Blood Vessels 13: 350-360 (1976)

Comparison of the Excitatory and Inhibitory Effects of Angiotensin and Vasopressin on Mammalian Portal Vein1 A. H. W esto n 2 and K. G o len h o fen D epartment o f Physiology, University of Marburg, Marburg/Lahn

K ey Words. Portal vein • Angiotensin • Vasopressin • Membrane potential Abstract. Angiotensin produced inhibitory and excitatory responses in guinea pig portal vein and excitatory responses in rabbit and rat portal vein. Vasopressin produced inhibitory responses in guinea pig and rabbit portal vein and excitatory and inhibitory responses in rat portal vein. In guinea pig portal vein, inhibition by angiotensin and vasopressin was associated with an increase in membrane potential which was, on average, smaller than that produced by isoprénaline, and with a sup­ pression of spike discharges. Tachyphylaxis of the responses to angiotensin and vasopressin in portal vein was not observed and their effects were unaffected by tetrodotoxin, atropine and phentolamine plus propranolol.

In tr o d u c tio n

Both angiotensin and vasopressin have excitatory actions on a wide variety of arterial smooth muscle preparations [B o h r , 1974; A ltur a e t a l., 1965; U c h id a e t a l., 1967], However, studies of their effects in venous smooth muscle have shown that, although angiotensin produces excitation of rat and rabbit portal vein, the action of vasopressin in these tissues is inhibitory [H u g h e s and V a n e , 1967; V o th e t a l., 1969],

Received: June 23, 1975; accepted: March 22, 1976.

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1 This work was supported by grants from the Deutsche Forschungsgemein­ schaft (Go 130/14) and the Alexander-von-Humboldt-Stiftung. 2 Alexander von Humboldt Research Fellow.

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During a recent study of the action of several agonists on vascular smooth muscle, it was found that low concentrations of vasopressin produced an initial excitatory response in rat portal vein whilst angio­ tensin had potent inhibitory effects in guinea pig portal vein. In view of these findings, it was decided to investigate in more detail the effects of angiotensin and vasopressin on the portal veins of the rat, rabbit and guinea pig. A preliminary account of some of these findings has been made [G o l e n h o fe n and W e s t o n , 1975].

Rats (250-350 g), guinea pigs (300-500 g) and rabbits (3-4 kg) of either sex were killed by stunning and bleeding and the portal veins were removed. A whole vein from each animal was mounted on the same glass holder and the three tissues were placed at resting tensions of 0.5 p in a thermostatically controlled tissue bath at 35 °C. Isometric mechanical changes were measured using a mechano-electrical transducer and a potentiometric recorder. Extracellular electrical activity was measured using a perfused glass capillary [G olenhofen and v. L oh , 1970a] whilst intracellular electrical changes were measured with the technique of partial fixation as described by G olenhofen and v. L oh [1970b] using glass microelectrodes (resistance 30-100 ML?) containing 3 M potassium chloride solution. Tension development was measured isometrically from the free end of the tissues. Electrical and mechanical activity were recorded using an oscilloscope and pen recorder. Fast electrical events recorded intracellularly were reduced in amplitude by about 15% on the pen recorder read-out. Responses to angiotensin, vasopressin and isoprenaline in the glass capillary were obtained by continuous perfusion o f the capillary with a constant concentra­ tion of the drug; thus responses occurred under equilibrium conditions. In the intra­ cellular experiments, the drugs were injected directly into the recording chamber. Because of continuous perfusion with physiological salt solution, the concentration of the drug was quickly reduced and the effects produced probably did not occur under equilibrium conditions. However, in both types of experiment, antagonists were always added to the physiological salt solution reservoir and the tissue perfused for 30 min before responses to the agonists were re-examined. The physiological salt solution used throughout had the following composition (mmol/1): Na* 137, K* 5.9, Ca+* 2.5, M g~ 1.2, CL 124, HCO,- 25, H ,P 0 4- 1.2, glucose 11.5. The solution was gassed with 95% 0 2, 5% C 0 2 and had a pH of 7.4. The following drugs were used: angiotensin, Ciba; atropine, Merck; noradrenaline, Hoechst; phentolamine, Ciba; propranolol, Rhein-Pharma; tetrodotoxin, Calbiochem; lysine-vasopressin, Sandoz and Calbiochem.

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Methods

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W eston/G olenhofen

Results

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Effects of Angiotensin and Vasopressin on Mechanical Activity Figure 1 shows typical spontaneous mechanical activity of rat, guinea pig and rabbit portal vein preparations mounted in the same tissue bath. In the rat and guinea pig, the activity consisted of regular phasic tension changes of frequency 0 .5 -1 .5/min. In the rabbit, mechanical activity was irregular in frequency and amplitude and, in contrast to the rat and guinea pig appeared more tonic. Preliminary experiments showed that both angiotensin and vasopressin had inhibitory actions in some tissues. For this reason, isoprenaline, whose /J-adrenoceptor-mediated inhibitory action had previously been studied in portal veins [J o h a n sso n et al., 1967; H o lm an et al., 1968; v. L o h , 1971], was used as a standard inhibitory agent for comparative purposes. In addition, vasopressin from two sources was used. Vasopres­ sin (Sandoz) contained a preservative and comparative experiments were made with lyophilized vasopressin (Calbiochem). No differences were found between the effects of vasopressin from the two sources and the results of all experiments with the drug have been pooled. The effects of angiotensin, vasopressin and isoprenaline were ex­ amined in 24 experiments and typical results are shown in figure 1. Vasopressin (0.005-0.1 IU/ml) produced an inhibitory response in both guinea pig and rabbit portal veins which persisted for as long as it was in contact with the tissue. In the guinea pig, phasic mechanical activity was reduced in frequency and amplitude whilst, at higher concentrations, spontaneous activity was completely abolished. In the rat, lower con­ centrations of vasopressin (0.005-0.05 IU/ml) produced a transitory excitatory response consisting of an increase in the amplitude and in the frequency of phasic contractions. Higher concentrations (up to 0.5 IU/ml) inhibited mechanical activity as described for guinea pig portal vein. In the guinea pig, angiotensin (5 x HT8-10~6 g/ml) reduced the am­ plitude of mechanical activity and complete inhibition of activity was sometimes observed. Higher concentrations (1(T6-1 0 4 g/ml) sometimes produced a transient excitatory response followed by inhibition (fig. 2) or, occasionally, an excitatory response alone. In the continued presence of angiotensin, normal mechanical activity usually returned about 3 min after the initial exposure to the drug. In the presence of concentrations which caused an initial excitatory response, the amplitude and duration of spontaneous activity following the inhibitory response were sometimes

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- Angiotensin 10" g/ml -

^Vasopressin 0 005 IU/ml -

Rat

Ral

Rabbit

Guineapig

B

»Vasopressin 005 IU/ml
|ti—11U I }hM | || |l 0

I— Vasopressin 0.05 IU/ml—t

s y |( || h * ------- 1»li.Kt f >11|) ]«w / U i j L ______ «jüsA JÜ l K. » i____i

Fig. 2. Effects of isoprénaline (A), angiotensin (B, C) and vasopressin (D) on electrical and mechanical activity in guinea pig portal vein. Extracellular recording of electrical activity in the perfused capillary. Preamplifier time constant = 0.02 sec. Continuous recording from the same preparation.

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tmin

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355

Electrophysiological Experiments The effects of angiotensin and vasopressin on guinea pig portal vein were further investigated using extra- and intracellular techniques of electrical recording. At first, a series of experiments was performed using the perfused capillary in which total electrical activity together with ten­ sion was measured. As shown in figure 2, angiotensin, vasopressin and iscprcnaline produced qualitatively similar effects, spike discharges being reduced in good correlation with mechanical inhibition. For a more precise measurement of the electrical events, membrane potential was measured intracellularly. Long-term intracellular penetra­ tions (15-30 min) of guinea pig portal vein were found to be relatively difficult. However, it was considered that only from such experiments could a reliable comparison be made of the effects of angiotensin, vaso­ pressin and the isoprénaline standard. In such experiments, however, the vein was sometimes so tightly stretched over the perspex cone that recorded electrical activity of the impaled cell was not always perfectly synchronized with the mechanical activity of the whole tissue. Typical effects of angiotensin, vasopressin and isoprénaline are shown in figure 3. The hyperpolarization produced by isoprénaline (mean

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increased (fig. 2). In the rat and rabbit, angiotensin (10~8-10~s g/ml) produced only excitatory responses. The excitatory and inhibitory re­ sponses of angiotensin and vasopressin were unaffected by tetrodotoxin (10~5 mol/1), atropine (10 “ mol/1) and phentolamine plus propranolol (each 10 0 mol/1). There were no significant changes in the responses of veins to angio­ tensin or vasopressin following repeated exposure to these agonists at approximately 5-min intervals, the tissue being washed after each ex­ posure. If tachyphylaxis is defined as a change in the response to an agonist following repeated exposure to that agonist, tachyphylaxis of the responses to angiotensin or vasopressin was not observed. Isoprénaline (10 8- 10 •’ mol/1) inhibited the mechanical activity in all preparations (fig. 1). In the guinea pig, the inhibitory response to isoprénaline was sometimes preceded by a phase of excitation whilst in the rat any excitatory response usually occurred after the initial inhibitory phase. The inhibitory responses in rat, guinea pig and rabbit portal vein were abolished by propranolol (10 e mol/1) in whose presence iso­ prénaline produced only excitatory responses which were abolished by phentolamine (10~° mol/I).

W eston/G olenhofen

356 Membrane potential

* m

50 mV

k \.

12 .

Tension

-A -» >

t _e Isoprenaline 10 °mol/l

50 mV

A-— /s._Av. t _7 Angiotensin 5x10 g/ml

50 mV

— An_] Angiotensin I0'®g/ml

50mV

/V

i'^V___

Vasopressin 0.05 IU/ml

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Fig. 3. Effects of isoprenaline (A), angiotensin !B, C) and vasopressin (D) on electrical and mechanical activity in guinea pig portal vein. Continuous intra­ cellular recording from the same cell. Spike amplitude reduced by about 15%.

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Table /. Effects o f angiotensin, vasopressin and oxytocin on mechanical activity in mammalian portal vein Portal vein

Angiotensin

Vasopressin

Oxytocin

Rat

excitation 1 ,6 ,7

inhibition 5 ,7 inhibition with initial excitation 1

inhibition 5 ,7

Guinea pig

inhibition with occasional excitation 1

Inhibition 1,7

inhibition 7

Rabbit

excitation 1 ,2 ,4

inhibition 1 ,3 ,7

inhibition 3 ,7

1 = Present study; 2 = C u t h b e r t and S u t t e r , 1965; 3 = H u g h e s and V a n e , 1967; 4 = S o m l y o and S o m l y o , 1968; 5 = V o t h et a i, 1969; 6 = V o t h et al., 1971; 7 = C arruba et al., 1973.

10 mV, 10 experiments) was greater than that produced by either angio­ tensin (mean 3 mV, 9 experiments) or vasopressin (mean 4 mV, 11 ex­ periments). However, since there are obvious difficulties in selecting equi-effective drug concentrations and since these concentrations were obtained by dropwise addition of the drugs to the bath, the significance of these apparent differences cannot be evaluated from the present ex­ periments. The initial excitatory response seen with higher concentrations of both angiotensin and isoprénaline was found to be associated with depolarization and increased spike frequency.

The results of the present experiments, together with those of previous workers who have studied the actions of polypeptides on portal vein, are summarized in table I. In contrast to their excitatory actions on arterial smooth muscle [B o h r , 1974; A l t u r a et al., 1965], angiotensin and vasopressin exhibit a spectrum of excitatory and inhibitory actions on the smooth muscle of portal vein. The octapeptide oxytocin also appears

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Discussion

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to have actions similar to those of vasopressin to which it shows structural similarity. In the case of vasopressin, at least, this mixture of excitatory and inhibitory effects is not confined to venous smooth muscle since G illespie and M c G rath [1974] have reported excitatory effects of vasopressin in rat anococcygeus muscle but inhibitory effects in the cat anococcygeus. In the present experiments, angiotensin was found to inhibit spon­ taneous mechanical activity in guinea pig portal vein. In his review, B o h r [1974] cited only one observation (in renal vein, unpublished) of an angiotensin-induced inhibitory action and commented that this in­ hibition might be related to the fact that the renal vein is exposed to the highest concentrations of angiotensin in vivo. However, there seems little reason to suppose that such an explanation would also apply to guinea pig portal vein. In their experiments in this tissue, C ar r u ba et al. [1973] reported that angiotensin had little or no action and no explanation can be offered for this discrepancy. The inhibition by angiotensin observed in the present experiments was not dependent on the presence of rabbit and rat portal veins in the same tissue bath since inhibition always occurred in preliminary experiments carried out with individual tissues in isolation. Also, inhibition was always a feature of the action of angiotensin in the electrophysiological experiments. Since there was no difference between the effects of vasopressin plus preservative and lyophilized vasopressin on all tissues, it can be concluded that the inhibitory effects observed were caused by vasopressin itself. V oth el al. [1969] found that purified vasopressin had a much greater inhibitory effect on spontaneous activity in rat portal vein than vaso­ pressin plus preservative and it is possible that the nature of the preser­ vative in the two instances was different. There was little qualitative difference between the inhibitory actions of angiotensin and vasopressin and those of isoprenaline, the /1-adreno­ ceptor agonist used as a standard inhibitory agent. Inhibition by all three agents was associated with a reduction in spike activity. However, the intracellular recordings showed that the hyperpolarization produced by isoprenaline was always greater than that produced by angiotensin or vasopressin. This suggests that, within the limitations of the intracellular method already mentioned (see Methods), there is a difference between the action of isoprenaline and that of angiotensin and vasopressin. The observed isoprenaline-induced hyperpolarization mediated by /1-adrenoceptors is in agreement with previous results in rabbit [H o lm an

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et al., 1968] and in guinea pig portal vein (v. L o h , 1971], The depolari­ zation associated with inhibition of mechanical activity described by J o h a n s s o n et al. [1967] for rat portal vein was not observed (see also H o l m a n , 1969). Thus, the observations of B ü l b r in g and K ur iy a m a [1973] that, in guinea pig taenia coli, only «-adrenoceptor-mediated inhibition is associated with a hyperpolarization, do not apply to guinea pig portal vein. Tachyphylaxis of the excitatory response to angiotensin in arterial preparations is a common observation [B o h r , 1974], Although this phenomenon has also been observed in some veins [S h e p h e r d and V a n h o u t t e , 1975], it was not observed in portal vein in the present experiments and in those of V o t h et al. [1971] and C a r r u b a et al. [1973]. It thus seems possible that a detailed study of angiotensin tachyphylaxis in arteries and its failure to occur in portal vein would provide useful information about the mechanisms involved in the genera­ tion of such tachyphylaxis.

A ltura, B. M.; H ershey, S. G., and Z weibach , B. W.: Effects of a synthetic analogue of vasopressin on vascular smooth muscle. Proc. Soc. exp. Biol. Med. 119: 258-264 (1965). Bohr , D. F .: Angiotensin on vascular smooth muscle. Handbook of experimental pharma­ cology, p. XXXVII (Springer, Heidelberg 1974). B ülbring , E. and K uriyama, H.: The action of catecholamines on guinea-pig taenia coli. Proc. R. Soc. B 867: 115-121 (1973). C arruba, M.; M andelli, V., and M antegazza , P.: The effect o f angiotensin II and other vasoactive drugs on isolated portal vein preparations, Archs int. Pharmacodyn. Ther. 201: 224-233 (1973). C uthbert, A. W. and S utter, M. C.: The effects o f drugs on the relation between the action potential discharge and tension in a mammalian vein. Br. J. Pharmac. Chemather. 25: 592-601 (1965). G illespie, J. S. and M c G rath , J. C.: The response of the cat anococcygeus muscle to nerve or drug stimulation and a comparison with the rat anococcygeus. Br. J. Phar­ macol. 50: 109-118 (1974). G olenhoeen, K. und Loh , D. v.: Elektrophysiologische Untersuchungen zur normalen Spontanaktivität der isolierten Taenia coli des Meerschweinchens. Pflügers Arch, ges. Physiol. 314: 312-328 (1970a). G olenhoeen, K. und Loh , D. v.: Intracelluläre Potentialmessungen zur normalen Spon­ tanaktivität der isolierten Portalvene des Meerschweinchens. Pflügers Arch. ges. Physiol. 319: 82-100 (1970b).

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References

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G olenhofen, K. and W eston, A. H.: Excitatory and inhibitory effects of angiotensin and vasopressin on vascular smooth muscle. J. Physiol., Lond.246: 54-55P (1975). H olman, M. E.: Electrophysiology o f vascular smooth muscle. Ergebn. Physiol. 61: 137-177 (1969). H olman, M. E.; K asby, C. B.; S uthers, M. B.. and Wilson, J. A. F.: Some properties of the smooth muscle of rabbit portal vein. J. Physiol., Lond. 196: 111-132 (1968). H ughes, J. and Vane, J. R .: An analysis of the responses o f the isolated portal vein of the rabbit to electrical stimulation and to drugs. Br. J. Pharmac. Chemother. 30: 46-66 (1967). Johansson, B .;J onsson, O .; A xelsson, J., and W ahlström, B.: Electrical and mechanical characteristics of vascular smooth muscle response to norepinephrine and isoproterenol. Circulation Res. 21: 619-633 (1967). L oh , D. v .: The effect of adrenergic drugs on spontaneously active vascular smooth muscle studied by long-term intracellular recording o f membrane potential. Angiologica 8: 144-155(16-27) (1971). S hepherd, J. T. and Vanhoutte, P. M.: Veins and their control (Saunders, London 1975). Somlyo, A. V. and Somlyo, A. P .: Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J. Pharmac. exp. Ther. 159: 129-145 (1968). U chida , E.; Bohr , D. F., and H oobler, S. W.: A method for studying isolated resistance vessels from rabbit mesentery and brain and their responses to drugs. Circulation Res. 21: 525-533 (1967). Voth , D.; A gsten, M.; Schipp , R. und L übcke, H.: Untersuchungen zur Wirkung von Angiotensin II auf einen autorhythmischen glatten Gefässmuskel (Pfortader der Ratte). Arch. Kreislaufforsch. 65: 41-57 (1971). Voth , D .; Schipp , M.; Agsten, M .; Schürmann , K.; K ohlhardt , M. und D udek, J.: Untersuchungen über den Einfluss des Kationenmilieus und verschiedener Pharmaka auf die Kontraktilität und Autorhythmik eines spontanaktiven glatten Gefassmuskels in vitro. Arch. Kreislaufforsch. 60: 364-387 (1969).

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Prof. Dr. med. K. G olenhofen, Institut für Physiologie der Universität, Deutschhaus­ strasse 2, D-355 Marburg!Lahn (FRG)

Comparison of the excitatory and inhibitory effects of angiotensin and vasopressin on mammalian portal vein.

Angiotensin produced inhibitory and excitatory responses in guinea pig portal vein and excitatory responses in rabbit and rat portal vein. Vasopressin...
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