine, (-))-arterenol and (-)-isoproterenol bitartrates, P-estradiol 3-benzoate and DL-propranolol hydrochloride (Sigma Chemical Co., St. Louis, MO); cocaine hydrochloride (Merck, Germany); phentolamine

A

INTIMA

b.

MEDIA

C.

ADVENTITIA

Fig.

LAYER LAYER

aorta

As shown in Fig. l(a), Bothrops jararaca aorta is similar to the mammalian aorta; it has three layers: intima, media and adventitia. The intima consists of a lining endothelium plus a thin layer of subendothelium connective tissue. Elastic fibers are concentrated in the thin intima and the thick media layers (Fig. lc); smooth muscle fibers are in the media layer, whereas the collagenic fibers are most abundant in the adventitia layer (Fig. lb). Media and adventitia layers also contain fibroblasts and amorphous substances.

Table

Hisiology

a.

chloride

a.

COLLAGENIC

b.

SMOOTH

FIBERS

MUSCLE

a.

ELASTIC

FIBERS

FIBERS

LAYER

1. Photomicrographs of sections of aorta from Bothrops jararaca snake. A: hematoxylin-eosin, B: Masson’s trichrome, C: Weigert’s resorcine methods ( x 990).

Catecholamines on snake aorta Table I. Electrolyte concentration of Bothrops jararaca plasma and Krebs solution Concentration 5. jarwm~ plasma*

Electrolyte Nat

(mM) Krebs solution

175.00** (166.96383.04) 4.94** (4.57-5.31) 2.74’** (2.562.82) 1.20 (1.13-1.27) 155.58” (108.67-122.49)

K’ Ca*+ Mg2+ cl-

142.00

199

Table 2. Contractile effects of adrenaline (AD), noradrenaline (NA) and isoprenaline (ISO) on Bothrops jaruraco isolated aorta, in the absence or presence of adrenergic uptake blockers Grouts

AD

PD* NA

IS0

Control

7.05 i 0.09 (10)

6.63 +-0.14 (16)

5.14_40.08 (13)

7.62 ri: 0.09* (6) 1.77 & 0.22, (10)

7.72 F O.l3* (7) 6.98 F 0.10 (11)

5.14 + 0.09 (6) 5.09kO.13 (6)

7.07 * 0.10 (8) 7.07 f 0.10 (8)

6.41 & 0.08 (6) 6.46+0.14 (6)

5.15 50.12 (7) 4.95+0.11 (7)

6.00 2.00

Cocaine 1 x 10-4M

1.20

3 x 10-4M j?-Estradiol

128.00

Ix

*Mean value and 95% confidence intervals. Values were significantly different from those found in Krebs solution (t-test, **P < 0.01, ***P < 0.02).

shown in Fig. 3, isoprenaline never produced relaxation of ~~~~r#~~juraraca pre-contracted aorta, even in the presence of phentolamine (3 x IO-‘M), an antagonist of alpha-adrenoceptors.

W4M

I x IO-‘M

Values represent mean + SEM. Numbers of experiments given in parenthesis. *Values are significantly different from those of the respective control group (t-test, P i 0.01)

were able to block the contractile response to isoprenaline.

Action of uptake-blocking agents

DISCUSSION

As shown in Table 2, cocaine shifted significantly the con~ntration-response curves of noradrenaline and adrenaline to the left, while those for isoprenaline were not affected. /I-Estradiol had no effect on noradrenaline, adrenaline or isoprenaline responses.

As Na+, K+, Ca2+ and Cl-

of from those in Krebs solution and these ions are important in smooth muscle contraction, it proved worthwhile to elaborate an appropriate physiological solution for working with Bothrops jararaca aorta. Action of adrenoceptor antagonists The first technique used to subclassify or differentiate adrenoceptors was based on a comparison of the Phentolamine (lo-’ M), a nonspecific alphaorder of potency of a series of agonists. In this work, adrenoceptor antagonist, displaced the concenmaking this kind of comparison on the snake aorta, tration-response curve of noradrenaline (1S-fold), the relative potency of sympathomimetic amines adrenaline (S-fold) and isoprenaline (Cfold) to the right, in a parallel way. In these conditions, the ECSO was found to be adrenaline > noradrenaline > isoprenaline. This order agrees with that suggested values were significantly higher than in the control, by Ahlquist (1948) for activation of alphawhereas the maximal responses were not affected adrenoceptors. The effects of the alpha-blocking (Table 3).

Propranolol, a beta-adrenoceptor antagonist, had no effect on the concentration-response curve of isoprenaline. Only concentrations as high as 10F4 M

IS0

-8

-9 l

-

Ad

Y

NA

-

concentrations

Bothrops juraraca plasma differed significantly

-7 .

-6 l

-5 .

.

IS0

l

-*

-7

-6

CONCENTRATION

-5

(Lag

-4

-3

Mf

Fig. 2. Mean concentration-response curves of adrenaline (AD, N = lo), noradrenaline (NA, N = 16) and isoprenaline (ISO, N = 13) on the isolated aorta from Bothrops jararacu. Vertical bars represent SEM.

Phent Fig. 3. Lack of relaxing effect of isoprenaline (HO) on Bothropsjaruraca isolated aorta, pm-contracted with BaCl, (3 x IO-‘M) and pretreated with phentolamine (Phent) (3 x lo-’ M). Doses of IS0 indicated as the logarithm of the molar concentration.

N. YAMANOUYEet al.

200

Table 3. soso values for adrenaline (AD), noradrenaline (NA) and isoprenaline (ISO) in the absence (control) and in the presence of phentolamine (lo-’ M), on the Bothrops jararaca isolated aorta. These experiments were performed in the presence of cocaine (IO-’ M) Group

AD

soSO* NA

IS0

Control

2.63 x 10-a (2.52 x lO~‘~2.75 x 10-a) N=6 2.08 x lo-‘** (1.39 x IO-‘-2.77 x lo-‘)

2.50 x 10-a (6.40 x 10m94.36 x 10m8) N=7 3.85 x lo-‘** (2.65 x lO~‘~5.05 x IO-‘)

1.28 x 10-6 (3.68 x IO-a-1.09 x 10-5) N=6 3.04 x 10-S** (2.03 x 10~s4.05 x 10-j)

Phentolamine

N=R

N=7

N=R

‘Mean values and 95% confidence intervals. N = number of experiments. **Values were significantly different from those of the respective control group (r-test, P < 0.01).

agent phentolamine also indicated that alpha-adrenoceptors were present in the aorta of this snake. This kind of receptor has been found in the cardiovascular system of all vertebrates so far studied. On the other side, the beta-adrenoceptors were found, in large arteries, only in some species like teleost fishes and fowl (Forster, 1981; Bolton and Bowman, 1969), and not in others, like lizards (Burnstock and Kirby, 1968; Kirby and Burnstock, 1969a). In the snake aorta, isoprenaline never produced relaxation, only contraction, probably by acting on alpha-adrenoceptors. Somlyo (1967) showed that, in chicken, propranolol increased isoprenaline contractile response, but in Bothrops juraraca aorta this was not observed; on the contrary, it blocked its contractile response, but this may be an unspecific effect. All these findings are indicative of the absence of beta-adrenoceptors in the aorta of Bothrqs jururucu. Since it has been shown that the endothelial cells are very important for some drugs that produce relaxation (Furchgott and Zawadzki, 1980) it could be supposed that the lack of relaxation observed in the snake aorta was due to the absence of the endothelium. However, it is already known that isoprenaline produces relaxation in an endotheliumindependent way (Vanhoutte and Rimele, 1982). The present data also agree with those found in the lizard (Burnstock and Kirby, 1968; Kirby and Burnstock, 1969a) and are consistent, since it is assumed that snakes are a monophyletic group that has originated from lizard or from some pre-lizard ancestors (Rieppel, 1979). It seems that the absence of betaadrenoceptors does not mean an evolutionary process, but a physiological adaptation, since in lizards and snakes, where the beta-adrenoceptors are absent, blood pressure is very low (Breno et al., 1984; Kirby and Burnstock, 1969b; Picarelli, 1987). The hypotensive effect evoked by isoprenaline on Bothrops jaruracu blood pressure (Breno and Picarelli, personal communication) as well as that seen on turtle blood pressure (Baysal and Vural, 1975; Zehr et al., 1981) may be due to action on the heart; however, the presence of beta-adrenoceptors in the resistance vessels cannot be excluded. The adrenergic innervation of the snake, a long animal, shows a physiological adaptation. Fluorescence histochemical studies, have shown that, in this animal, the anterior arteries and veins have little adrenergic innervation in contrast to the extremely dense innervation of the arteries and veins posterior to the heart (Donald and Lillywhite, 1988). This adaptation would be a

regulatory process to counteract the gravitational disturbance imposed by vertical posture or movements in the air. In mammalian tissues, cocaine and /I-estradiol are inhibitors of the mechanisms of catecholamine neuronal and extraneuronal uptake, respectively (Iversen, 1974). Testing the effect of these drugs on Bothrops jararacu aorta, exclusively neuronal uptake was found. However, besides neuronal uptake, extraneuronal uptake is an important mechanism for elimination of noradrenaline in the mammalian aorta (Kalsner, 1975; Henseling, 1978). Noradrenaline and adrenaline caused a prolonged hypertensive effect in snakes (Picarelli, 1987) as compared with mammals. This effect could be explained by a deficient mechanism for concluding the action of catecholamines, since just one type of neuronal uptake is present in this preparation. This indicates that the extraneuronal uptake appeared in a later step of evolution, perhaps with the purpose of making the catecholamine metabolizing system more efficient. The characterization of the adrenoceptors in the snake aorta has been based on their responses to agonists and blocking agents which are known for their selective effects on the mammalian receptor systems. Taking these drugs as tools in the comparative analysis of the adrenoceptors in the lower vertebrates, the present findings provide important information on similarities and differences in the adrenoceptor patterns in the vascular systems of lower and higher vertebrates. Acknowledgements-The authors wish to thank Dr H. Hayashi for the histological data, Carlos Jared for the photographs, Mrs Lindonea dos Santos, Lucia Manoel Chagas, Mafalda Morando de Lima, Maria Lucia C.. P. Martins and Maria Zelma da Silva for their technical assistance and Miss Wanda Regina Carrella da Silva for typing the manuscript. N.Y. received support from FEDIB (Fundo Especial de Pesquisa do Instituto Butantan).

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