J. Physiol. (1978), 283, pp. 41-51 With 4 teXt-ftgure Printed in Great Britain

41

INVOLVEMENT OF UPTAKE1 AND UPTAKE2 IN TERMINATING THE CARDIOVASCULAR ACTIVITY OF NORADRENALINE IN NORMOTENSIVE AND GENETICALLY HYPERTENSIVE RATS

BY CHRISTOPHER BELL AND ROSLYN KUSHINSKY From the Department of Physiology, University of Melbourne, Parkville, 3052 Victoria, Australia

(Received 16 February 1978) SUMMARY

1. In pithed, pancuronium-treated rats, inhibition of Uptake, with desmethylimipramine (2.5 mg/kg i.v.) increased the time course of the pressor response to vasomotor nerve stimulation (1 and 5 Hz for 5 sec) but not the time course of the pressor response to injected noradrenaline (10 and 50 ng i.v.). 2. Subsequent inhibition of Uptake2 with metanephrine (3 mg/kg, i.v.) did not affect the time course of responses to either vasomotor nerve stimulation or injected noradrenaline. 3. It is concluded that Uptake1 but not Uptake2 is important in terminating the activity of noradrenaline released from rat vasomotor nerves and that neither process inactivates intralurninal catecholamine. 4. By contrast, chronotropic responses of rat atria to adrenergic nerve stimulation were prolonged by blockade of both Uptake1 and Uptake2, in agreement with previous evidence for involvement of both processes in terminating sino-atrial adrenergic responses. 5. Animals from the Otago strain of genetically hypertensive animals were identical to normotensives in their vascular handling of catecholamine. However, they lacked an atrial Uptake2 process. 6. This defect may be related to the high resting heart rate and abnormal cardiac catecholamine turnover observed to exist in the Otago hypertensive rats. INTRODUCTION

The development of high arterial pressure in genetically hypertensive rats is reduced considerably by neonatal destruction of the peripheral adrenergic nervous system (Smirk, 1970; Clark, Laverty & Phelan, 1972; Folkow, Hallback, Lundgren & Weiss, 1972). It seems that there may be chronic adrenergic overactivity during the development of hypertension, as plasma levels of catecholamines and dopamine /1-hydroxylase are raised (Grobecker, Roizen, Weise, Saavedra & Kopin, 1975; Roizen, Weise, Grobecker & Kopin, 1975; Nagatsu, Ikuta, Numata, Kato, Sano, Nagatsu, Umezawa, Matsuzaki & Takeuchi, 1976; Nakamura, Suzuki & Nakamura, 1976), there is increased adrenergic drive to the heart (Pfeffer & Frohlich, 1973) and

42 C. BELL AND R. KUSHINSKY turnover rates for cardiac noradrenaline are normal or elevated in the face of elevated blood pressure (Phelan, 1970; Louis, Krauss, Kopin & Sjoerdsma, 1970; Yamori, 1976). In addition, the vascular beds of these animals have been reported to be abnormally responsive to applied noradrenaline (McGregor & Smirk, 1968; Lais & Brody, 1975). All these phenomena could be secondary to deficient catecholamine inactivation in genetically hypertensive animals. We have therefore investigated the ability of the cardiovascular system to inactivate noradrenaline in one such strain and in a genetically related strain of normotensive animals. METHODS

Animals The rats used were bred in this Department and were F2-F3 descendants of a breeding stock obtained from Otago in 1975, which comprised genetically hypertensive rats of the fortieth hypertensive generation and normotensive animals from the same original Wistar colony (Phelan, 1968). Both strains were maintained by random breeding within each successive generation. Systolic blood pressures of intact animals were recorded under ether anaesthesia using an occlusive tail cuff (Byrom & Wilson, 1938) and a photoelectric pulse detector (Grass Instruments model PTTI).

Pithed rat8 Mean blood pressures were recorded from one carotid artery of artificially respired, pithed rats weighing 250-350 g with a Statham P23 AC pressure transducer and a rectolinear chart recorder. Pressor responses were obtained to intravenous injections of noradrenaline bitartrate (10 and 50 ng) and to electrical stimulation of the spinal cord and hence the sympathetic outflow via the pithing rod, using a s.c. needle in one hindlimb as the indifferent electrode (Gillespie & Muir, 1967). The stimulation parameters were biphasic square wave pulses of 05 imsec duration delivered at 1 and 5 Hz and supramaximal voltage in 5 sec trains. All animals were treated with the muscle relaxant pancuronium (Pavulon, 2 mg/kg i.v.) in order to minimize artifacts due to skeletal muscle activation and with atropine sulphate (5 mg/kg s.c.) to prevent vagal activation.

Isolated atria Chronotropic responses of isolated, paired atria to intramural adrenergic nerve stimulation were studied by the method reported in a previous paper (Bell & Grabsch, 1976). 5 sec trains of 0-5 msec monophasic square-wave pulses were delivered at frequencies of 1 and 5 Hz and supramaximal voltage. Uptake inhibitors Inhibition of Uptake, and Uptake2 was achieved by administration of desmethylimipramine (DMI, Pertofran; Ciba-Geigy) and metanephrine hydrochloride (Sigma) respectively. Both inhibitors caused, in pithed animals, small (5-15 mmHg) increases in blood pressure of variable time course. Following inhibitor administration time was allowed for reestablislument of a stable blood pressure before further responses to nerve stimulation or noradrenaline were elicited. This period was of the order of 5 min. Isolated atrial preparations were allowed to equilibrate with the inhibitors for 20 min before responses to nerve stimulation were obtained. Neither inhibitor had any effect on the resting force or rate of contraction of the atria. Statitdic

The statistical significance of differences between means was assessed using two-tailed Student's t tests, on the assumption that no difference existed.

NORADRENALINE INACTIVATION IN NORMAL AND GH RATS

43

RESULTS

Blood pressures The blood pressures of normotensive and hypertensive rats before pithing were 130 + 3*0 mmHg and 183 + 3-3 mmHg respectively (means+ s.E. of mean). After pithing, the blood pressures of hypertensive animals remained higher than those of normotensives (69-4 + 2-9 and 52-0 + 2-4 mmHg respectively, P < 0.001), in agreement with previous evidence for a structural component to the hypertension in mature animals (Clark, 1971; Folkow et al. 1972). A

DMI B

rig. 1. Blood pressure recordings from (A) a pithed, normotensive rat and (B) a pithed, genetically hypertensive rat; pre-treated with pancuronium and atropine. Pressor responses were obtained to i.v. injections of 10 ng NA (at arrows) and to electrical stimulation of the spinal cord for 5 sec at 1 Hz with 0-5 msec pulses (at black dots). During responses to electrical stimulation the chart recorder speed was increased fivefold so as to facilitate measurement of response time course. Between the first and second panels, Uptakel was inhibited by systemic administration of DMI (2.5 mg/ kg). Note that pressor responses to similar stimuli were larger in the hypertensive than in the normotensive animal, and that DMI increased the amplitude of responses to both noradrenaline and spinal cord stimulation but prolonged only responses to spinal cord stimulation. Calibration bars represent: (vertical) 50 mmHg, (horizontal) 20 see (fast speed) and 100 sec (slow speed).

Pressor responses to spinal cord stimulation and injected noradrenaline Stimulation of the spinal cord produced a rapid increase in blood pressure which comprised an initial fast and then a slower phase of decay on cessation of stimulation (Fig. 1). The amplitude or shape of either phase was not altered appreciably by acute bilateral adrenalectomy, but both phases were abolished or nearly abolished following systemic administration of the adrenergic neurone blocking drug guanethidine (Ismelin, 2 mg/kg). We therefore concluded that the response elicited by spinal

C. BELL AND R. KUSHINSKY stimulation under the conditions of our experiments was attributable primarily to vasomotor nerve stimulation and that adrenal medullary secretion was not an important contributing factor. Intravenous injections of noradrenaline (NA) produced increases in blood pressure which were of longer duration than those resulting from spinal cord stimulation and which did not exhibit two decay phases (Fig. 1). In hypertensive animals the amplitude of responses both to vasomotor nerve stimulation and to NA were significantly (P < 0.001) greater than in normotensives (Fig. 1), agreeing with previous reports of vascular hyperreactivity for this strain 44

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Fig. 2. Amplitudes, rise times and decay time constants of pressor responses to spinal cord stimulation (1 and 5 Hz) and intravenous noradrenaline (10 and 50 ng) in pithed, normotensive (N) and genetically hypertensive (G) rats under control conditions (black columns) and after inhibition of Uptake, by administration of 2-5 mg/kg DM I..v. (open columns). The short vertical lines represent 1 s.E. mean and the asterisks denote values significantly different from controls (* P < 0-02, ** P < 0.001).

NORADRENALINE INACTIVATION IN NORMAL AND GH RATS 45 (Laverty, 1961; McGregor & Smirk, 1968; Jones, Young & Phelan, 1977). On the other hand the time courses of all responses were similar to those in normotensives. For quantitative assessment of responses the following parameters were measured: peak amplitude, rise time to peak amplitude and time constant of decay (taken as time to reach one third of the peak amplitude). Control values obtained for these parameters in normotensive and hypertensive animals are shown in Fig. 2. Effect of Uptake, inhibition on pressor responses to spinal cord stimulation and injected NA

Uptake, was inhibited by systemic administration of DMI, 2-5 mg/kg, in a series of twelve male and six female normotensive animals: as no differences in responses or in the effects of DMI were noted between the sexes, the results from both have been pooled (Fig. 2). Inhibition of Uptake, increased the amplitude of responses to stimulation at 1 Hz but not that of responses to stimulation at 5 Hz. The rise times of these responses were not significantly affected, but the time courses of decay were greatly prolonged at both 1 and 5 Hz. The amplitudes of responses to 10 and to 50 ng NA were enhanced, if anything, more than was that to stimulation at 1 Hz. By contrast, however, the time courses of NA responses were not prolonged significantly. The effect of Uptake1 inhibition on pressor responses was also examined in eight male and six female hypertensive animals. As in the normotensives, DMI greatly prolonged the decay times of responses to nerve stimulation, but not of those to NA; and increased the amplitude of responses both to NA and to stimulation at 1 Hz (Fig. 2). Effect of Uptake2 inhibition on pressor responses to spinal cord stimulation and injected noradrenaline The effect on responses of inhibition of Uptake2 by systemic administration of metanephrine, 3 mg/kg, was examined in two male and four female normotensive animals. Because of the possibility that inhibition of one uptake process might lead to a compensatory increase in efficiency of the other (Iversen, 1968), Uptake1 was inhibited with DMI before metanephrine administration. The results of these experiments are shown in Fig. 3. No consistent changes in amplitude or duration of any response were produced by inhibition of Uptake2, with the exception of the time course of decay following vasomotor nerve stimulation, which were shortened by a variable amount (4-60 %) in five out of six rats. Despite this trend, enhancement of the decay time course in the remaining rat prevented the shortening reaching statistical significance (1 Hz: t = 2-25, P < 0-08 > 0 05; 5 Ht t = 2-55, P < 0-06 > 0.05). Although the mean values for the other parameters showed some variable degree of shift after metanephrine, these were not associated with any consistent alteration, the values being increased in some animals and decreased in others. Effect of Uptake2 inhibition on responses to adrenergic nerve stimulation of isolated atria In view of the apparent absence of involvement of Uptake2 in terminating vascular responses to catecholamines we examined responses to adrenergic nerve stimulation of isolated atria, where Uptake2 has been shown previously to be concerned in termination of the response (Bell & Grabsch, 1976).

C. BELL AND R. KUSHINSKY Resting heart rates in atria from a series of eight normotensive and eight hypertensive female rats were similar: normotensive, 231 + 16; hypertensive, 238 + 23 beats. min-'. Following inhibition of Uptake, with 2 /sM-DMI, the time courses of responses in normotensive atria to stimulation at 1 and 5 Hz were similar to those previously reported for atria from female Sprague-Dawley rats in the presence of 46

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Fig. 3. Amplitudes, rise times and decay time constants of pressor responses to spinal cord stimulation (1 and 5 Hz) and i.v. NA (10 and 50 ng) in pithed, normotensive rats after inhibition of Uptake, by administration of DMI, 2-5 mg/kg, i.v. (black columns) and after subsequent inhbition of Uptake2 by administration of 3 mg/kg metanephrine i.v. (open columns). The short vertical lines represent 1 S.E. mean.

DMI by Bell & Grabsch (1976) (Fig. 4). Subsequent inhibition of Uptake2 with 4 /LM-metanephrine produced further prolongation of responses by 30-40 % (Fig. 4). The time course and amplitude of responses of hypertensive atria to stimulation in

.N ORADREN2ALINE INACTIVATION2 INN.ORMAL A.NVD GH RATS 47 the presence of DMI were not significantly different to those of normotensive atria. However inhibition of Uptake2 did not produce any prolongation of responses over that seen in the presence of Uptake, inhibition alone (Fig. 4). 200

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Fig. 4. Time course of recovery (half-decay time) of chronotropic responses to intramural adrenergic nerve stimulation of atria from normotensiv'e (N) and genetically hypertensive (G) rats after inhibition of Uptake, with 2 /M-DAII (black columns) and after subsequent inhibition of Uptake2 with 4 /iM-metenaphrine (open columns). The short vertical lines represent 1 S.E. mean and the asterisks denote values significantly different after Uptake2 inhibition (* P < 0-05. ** P < 0 01). DISCUSSION

We observed that inhibition of Uptake1 Awith DMI1 considerably prolonged pressor responses to spinal cord stimulation and hence to vasomotor nerve activation, indicating that reuptake of released NA into the noradrenergic axons is important in terminating responses to vasomotor activation at low stimulus frequencies. A similar conclusion was reached in a previous paper with regard to the role of Uptake1 in terminating the action of neurogenic NA in the sinoatrial node (Bell & Grabsch, 1976). By contrast inhibition of Uptake1 did not prolong pressor responses to injected NA, indicating that, neuronal uptake does not, participate in terminating the effect of circulating NA on vascular smooth muscle. This is not surprising in view of the other mechanisms for amine inactivation which an intraluminal NA molecule would encounter within the arterial media before it gained access to the vasomotor axons at the medio-adventitial junction (Avakian & Gillespie, 1968; de la Lande, Hodge, Lazner, Jellett, & Waterson, 1970). On the other hand, we observed that in the presence of DMI the peak amplitudes of responses to NA xere enhanced by at least as much as were those to nerve stimulation. In view of the data on the time course of responses discussed above, this is unlikely to reflect any role for Uptake1 in limiting response amplitude. Such a

C. BELL AND B. KUSHINSKY conclusion is confirmed by the observation that the amplitudes of responses to vasopressin are also increased to a similar extent by DMI (C. Bell, unpublished observations). The enhancement could be attributed to a direct effect of DMI on muscle cell excitability (Bennett, 1973), or conceivably to slight muscle depolarization by the accumulation in the synaptic cleft of spontaneously released transmitter (Bell, 1969; Hirst, 1977) normally removed by Uptake,. In this context it is relevant to note that most conclusions about the importance of Uptake1 in neurogenic NA inactivation have been based on enhanced magnitude of contractile responses following Uptake inhibition (Trendelenburg, 1959; Haefely, Hurlimann & Thoenen, 1964; Sigg, Soffer & Gyermek, 1963; Pals, Fulton & Masucci, 1968 and others), although it has also been suggested that the area under the response might provide a more accurate gauge of the time course of transmitter action (Haefely et al. 1964; see also Glover & McCulloch, 1970). Our results, however, suggest that changes in response amplitude may be misleading and that changes in time course alone give a better indication of the role of Uptake1 in normal transmission.

48

Despite the apparent importance of Uptake, in terminating mechanical responses of smooth and cardiac muscle to adrenergic nerve stimulation, this is not reflected, for those tissues wowhich have been studied, in dramatic changes of time course of individual excitatory junction potentials (Bell, 1967; Holman, 1967; Bennett, 1973). It seems likely, therefore, that the relationship between electrical and mechanical events is not as rigid as envisaged by some workers.

It has been shown in the rat heart that Uptake1 becomes saturated at extracellular NA concentrations of above 02 #ug/ml. (Iversen, 1968; Lightman & Iversen, 1969). In view of the prolongation of responses to vasomotor nerve stimulation which we observed following inhibition of Uptake1, it was of interest that the time course of responses to repetitive stimulation at 5 Hz was not appreciably different from that to stimulation at 1 Hz and that the degree of prolongation produced by DMI was at least as great at 5 as at 1 Hz. If the situation in blood vessels is similar to that existing in the heart, then these results imply that the concentration of NA attained near the vasomotor axon membrane during physiological frequencies of activation is always less than about 0-2 ,tg/ml. Inhibition of Uptake2 after inhibition of Uptake, produced no further prolongation of pressor responses to nerve stimulation or to injected NA: in fact in the majority of rats the reverse w'as true. This suggests a lack of involvement of Uptake2 in terminating the biological activity of either extraluminal or intraluminal NA in the vasculature. There is published evidence to indicate that Uptake2, as well as removing NA from sites of biological action in some situations, can itself prolong responses to NA by initial binding and subsequent. slow release of transmitter in the region of the receptors (Avakian & Gillespie, 1968; Gillespie, 1968; Draskoczy & Trendelenberg, 1970). Such a phenomenon might underlie our observation that blockade of Uptake2 tended to shorten responses to vasomotor nerve stimulation. In tile experiments performed on the atria, by contrast, we obtained evidence that both Uptake processes participate in inactivating the NA released by transmural stimulation.

It seems unlikely that the increased responsiveness shows n by the vasculature of Otago hypertensive rats to catecholamines (Laverty, 1961; McGregor & Smirk, 1968) is due to deficient inactivation of amines since we have shown that the time courses of vascular responses to vasomotor stimulation and NA were similar in normotensive and hypertensive rats both before and after blockade of Uptake1. However we did observe that the atria of hypertensive animals lack the Uptake2 mechanism present

NORADRENALINE INACTIVATION IN NORMAL AND GH RATS 49 in normotensive rats. This deficiency may be linked to the high turnover rate of cardiac NA (Phelan, 1970) and the elevated heart rate (Clark, 1971) reported for young rats of this strain. It is of considerable interest to note that in the Kyoto strain of spontaneously hypertensive rats, where there is also evidence of excessive adrenergic drive to the heart (Pfeffer & Frohlich, 1973; Numao, Suga & Iriuchijima, 1975; Hallbaick, Isaksson & Noresson, 1975), Salt & Iversen (1973) have found a deficiency of the Uptake, process in the atria. The early 'labile' or 'borderline' stage of essential hypertension is in many patients characterized by elevated cardiac output but little change in calculated total peripheral resistance (Bello, Sevy & Harakal, 1965; Finkielman, Worcel & Agrest, 1965; Frohlich, Kozul, Tarazi & Dustan, 1970; Tarazi, Ibrahim, Dustan & Ferrario, 1974). This enhancement of cardiac output appears to be sympathetically mediated (Frohlich et al. 1970; Conway, 1970). By contrast, in established hypertensives peripheral resistance is raised considerably but cardiac output is usually normal or below normal (Bello et al. 1965). Such observations, together with theoretical considerations and data from experimental models, have led to the proposal that it is elevation of cardiac output which is the primary stimulus for development of hypertension (Guyton, Coleman, Bower & Granger, 1970; Ledingham, 1971). Interestingly enough, young Kyoto hypertensive rats exhibit during the phase of developing hypertension a haemodynamic pattern similar to that seen clinically, with an initial elevation of cardiac output and only later elevation of peripheral resistance (Albrecht, Vizek & Krecek, 1972; Pfeffer & Frohlich, 1973). Our results, and those of Salt & Iversen (1973), are compatible with the view that raised cardiac output in young genetically hypertensive animals could be due to faulty cardiac inactivation of

catecholamines. This work was supported by the National Heart Foundation of Australia. We wish to thank Miss J. Thompson for able technical assistance and Ciba-Geigy for generous drug donations. REFERENCES ALBRECHT, I., VIZEK, M. & KIE6EK, J. (1972). The hemodynamics of the rat during ontogenesis with special reference to the age factor in the development of hypertension. In Spontaneou8 Hypertension: Its Pathogenes~i and Complication8, ed. OKAMOTO, K., pp. 121-127. Tokyo: Igaku Shoin. AVAIAN, 0. V. & GILLESPIE, J. S. (1968). Uptake of noradrenaline by adrenergic nerves, smooth muscle and connective tissue in isolated perfused arteries and its correlation with the vasoconstrictor response. Br. J. Pharmac. Chemother. 32, 168-184. BELL, C. (1967). Effects of cocaine and of monoamine oxidase and catechol-O-methyl transferase inhibitors on transmission to the guinea-pig vas deferens. Br. J. Pharmac. Chemother. 31, 276-289. BELL, C. (1969). Transmission from vasoconstrictor and vasodilator nerves to single smooth muscle cells of the guinea-pig uterine artery. J. Physiol. 205, 695-708. BELL, C. & GRABScH, B. (1976). Involvement of Uptake2 in the termination of activity of neurogenic noradrenaline in the rat isolated atrium. J. Physiol. 254, 203-212. BELLO, C. T., SEVY, R. W. & HARAKAL, C. (1965). Varying hemodynamic patterns in essential hypertension. Am. J. med. Sci. 250, 24-35. BENNETT, M. R. (1973). An electrophysiological analysis of the uptake of noradrenaline at sympathetic nerve terminals. J. Physiol. 229, 533-546. BYROM, F. B. & WILsoN, C. (1938). A plethysomographic method for measuring systolic blood pressure in the intact rat. J. Physiol. 93, 301-304.

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MCGREGOR, D. D. & Sra, F. H. (1968). Vascular responses in mesenteric arteries from genetic and renal hypertensive rats. Am. J. Physiol. 214, 1429-1433. NAGATSU, T., IxUTA, K., NumATA, Y., KATO, T., SANo, M., NAGATSU, I., UMEZAWA, H., MATSuzAXI, M. & TAKEUCHI, T. (1976). Vascular and brain dopamine fl-hydroxylase activity in young spontaneously hypertensive rats. Science, N.Y. 191, 290-291. NAK.MURA, K., Suzu, T. & NAx.A&uR, K. (1976). Changes in adrenal function in spontaneously hypertensive rats. Jap. Heart J. 17, 416-418. NuM.o, Y., SUGA, H. & IRCHIJIMA, J. (1975). Hemodynamics of spontaneously hypertensive rats in conscious state. Jap. Heart. J. 16, 719- 730. PALS, D. T., FULTON, R. W. & MA.succi, F. D. (1968). Angiotensin, cocaine and desipramine: comparison of effects on blood pressure responses to norepinephrine, tyramine and phenylephrine in the pithed rat. J. Pharmac. exp. Ther. 162, 85- 91. PFEFFER, M. A. & FRORmIcH, E. D. (1973). Hemodynamic and myocardial function in young and old normotensive and spontaneously hypertensive rats. Circulation Res. 32-33, Suppl. I, 2835. PrE;AN, E. L. (1968). The New Zealand strain of rats with genetic hypertension. N.Z. med. J. 67, 334-344. PWELAN, E. L. (1970). Genetic and autonomic factors in inherited hypertension. Circulation Re8. 26-27, Suppl. II, 65-74. ROIzEN, M. F., WEIsE, V., GROBECKER, H. & KoPrm, I. J. (1975). Plasma catecholamines and dopamine-fl-hydroxylase activity in spontaneously hypertensive rats. Life Sci., Oxford, 17, 283-288. SALT, P. J. & IvERSEN, L. L. (1973). Catecholamine uptake sites in the rat heart after 6-hydroxydopamine treatment and in a genetically hypertensive strain. Naunyn-Schmiedeberg's Arch. Pharmacol. 279, 381-386. SIGG, E. B., SOFFER, L. & GYERMEK, L. (1963). Influence of imipramine and related psychoactive agents on the effect of 5-hydroxytryptamine and catecholamines on the cat nictitating membrane. J. Pharmac. exp. Ther. 142, 13-20. SMIRK, F. H. (1970). The neurogenically maintained component in hypertension. Circulation Res. 26-27, Suppl. II, 55-63. TARAzI, R. C., IBRAHIM, M. M., DUSTAN, H. P. & FERRARIO, C. M. (1974). Cardiac factors in hypertension. Circulation Rem. 34-35, Suppl. I, 213-221. TRENDELENBU`RG, U. (1959). The supersensitivity caused by cocaine. J. Pharmac. exp. Ther. 125, 55-65. YAMORI, Y. (1976). Neurogenic mechanisms of spontaneous hypertension. In Regulation of Blood Pressure by the Central Nervous System, ed. ONESTI, G., FERNANDES, M. & KIM, K. E., pp. 6576. New York: Grune & Stratton.