The vasomotor innervation of the inferior vena cava of the domestic fowl (Gallus gallus domesticus)—II. Functional observations

Gen. Pharmac., 1975, Vol. 6, pp. 49 to 51. Pergamon Press. Printed in Great Britain

THE VASOMOTOR I N N E R V A T I O N OF THE INFERIOR VENA CAVA OF THE DOMESTIC FOWL (GALLUS GALLUS DOMESTICUS)--II. F U N C T I O N A L OBSERVATIONS TERENCE BENNETTAND TORBJORN MALMFORS Department of Physiology, Nottingham University Medical School, University Park, Nottingham, NG7 2RD, and Department of Toxicology, Astra Pharmaceuticals AB, S-151 85 SOdertiilje, Sweden (Received 27 March 1974) Abstract--1. The effects of drugs and nerve stimulation on isolated inferior venae cavae from chicks were studied. 2. The vessel has a well-developed noradrenergic vasomotor innervation, but only a weak cholinergic innervation. 3. The effects of noradrenergic nerves are mediated via a-receptors; fl-receptors are absent from the vessel. INTRODUCTION THERE have been relatively few functional observations on the innervation of the vasculature in birds (see Jones & Johansen, 1972; Bennett, 1974), particularly veins. In a study of the cardiovascular adjustments to "diving" in ducks, Djojosugito et al. (1969) found that a neurogenic venoconstriction formed part of the response. These workers did not, however, characterize the venoconstriction pharmacologically. Bolton & Bowman (1969) investigated adrenoceptors in the cardiovascular system of the domestic fowl, and concluded that the fowl and mammalian systems were basically similar. It was recently demonstrated (Bennett & Malmfors, 1970; Bennett et aL, 1974) that the inferior vena cava in the domestic fowl is densely innervated, and possesses a well-developed longitudinal muscle coat. These features indicated that this vessel might be a suitable model to use in an investigation of the physiology and pharmacology of the motor innervation of avian venous smooth muscle. In the present work observations have been made on inferior venae cavae from chicks of different ages, since earlier findings (Bennett, 1971; Bennett et aL, 1973) indicated that noradrenergic vasomotor nerves in young chicks differ in some ways from those in older animals. MATERIALS AND METHODS White Leghorn chicks, 2 and 6 weeks old were used in this study; animals were killed by decapitation under light ether anaesthesia. The length of the inferior vena cava between the liver and the adrenal glands was removed and slit open. The tissue was mounted longitudinally in a 50ml organ bath containing a saline solution (Ginsborg, 1960) of the following composition (mM): NaCI, 150; NaHCOs, 20; KC1, 5; CaCll, 5"0; MgCli, 5'0, with glucose 2 g/1. The solution was bubbled with 95 per cent oxygen and 5 per cent carbon dioxide, and kept at 37°C. Longitudinal contractions of the

inferior vena cava were recorded through a Statham force-displacement transducer coupled to a Grass model 5 polygraph. Electrical stimulation was effected through platinum electrodes encircling the preparation; with the stimulus parameters used (60 V strength, 0.2 msee pulse duration, 0.1-60 Hz, for 10 see every 5 min) no direct muscle stimulation occurred. All preparations were allowed to equilibrate for I hr before experiments were begun. Some animals were injected intravenously with reserpine (5 mg/kg) on the two days prior to being killed. Drugs used were:--L-noradrenaline bitartrate, Ladrenaline bitartrate, L-isoprenaline hydroehloride, tyramine hydrochloride, dopamine hydrochloride, phenoxybenzamine hydrochloride, propranolol hydrochloride, cocaine hydrochloride, desmethylimipramine hydrochloride, guanethidine sulphate, dex-amphetamine sulphate, reserpine (Serpasil), acetylcholine chloride, neostigmine, methylsulphate, hyoscine hydrobromide, perttolimum tartrate and tetrodotoxin. All stock solutions of cryatalline drugs were freshly prepared; doses referred to in the text are in terms of the forms given above, except for noradrenaline, adrenaline and isoprenaline which are expressed in terms of the bases. RESULTS Preparations from animals in both age groups were usually quiescent, but spontaneous activity was occasionally seen (Fig. 1). This activity took the form of rhythmical contractions that were unaffected by phenoxybenzamine (1 × 10 -e g/ml), propranolol (5 × 10 -~ g/ml), guanethidine (5 × 10 -6 g/ ml), hyoscine (1 × 10 -e g/ml) or tetrodotoxin (5 × 10 -~ g/ml). Responses o f the inferior vena cava to drugs Noradrenaline (NA) and adrenaline (A) (1 × 1 0 - s to 1 × 10 -s g/ml) caused a sustained contraction of the inferior vena cava. In animals from both age groups the inferior venae cavae were more sensitive to A than NA, but there were no marked differences in the catecholamine sensitivities of the tissues taken from chicks of different ages. In the presence of

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desmethylimipramine (DMI) (1 x 10-6 g/ml), the differential sensitivity of the inferior vena cava to A and N A was abolished. Isoprenaline (1 x 10 -9 to 5 x 10-*g/ml) had no effect on the inferior vena cava, even when the tone of the tissue was raised with barium chloride. Dopamine (1 x l0 -7 to 5 x l0-6g/ml) caused a contraction that was reduced, but not abolished by pretreatment D M I (1 x 10 -6 g/ml). Tyramine (5 x 10 -6 g/ml) caused a marked conreaction of the inferior vena cava; this effect was prevented by prior treatment with D M I (1 x 10 -6 g/ml). Tyramine had little effect on inferior venae cavae taken from reserpinized chicks. The excitatory effects of all the above drugs were abolished by phenoxybenzamine (1 x 10 -6 g/ml), but were unaffected by propranolol (5 x 10 -v g/ml). Acetylcholine (1 x 10 -6 to 5 x 10 -6 g/ml) caused the inferior vena cava to contract; this effect was blocked by hyoscine (5 x 10 -7 g/ml). Responses o f the inferior vena cava to nerve stimulation Transmural electrical stimulation of the inferior vena cava caused a pronounced shortening of the vessel. With the stimulation parameters used (see Materials and Methods), the response was completely abolished by tetrodotoxin (5 x 10 -7 g/ml), indicating that it was due to stimulation of vasomotor nerves (see Gershon, 1966). Inferior venae cavae from 2-week old chicks were much smaller than those from 6-week old chicks, thus the force developed when they contracted was much less (Fig. 2). With preparations from animals in either age group, the durations and amplitudes of the responses to electrical stimulation increased with increasing stimulation frequency (Figs. 2 and 3). The increase in the durations of the responses was particularly marked at high stimulation frequencies. Pentolinum (1 x 10 -e g/ml) had no effect on the nerve-mediated responses. Cocaine (5 x 10 -7 g/ml), D M I (1 x 10 -6 g/ml) or dex-amphetamine (1 × 10 -7 g/ml) caused a marked increase in the amplitudes and durations of nervemediated responses (Fig. 4). Guanethidine (5 x 10 -e g/ml) had no direct excitatory effect on the inferior vena cava, but, following nerve stimulation in the presence of the drug, the tone of the tissue remained elevated and the nerve-mediated response was usually abolished (Fig. 5). Even following repeated washing there was no marked decrease in tone and no reapparance of the response to nerve stimulation (Fig, 5). Under these conditions dex-amphetamine (1 x l0-Tg/ml) had no effect on the tone, but over a period of about 40 min in the presence of the drug, the responses to nerve stimulation gradually re-appeared, and were finally greater in amplitude and duration than the responses seen before treatment with guanethidine (Fig. 5). When the dex-amphetamine was washed

from the bath the enhancement of the response was lost (Fig. 5). The block of nerve-mediated responses by guanethidine was also antagonized by cocaine (5 x 10 -e g/ml) and D M I (1 x 10 -6 g/ml), but not as effectively as by dex-amphetamine. Addition Of phenoxybenzamine (1 x 10 -6 g/ml) to the organ bath usually caused a rapid and almost complete block of the response to nerve stimulation (Fig. 6). When a response persisted, it was unaffected by prolonged treatment with propranolol (5 x 10 -7 g/ml) but was rapidly abolished by hyoscine (5 × 10 -7 g/ml) (Fig. 6). Hyoscine (5 x 10 -7 g/ml) also caused a decrease in the amplitude of the nervemediated response under normal conditions, but neostigmine (5 x 10 -7 g/ml) had no obvious effect on the amplitude of the response to nerve stimulation. Acetylcholine (1 x 10 -9 to 5 x 10 -6 g/ml) caused contraction of the inferior vena cava, as described above. Application of low doses of acetylcholine (1 x 10 -9 to 1 x 10 -8 g/ml) to the inferior vena cava had no obvious modulatory effects on the responses to nerve stimulation (Fig. 7). However, higher doses of the drug caused marked increases in tone and the nerve-mediated responses were then depressed. DISCUSSION In spite of the fact that previous findings (Bennett, 1971 ; Bennett et al., 1973) indicated a more efficient uptake of catecholamines by noradrenergic nerves in older chicks, the present observations do not demonstrate any functional differences between the vasomotor innervation of the inferior vena cave in young and old animals. However, it was found that the vessels from animals in both age groups were more sensitive to A than NA, but that this differential sensitivity was abolished by the catecholamine uptake inhibitor DMI. These findings indicate that the noradrenergic vasomotor nerves are effective in reducing the concentrations of A and N A at the receptor level and furthermore, that the nerves show a greater affmity for N A than A (see Iversen, 1967). From the present work it appears that 13adrenoceptors are absent from the inferior vena cava of the domestic fowl. Bolton & Bowman (1969) have previously demonstrated that the distribution of I~adrenoceptors, in different areas of the fowl cardiovascular system, is not uniform. The differences in in receptor populations of the various regions of the cardiovascular system may account for the differences in responses to the sympathomimetic amines dopamine and tyramine (see Bolton & Bowman, 1969), although the effects of these two drugs was in line with their suggested mode of action (see Iversen, 1967). In the absence of 13-receptors, it seems that the effects of noradrenergic nerves on the inferior vena cava must be mediated via a-receptors. Support for this belief was provided by the finding that the a-receptor blocking drug, ohenoxybenzamine,