Distribution of components of 3H-noradrenaline uptake in the wall of the rabbit aorta

EUROPEAN JOURNAL OF PHARMACOLOGY 19 11972) 239-245. NORTH-tlOLLAND PUBLISHINGCOMPANY

DISTRIBUTION OF COMPONENTS OF 3 H - N O R A D R E N A L I N E U P T A K E IN T H E W A L L O F T H E R A B B I T A O R T A J.A. BEVAN, R.D. BEVAN, J.V. OSttER and C. SU Departments o f Pharmacology and Patholog3,, Division o f Neuropathology. UCLA School o f Medicine. Los Angeles, Cbl(lbmia 90024. U.S.A.

Received 1 November 1971

Accepted 4 May 1972

J.A. BEVAN, R.D. BEVAN, J.V. OSHER and C. SU, Distribution o f components oJ .3 H-noradrenalme uptake in the wall oJ the rabbit aorta. European J. Pharmacol. 19 (1972) 000-000. An analysis of the distribution of tritiated noradrenaline (31t-NA) in the wall of the rabbit thoracic aorta has been studied by a frozen-section technique. Some of the components of this uptake were defined by relating tritium distribution to histological structure and by determining the effect of tissue washing, electrical stimulation of the intramural nerves and pretreatment with phenoxybenzamine (PBZ) in a concentration that inhibits maximally 3H-NAbinding. 5 components of 3H-NAdistribution in the vessel wall were defined; that (i) in the extracellular space, (ii) loosely bound to all tissues, (iii)specifically bound tn sympathetic nerves, (iv)bound to non-nervous tissues at sites sensitive to PBZ, (v) bound to all tissues at sites insensitive to PBZ. Noradrenaline Blood v e s s e l Aorta

Distribution Phenoxybenzamine

Neuronal binding Extraneuronal binding

1. INTRODUCTION

2. MATERIALS AND METHODS

Noradrenaline is bound to all cellular elements of the arterial wall (Avakian and Gillespie, 1968; Maxwell et al., 1968; Nedergaard et al., 1969; Gillespie et al., 1970; Nedergaard and Bevan, 1971). However, no quantitative study has been made of the distribution of noradrenaline through the depth of the wall of the blood vessel and its constituent components. In this paper, the distribution profile of baH noradrenaline (3 H-NA) in the wall of the rabbit aorta studied by a frozen-section technique, is related to anatomical structure of the vessel, and using various treatments analyzed into some of its components. Preliminary reports of this work have been previously published (Bevan et al., 1969;Bevan et at., 1970).

The thoracic aortae of rabbits weighing 2 . 3 - 2 . 7 kg were used. After the animal had been stunned and bled, the aorta was cannulated with polyethylene tubing and removed. It was then immersed in cold Krebs solution and carefully trimmed of loose fat and connective tissue. Standard helical strips 2 3 cm long and 4 - 5 mm wide were cut. A strip was mounted on a ring tissue holder and one end fixed to the holder with heavy thread. This holder, like all plastic elements of the superfusion apparatus (fig. 1 ) was made from Kel-F plastic. This material is rigid and withstands frequent freezing to low temperatures. Hanging loosely from the ring tissue holder, the strip was allowed to equilibrate in Krebs bicarbonate

240

ZA. Bevan el al., Distribution o f stI-NA in aorta

AORTA STRIP ~~REBS ELECTRODE ~'/'//~, RING TISSUEH O L D E R ~ ~

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solution equilibrated with 95% 02 and 5% CO2 at 39°C for 1 hr. It was then incubated in Krebs solution containing 3H.NA (4.4,uCi/ml, 5 X 10-7 M, 10-7 g/ml) or d,l-3H-isoprenaline (3.0#Ci/ml, 5 X 10-7 M). When appropriate, phenoxybenzamine was added to the solution during the latter 30 min o f the equilibration period and during the subsequent incubation, washing and superfusion periods (see below). Tissues attached to the ring tissue holder, with or without an interim period of washing, were then placed in the superfusion apparatus (fig. I). The intimal surface of the strip was placed downwards on the plane-surfaced platinum disc (3 cm in diameter) which formed a tilted floor to the apparatus. This disc acted as one electrode for transmural stimulation of the intramural nerves. The free end of the strip was attached to the strain gauge using a suture of braided silk, and placed under 1.0 g resting tension. Since radioactivity profiles of different strips were to be compared, it was essential that strips were m o u n t e d under identical tensions. The tissue was superfused with warm, oxygenated Krebs solution, which sometimes contained SH-NA and other drugs and the superfusate was collected from the lower edge of the apparatus. A platinum wire electrode for transmural stimulation was lowered onto the tissue until a meniscus of Krebs solution formed between the wire and the strip. The tissue was

stimulated with biphasic square wave pulses of 0.3 1.0 msec duration and 4 0 V amplitude at a frequency of 10 Hz. The tissue still mounted in tire superfusion apparatus, was frozen by immersion in isopentane cooled to the temperature of liquid nitrogen, and then placed m a cryostal at 20°C. In order to ensure that tire artery was sectioned parallel to its snrlhce, the central part of the strip was fixed to the surface of a piece of liver previously frozen onto the tissue mount and cut using the nricrotome blade in situ. This fixation was achieved using precooled Krebs applied with a capillary pipette. The mounted strip was trimmed to a desired shape, usually 5 nnn square, and the newly cut edge fixed and buttressed with frozen Krebs sohition. Serial slices were cut usually of 24/2m thickness, placed in vials and digested in 0.5 ml Sohiene 100 (Packard, Downers Grove, 111., U.S.A.) for 8 hr. 10 ml of toluene-based phosphor solution was added io each vial, and its radioactivity was determined by scintillation spectrometry. Distribution profiles of radioactivity expressed as uptake ( m l / g ) w e r e constructed. Tissue weight was calculated from the dimensions of the section, assuming fire tissue specific gravity to be 1.05. Profiles determined from various segments were superimposed, using the adventitio-medial junction as a reference point. In order to determine the nature of the tightly bound tritiated material in the aorta, segments incubated in 3H-NA for 1 hr and then soaked in nonradioactive Krebs solution for 3 hr were homogenized in 5e~ trichloracetic acid and centrifuged. To 5 ml of the supernatant, 0.5 ml 2% EDTA and 0.2 ml 10% ascorbic acid were added. After adjusting the pH to 8.4, the mixture was passed through a 6 X 20 irrm alumina column. The effluent and 2 ml distilled water wash were combined and their activity expressed as non-catechol metabolites. The alumina was then eluted with 4 ml 0.2 N hydrochloric acid, and an aliquot of the eluate counted to give total catechol content; the remainder was extracted with ethyl acetate to obtain the deaminated catechol content. The dill ference between total catechols and deaminated catechols was expressed as intact NA (Su and Bevan, 1970). In some experiments alternate frozen sections (cut at 1 2 ~ m ) were analysed for their tritium content, and by light microscopy. These latter interleaved sec-

J.A. Bevan et al.. Distribution of 3H-NA in aorta

tions were stained with hematoxylin and eosin for light microscopy. A consistent change in translucency of the sections was seen approximately half way through the thickness of the vessel wall; the section first showing this change was recorded. Drugs used were phenoxybenzamine hydrochloride. The isotopes d,l-3H-isoprenaline hydrochloride and 1-7-aH-noradrenaline were obtained from Amersham/Searle, Arlington Heights, 111.,U.S.A.

3. RESULTS 3.1. Distribution o f bound 3H-NA in the vessel wall The distribution of bound tritiated material (i.e. material retained after prolonged washing in nonradioactive Krebs solution) though the thickness of the wall of five rabbit aortae is shown in fig. 2. These 5 segments were selected from a series of 25 because the wall thickness was the same and the sectioning technically satisfactory. These vessels were incubated in 3H-NA (5 × 10-7 M) for 1 hr and then washed for 3 hr before being frozen and sectioned. NA content is

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IOOu Fig. 2. Distribution of 3H-NA uptake through thickness of segments of rabbit aorta after exposure to 3H-NA t5 X 10-7 M) for 1 hr followed by 3 hr washing (o,n =5). Adventitia is to the left and media to the right. During the last 2 hr of washing segments represented by (o) were subjected to transmural stimulation (n=3). Segments represented by (A) were pretreated with phenoxybenzamine (10-4 M) for ga hr prior to procedure (n=3). (A)-o is greater than o (p < 0.05); (x)-o is greater than • (p < 0.05).

241

expressed as ml of bath solution cleared per g wet tissue weight. The clearance was greatest {about 5 ml/g) near the middle of the vessel, and fell off rapidly to values between 0.25 and I.Oml/g at the inner and outermost surfaces. In fig. 2, the tritium distribution curve is shown also for aorta segments presoaked for 30 min in phenoxybenzamine (PBZ, 10-4 M) and then exposed to 3H-NA and washed as before but in the presence of PBZ. This concentration of PBZ was found previously to inhibit maximally 3 H-NA uptake in the intact rabbit aorta strip and in separated adventitial and medial preparations (Nedergaard and Bevan, 1971). Treatment with PBZ inhibited the large uptake of 3H-NA in the middle part of the tissue, tile distribution curve being fairly fiat throughout the thickness of the vessel wall. At the outer and inner surfaces, the tritium content was unaltered by PBZ. 3.2. Nature o f the tritiated material bound to the vessel wall Eleven preparations were exposed to 3H-NA and then washed, under conditions identical to those used in the distribution study (see above). The tissues were then homogenized and the bound tritiated material assayed for 3H-noradrenaline, 3H-deaminated catechols and 3H-noncatechols. The preparation retained 18,5 -+ 0.5 (mean +- S.E.M.)/aCi of tritium per g wet weight, equivalent to 2.11 × 10-9 mole of 3H-NA per g. Of this 89.3 +- 0.4% (mean +- S.E.M.) represented intact Ha-NA, 8.8 +- 0.3% the metabolic products of catechol-O-methyl transferase (COMT) or COMT in combination with monoamine oxidase (MAO). A small fraction (1.8 -+ 0.2%) could be identified with the products of MAO alone. In view of this finding, in this paper the total tritium content of each section will be assumed to represent 3H-NA and will be referred to as such. 3.3. The location o f the adventitio-medial /unction In 9 tissue segments, the site of the adventitiomedial junction was determined by combining the isotope frozen section technic with light microscopy. Alternate 12/am sections o f an artery previously exposed to 3H-NA and then washed as above were assayed for tritium and stained with hematoxylin and eosm for microscopic examination. The site of the adventitio-medial junction was invariably closer to

242

J.A. Ber'an et al.. Distribution of 3H-NA in aorta

Table 1 Site of adventitio-medial junction of aorta in relation to peak tritium uptake. Alternate 12u sections of a segment of aorta previously exposed to 3tt-NA and then washed, were assayed for tritium content and stained with hematoxylin and eosin. A total of 9 segments (tissue number) were studied. The percentage of each section occupied by smooth muscle was estimated from the stained material. The section which on cutting showed a characteristic change in translucency was noted. Numbers in the table are percentages of section occupied by smooth muscle: + refers to sections showing characteristic change in translucency. Section numbers refer to sequential 12,zm sections cut towards the intima: section 1 being that showing peak tritium uptake. Section number

Tissue number 1

2

3

4

5

6

0

0 + 90

15 + 100

0 + 100

0

75

1

2 3 4

3.5. Distribution o / t o t a l 3H-NA uptake m vessel wall The distribution of 3H-NA through the wall of the rabbit aorta after it had been soaked for l h r in 3H-NA (5 × 10 -'z M) and not subsequently washed is shown in fig. 3. The general pattern of the distribution is similar to that for the bound material in fig. 2. At most levels, the uptake o f 3H-NA exceeded the a m o u n t present as free amine in the extracellular space (fig. 3). The value of this space previously determined using ' 4C-inulin and a similar frozen section technique was taken as 0.4 and 0.6 mt/g for the media and adventitia respectively (Tbrbk el al., 1970). After the tissue had been washed for 10 rain,

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0 50 + 0 + + 85 100 90

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"~8the intimal surface than the peak tritium activity (see table 1). The change in translucency o f the frozen sections previously noted to occur a p p r o x i m a t e l y in the middle o f the vessel coincided with the site o f the adventitio-medial j u n c t i o n (table 1 ). 3.4. The e/yect oJ transmural nerve stimulation The effect o f transmural nerve stimulation for 2 hr on the distribution of b o u n d 3H-NA was d e t e r m i n e d in segments from 3 vessels. Stimulation was started 1 hr after removing the tissue from the 3H-NA solution. The mechanical response of the tissue ceased after 2 0 - 3 0 rain of stimulation. The mean 3H-NA distribution curve is included in fig. 2. The characteristic peak of 3H-NA was absent, though the level of activity in the inner 2/3 of the media and the outer 1/2 of the adventitia was unaffected. If it is assumed that aH-NA is lost only from nervous tissue as a result of stimulation, and the adventitio-medial j u n c t i o n has the same position relative to the section with peak tritium c o n t e n t as found above, then these results suggest that NA-concentrating nervous tissue is found in the outer layers o f the media. The presence of adrenergic nerve terminals in the outer media has been recently noted on fluorescence m i c r o s c o p y (Waterson, personal c o n m m n i c a t i o n ) .

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I00~. Fig. 3. Distribution of 3H-NA uptake through thickness of segments of rabbit aorta after exposure to 31t-NA (5 X 10-7 M) for 1 hr (e,n=5); after exposure for 1 hr subsequent to pretreatment with phenoxybenzamine (10-4 M) for 1/zhr (A, n =3); and after exposure for l hr followed by a 10-min wash period (% n=3). Adventitia is to the left and media to the right. E.C.S. represents magnitude of extracellular space. (~)-e is greater than • (p < 0.05) ; (x) • is greater than (o+E.C.S.) (p < O.O5).

J.A. Bevan et al.. Distribution of aH-NA in aorta

when presumably a considerable proportion of the tritium in the extracellular space had washed out, the loss of tritium from most levels of the vessel wall was greater than the expected amount in the extracellular space. This was determined at each level by estimating whether uptake before washing minus the expected uptake of free amine in the extracellular space was significantly greater than uptake after washing for 10 rain. The concentration of 3H-NA in the vessel wall after PBZ-pretreatment ( 1 . 2 - 1 . 3 ml/g) is fairly constant throughout tile media and the inner 1/2 to 2/3 of the adventitia. 3.6. Distri'bulion oJ total 3H-isoprenaline h, ~essel wall The distribution of 3H-isoprenaline through tile wall of the rabbit aorta after the tissue was soaked for t hr in 3H-isoprenaline (5 X 10-7 M) is shown in fig. 4. The distribution of isoprenaline was studied because this amine, closely related to NA, is not specifically transported into nerve and muscle (Callingham and Burgen, 19661 and consequently its study might help in the interpretation of the distribution of 3 H-NA. 3H.isoprenaline was distributed fairly evenly through the media at approximately 1.4 ml/g. The uptake decreased towards the outer adventitia, and

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Fig. 4. Distribution of 3H-isoprenaline (3H-ISP) uptake through thickness of segments of rabbit aorta after exposure for 1 hr (e, n=5); after exposure for ! hr subsequent to pretreatment by phenoxybenzamine (10-4 M) for ½ hr (A, n=3); and after exposure for 1 hr followed by a 10-rain wash period (o, n=3). Adventitia is to the left and media to the right. E.C.S. represents magnitude of extracellular space. ( ~ ) - e is greater than • (p < 0.05).

243

there was no peak at the adventitio-medial junction. Washing the tissue for 10 min, did not change the shape of the distribution curve, but reduced the quantity of 3H-isoprenaline by an amount equal to the free amine present in the extracellular space. Pretreating the tissue with PBZ reduced the uptake of 3H-isoprenaline fairly evenly throughout tile tissue.

4. DISCUSSION The concentration of 3H-NA used in this study, 5 × 10-v M, was somewhat less than the peak transmitter concentration found in a vessel, with fairly dense innervation during sympathetic activity (Ljung, 1969). Thus these findings might be related to the intramural distribution of neurogenically released transmitter. At this concentration a significant binding to the media takes place whereas at lower concentrations (10 -8 M), most of the uptake is into the adventitia (Nedergaard and Bevan, 1971). This concentration of 3H-NA is approximately t00 times lower than the threshold concentration needed to cause an increase in vascular muscle fluorescence in vitro (Avakian and Gillespie, 1968). The outer layer of the media as defined by histological techniques corresponds with a change in translucency consistently seen during tissue sectioning and lies slightly towards the medial side of peak tritium activity. Thus the region of maximum density of 3 H-NA concentrating nerve terminals lies immediately outside the outer muscle lamina. Noradrenaline accumulation in the outer media, which can be depleted by nervous activity and prevented by PBZ pretreatment, probably represents a H-NA taken up into sympathetic nerve terminals. Noradrenaline with the same characteristics was found in the tuner half of the adventitia. Since adrenergic neurones lose their Schwann cell sheath only close to their nodal swellings (Verity and Bevan, 1968), this finding suggests that 3H-NA is taken up not only at varicosities and adjacent parts of the axons (Muryobayashi et al., 1968; Devine and Simpson, 1968), but also by ensheathed nerves. There was no activity peak corresponding to the position of the internal elastic lamina. The fiat distribution of tritium after pretreatment with PBZ suggests that this 3H-NA component is not associated with any specific cell type.

244

J.A. Bevan et al., Distribution o f 3H-NA in aorta

The noradrenaline distribution curve in the aorta after soaking in 3H-NA without washing is similar in shape to that for bound 3H-NA. Uptake by tissues in the middle of the media was approximately 1.75 ml/g. Since the mean loss from this part of the media upon washing for 1 0 m i n was 0.85 ml/g, approximately twice the volume of tire extracellular space, this suggests a loosely bound component of 3 H-NA uptake in equilibrium with the free amine in the space. Our results allow the subdivision of 3H-NA taken up into the wall of a blood vessel into 5 conrponents or compartments. This subdivision is based upon the effects of nerve stimulation, washing and exposure to PBZ in a concentration known to maximally inhibit 3 tt-NA binding: (1) free 3H-NA in the exlracellular space; (2)loosely bound 3H-NA, represented by the 3H.NA removed by washing for 10 min in excess of that free in the extracellular space. The magnitude and distribution of this component is unknown: (3) neuronally bound 3H-NA: its distribution is similar to the sympathetic nerves, is PBZ-sensitive and is released by nerve activity; (4) 3H-NA bound at extraneuronal sites blocked by PBZ: this component is found in the media and is not depleted by prolonged nervous activity. Its distribution suggests its association with muscle tissue. Since the medial binding of 3H.isoproterenol, an amine known not to enter the ceil, is also reduced by PBZ, this component may include extracellular sites; (5) 3H-NA bound at extraneuronal sites not blocked by PBZ. As with any subdivision based upon semi-empirical criteria, the distinction of these components may not be absolute. In particular, there may be overlap between component (2) and both components (4) and (5). Only complex kinetic analysis beyond the scope of the technic described in this paper could this distinction be absolutely maintained. However the magnitude of the loss after 10 min wash and the amount remaining after 3 hr washing is strongly suggestive of the proposed distinction. In the rabbit aorta, bath NE and therefore NE in the extracellular space appears to be in equilibrium with the c~-receptor, and there is evidence that the access of NE to this receptor may be rate limiting in the contractile response to NE (Bevan, 1960). The aorta takes about 5 - 1 0 min to reach an equilibrium contractile response to NE, thus if we were attempt-

ing to establish the component in direct equilibrium with the c~-receptor, it would probably be included in component (2). A comparison of these results with those of Avakian and Gillespie (1968) obtained using quantitative fluorescence microscopy is not warranted. The tritiated NA concentrations used were smaller by two orders of magnitude; the 24/a sections did not permit differentiation of cellular elements; and different vessels were studied. However, these results are consistent both quantitatively and qualitatively with 3 H-NA uptake studies into intact and whole adventitia and media preparations of aorta (Nedergaard and Bevan, 1971 ).

ACKNOWLEDGEMENTS This study was supported by grants from the USPHS (ttE-8359), The American Medical Association Education and Research Foundation, and the Los Angeles County Iteart Association (No. 408).

REFERENCES Avakian, O.V. and J.S. Gillespie, 1968, Uptake of noradrenaline by adrenergic nerves, smooth muscle and connective tissue in isolated perfused arteries and its correlation with the vasoconstrictor response, Brit. J. Pharmacol. 32, 168. Bevan, J.A., 1960, The use of the rabbit aorta strip in the analysis of the mode of action of epinephrine on vascular smooth muscle, J. PharmacoL 129, 417. Bevan, J.A., O.A. Nedergaard, J.V. Osher, C. Su, J. T6r6k and M. Verity, 1970, On the mechanism of neuromuscular transmission in blood vessels, Proc. IV Intern. Congr. Pharmacol. 2, 7. Bevan, J.A., J.V. Osher and R.D. Bevan, 1969, Distribution of bound norepinephrine in the arterial wall, European J. Pharmacol. 5,299. Callingham, B.A. and A.S.V. Burgen, 1966, The uptake of isoprenaline and noradrenaline by the perfused rat heart, Mol. Pharmacol. 2, 37. Devine, C.E. and F.O. Simpson, 1968, Localization of tritiated norepinephrine in vascular sympathetic axons of the rat intestine and mesentery by electron microscope radioautography, J. Cell Biol. 38, 184. Gillespie, J.S., D.N.H. Hamilton and R.J.A. Hosic, 1970, The extraneuronal uptake and localization of noradrenaline in the cat spleen and the effect on this of some drugs, of cold and of denervation, J. Physiol. 206,563. Ljung, B., 1969, Local transmitter concentrations in vascular smooth muscle during vasoconstrictor nerve activity, Acta Physiol. Stand, 77, 212.

J.A. Bevan et al., Distribution of 3H-NA in aorta Maxwell, R.A., S.B. Eckhardt and W.B. Westila, 1968, Concerning the distribution of endogenous norepinephrine in the adventitia and the media-intimal layers of the rabbit in the adventitia and the media-intimal layers of the rabbit aorta and the capacity of these layers to bind tritiated norepinephrine, J. Pharmacol. 161, 34. Muryobayashi, T., J. Mori, M. Fujiwara and K. Shimamoto, 1968, Fluorescence histochemical demonstration of adrenergic nerve fibers in the vagus nerve of cats and dogs, Jap. J. Pharmacol. 18,285. Nedergaard, O.A. and J.V. Bevan, 1971, Neuronal and nonneuronal uptake of adrenergic transmitter in the blood vessel, in: The Physiology and Pharmacology of Vascular Neuroef].~ctor Systems, eds. Bevan, Furchgott, Maxwell and Somlyo (Karger, Basel) p. 22.

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Nedergaard, O.A., A. Vagne and J.A. Bevan, 1969, Distribution of norepinephrine uptake within rabbit aorta between adventitia and media, Experientia 25, 150. Su, C. and J.A. Bevan, 1970, The release of 3H-norepinephrine in arterial strips studied by the technique of superfusion and transmural stimulation, J. Pharmacol. 172, 62. T6r6k, J., O.A. Nedergaard and J.A. Bevan, 1970, The distribution of the inulin space in the rabbit thoracic aorta, Experientia 25, 55. Verity, M.A. and J.A. Bevan, 1968, Fine structural study of the terminal effector plexus, neuromuscular and intermuscular relationships in the pulmonary artery, J. Anat., 103, 49.