Naunyn-Schmiedeberg's Arch Pharmacol (1983) 322:1-5 Naunyn-Schmiedeberg's

Archives of Pharmacology �9 Springer-Verlag 1983

Autoradiographic Study of Rat Hearts Perfused with 3H-Isoprenaline

Isabel Azevedo, H. B6nisch, W. Osswald, and U. Trendelenburg

Laboratorio de Farmacologia, Faculdade de Medicina, P-4200 Porto, Portugal Institut ftir Pharmakologie und Toxikologie der Universit/it WiJrzburg, Versbacher Strasse 9, D-8700 Wtirzburg, Federal Republic of Germany

Summary. B6nisch et al. (t974) identified kinetically two extraneuronal compartments into which 3H-isoprenaline distributes in the perfused rat heart: compartment III (char- acterized by a half time for the efflux of 3H-isoprenaline of about 10 min) had about the same size as compartment IV (half time for efflux: 23 min). These authors suggested that compartment III might be located in the vascular smooth muscle, while compartment IV might be located in myocar- dial cells. The present study was carried out to validate or refute this suggestion. Rat hearts were perfused for 5, 20 or 60 min with 1 ~tmol/1 3H-isoprenaline; additional hearts were perfused with I gmol/1 3H-isoprenaline for 30rain in the presence of either 20 gmol/1 corticosterone or 20~tmol/1 corticosterone plus 30 gmol/1 cocaine. COMT was inhibited in all experiments (by the presence of 100 gmol/1 U-0521).

Quantitative autoradiography revealed in all groups that the silver grain density (grains/mm z) was greater over small blood vessels (arterioles and venules) than over myocardial cells. However, total silver grains over myocardial cells greatly exceeded those over small blood vessels (by a factor of 6 to 9). Thus, the suggestion of B6nisch et al. (1974) is untenable. Autoradiographic results obtained with small specimens of ventricular muscle are representative of the whole heart, since "silver grains over total tissue" (per mm 2) were highly significantly correlated with the 3H-isoprenaline content of the homogenized hearts (in pmol/g).

While corticosterone reduced the accumulation of 3H- isoprenaline in myocardial cells, it failed to affect the appearance of silver grains over Purkinje cells. However, cocaine prevented this type of accumulation. Thus, uptake in Purkinje cells appears to resemble neuronal rather than extraneuronal uptake.

Key words: 3H-Isoprenaline - Extraneuronal distribution compartments - Perfused rat heart - Extraneuronal uptake - Autoradiography

Introduction

Studies of the distribution of 3H-(+)-isoprenaline in the perfused rat heart (B6nisch and Trendelenburg 1974; B6nisch et al. 1974; Uhlig et al. 1974; B6nisch 1978) led to the conclusion that, in the rat heart, there are two major extraneuronal compartments into which 3H-isoprenaline distributes: compartments III and IV. These two compart-

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ments differ from each other in two respects. Effiux of 3H-isoprenaline from compartment III has a shorter half time (10.1 rain) than from compartment IV (22.6 min; B6nisch et al. 1974). Moreover, the evidence indicated that compartment III had a high catechol-O-methyl transferase (COMT) ac- tivity, while the COMT activity of compartment IV was small or absent (B6nisch and Trendelenburg 1974; Uhlig et al. 1974). After loading of the hearts with 0.95gmol/1 of 3H-isoprenaline for 30rain, nearly equal amounts of the labelled amine had distributed into compartments III and IV (B6nisch et al. 1974). The distribution of 3 H-isoprenaline into both compartments involves translocation by the extra- neuronal uptake mechanism (uptake2; Iversen 1965), since the filling of these compartments is impaired by the inhibitor ofextraneuronal uptake, corticosterone (B6nisch et al. 1974).

On the basis of various observations, B6nisch et al. (1974) speculated that the COMT-containing compartment III might be associated with the vascular smooth muscle of the coronary bed, while compartment IV (which is largely devoid of COMT activity) is located in myocardial cells.

The present study was undertaken to determine, with autoradiographic techniques, whether the morphological counterparts of compartments III and IV can be identified. More precisely, rat hearts were perfused with 1 gmol/1 3H-isoprenaline, and it was determined whether the distri- bution of grains over vascular smooth muscle and myocardial cells reflected the relative sizes of compartments III and IV mentioned above.

Methods

Rat hearts were perfused with 1 ~tmol/1 3H-isoprenaline as described in detail by B6nisch and Trendelenburg (1974). To inhibit COMT 100 gmol/1 U-0521 were present in all experi- ments, from the beginning of the 20 min of pre-perfusion to the end of the experiment. In some experiments (see Results), also 20gmol/1 corticosterone or 20gmol/1 corticosterone + 30 gmol/1 cocaine was present throughout the experiment.

At the end of the experiment small tissue samples were removed from the anterosuperior zone of the interventricular septum for histological and autoradiographic study. The remaining tissue of the heart was homogenized and processed for radiochemical measurement of 3H-isoprenaline and its metabolite, 3H-OMI (3H-O-methylisoprenaline). For details of column chromatography, scintillation counting etc., see B6nisch (1978).

The ventricular tissue samples were fixed in 3 % glutaral- dehyde in cacodylate buffer, pH7.3, at 4~ for 18h, dehy-

drated th rough e thanol and embedded in paraffin. Sections o f 5 ~tm thickness were s tained with per iodic acid + Schiff and Harr is hematoxyl ine and coa ted by dipping in I l ford emuls ion K-5 di luted 1:1 with distilled water . Af te r 30 days o f ex- posure, the au to rad iographs were processed in K o d a k D-170 and examined by light microscopy. Silver grains were counted in 6 sections f rom each heart . C a m e r a lucida drawings were used to calculate the relat ive areas occupied by the different tissue consti tuents. The radioac t iv i ty was expressed as silver grain density per m m 2 of each structure, as counted in each drawing. The means for each s t ructure and exper iment were compared with the respective controls by Student ' s t - t es t

F o r de te rmina t ion o f sur face /vo lume rat ios ventr icular tissue samples, fixed as descr ibed above, s tayed in the fixative for 9 0 m i n ; small pieces were post-f ixed in 2 % osmium te t roxide in the same buffer for 1 h, dehydra ted th rough e thano l and embedded in Epon. F r o m these E p o n blocks semi- thin sections were obtained, s tained with to luidine blue in 1% borax and s tudied under the l ight microscope. These sections a l lowed us to choose the zones (conta ining small b lood vessels) to be thinly cut for u l t ras t ruc tura l study. These thin sections were s tained with uranyl acetate and lead ci trate and s tudied under a Siemens E lmiskope 101.

F o r different types o f cells, the sur face /vo lume ra t io was de termined as descr ibed by Weibel (1972); measurements were m a d e in 50 different micrographs , ampli f ied 18,000 times. A t ransparen t test area (18 x 15cm) conta in ing 50 paral lel and equidis tant lines (1.8 cm long) was super imposed on the micrographs . The n u m b e r o f intersect ions o f the lines

with cellular membranes (I) and the n u m b e r o f lines ending inside cells (P) were determined. The sur face /volume rat io (s/v) was given by Weibel (1972) as

4I s / v -

P

Substances Used in this Study. (+)-Isoprenal ine hydrochlor ide (C. H. Boehr inger Sohn, Ingelheim, F R G ) , (+)-3H-7-iso- prenal ine ( N E N , Dreieich, F R G ; specific activity 2.11 Ci/ m m o l ; ch romatograph ica l ly purif ied over A120 2 with elut ion with 0.2tool/1 acetic acid before use); Y,4 ' -d ihydroxy-2- methyl p rop iophenone (U-0521 ; U p j o h n Comp. , Ka lamazoo , MI, USA) , cor t icos terone (E. Merck, Darms tad t , F R G ; stock so lu t ion: 50mmol /1 in absolute ethanol) , cocaine hydrochlor ide .

Results

1. Volumetric Density of Different Structures

His to logy o f all 80 sections (about 6 per heart , 13 hearts) showed that 52 ~ o f the tissue consisted o f myocard ia l cells, 0.4 ~o of ar ter ioles and 1.5 ~ o f venules (see "Vv" in Table 1). The remainder o f the tissue is called "ex t ramuscu la r space" (Vv = 46 %) which encompasses not only the extracel lular space bu t also nerve endings, Schwann cells, mas t cells, capillaries, f ibroblasts etc., i.e. all those s tructures that were not readily identif iable under the l ight microscope.

Table 1. Silver grain density (silver grains/mm 2 ; means + S.E.) over the different structures of the rat heart and 3H-isoprenaline content (pmol/g; means + S.E.) of the whole heart. Rat hearts were perfused with 1 ~tmol/13H-isoprenaline, COMT was inhibited by the presence of 100 gmol/1 U-0521. After the end of perfusion with 3H-isoprenaline, tissues were removed for analysis. Experimental conditions: perfusion with 3H-isoprenaline for 5 min (group 1), 20 min (group 2) or 60 min (group 3). The hearts of groups 4 and 5 were perfused with 3H-isoprenaline for 30 min, either in the presence of 20 gmol/1 corticosterone (group 4) or in the presence of 20 gmol/1 corticosterone plus 30 gmol/1 cocaine (group 5). Vv = volumetric density (means • S.E.) of each structure as determined in all specimens (n = 80). Total tissue = total number of silver grains per mm 2 of whole cardiac tissue. Asterisks indicate significance of difference between two means *P< 0.05; ***P< 0.01; ****P< 0.001). Asterisks appearing in group 1 indicate differences between groups 1 and 2; those appearing in group 3 relate to differences between groups 2 and 3; those appearing in group 4 relate to differences between groups 2 and 4; asterisks appearing in group 5 indicate differences between groups 4 and 5. *** P < 0.01 for differences between groups 1 and 3. n = number of observations. For myocardial cells n equals the number of specimens. For extramuscular space, arterioles, venules and Purkinje cells n refers to the number of specimens in which these structures were identified (note that small blood vessels and Purkinje cells were identified in only a fraction of all specimens); for total tissue and 3H-isoprenaline content n refers to the number of hearts, n.d. = no labelled Purkinje cells were detected

Group Myocardial cells Extramusc. space Arterioles Vv=52 • 1 ~ Vv=46 • 1~ Vv=0.4 • 0.1~

Grains/mm 2 n Grains/mm 2 n Grains/ram 2 n

1 5889 • 383*3** 18 683 • 96*** 17 32186 • 4202*** 6 2 12816 • 602 14 2179 • 530 14 34348 • 6354 3 3 21473 • 1647"*** 18 3067 • 18 91943 • 12118* 8 4 8282 • 631"*** 18 943 • 182" 18 55416 • 20106 5 5 7548 • 726 12 221 • 101"** 12 30021 • 6944 2

Group Venules Purkinje cells Total tissue 3H-Isoprenaline Vv = 1.5 • 0.2 ~ content

Grains/mm 2 n Grains/mm 2 ~ Grains/mm 2 n pmol/g n

1 27287 • 4518" 9 n.d. 3945 • 449"*** 3 1562 • 142"*** 2 40794 • 2961 6 n.d. 8208 • 1280 2 4177 • 194 3 105361 • 14765*** 10 75140 • 2611 2 14465 • 2200 3 6722 • 220**** 4 45365 • 9959 7 52433 • 9146 4 5661 • 670 3 1450 • 215'*** 5 47233 • 3837 8 n.d. 4779 • 349 2 1536 • 227

Fig. 1. Autoradiographs of a rat heart perfused with 3H-isoprenaline (1 gmol/1) in the presence of the catechol-O-methyltransferase inhibitor U-0521 (100 ~tmol/1) for 5 rain. Silver grain density is higher over small blood vessels, both venules (V) and arterioles (A), than over myocardial cells (M). E, extramuscular space. Calibration bars = 100 ~tm

2. Autoradiography

Table 1 presents the grain density over the identified struc- tures as well as over the whole tissue (grain density per mm2). Most groups consisted of 3 perfused hearts. When the duration of the perfusion with i gmol/1 3H-isoprenaline was increased from 5 min (group 1) to 20 min (group 2) to 60 min (group 3), the grain density over the various structures tended to increase. The increase (from 5 to 60 min of perfusion) was 3.6-fold for myocardial cells, 4.5-fold for extramuscular space, 2.9-fold for arterioles, 3.9-fold for venules and 3.7-fold for total tissue. Considering the variability (especially for arterioles and venules, which were found in only a small proportion of all tissue samples) it is unlikely that any pro- nounced differences occurred between structures.

In all groups the small blood vessels exhibited the highest density of grains. Already after the shortest duration of perfusion (5 rain) the blood vessels were much more marked than the surrounding myocardium (Fig. 1). However, in spite of the high grain density over the small blood vessels, the absolute grain number over myocardial cells (calculated as "grain density times Vv") greatly exceeded the corresponding number of grains over the small blood vessels. In a total of 64 specimens grain densities were determined over myocardial cells and over blood vessels. The geometric mean of the ratio "density over blood vessels/density over myocardial cells" amounted to 4.22 (95 ~ confidence limits: 3.75 and 4.76), and the ratio clearly exceeded unity (P < 0.001). On the other hand, when these ratios were calculated for each of the five experimental groups, no significant differences were observ- ed between the different groups.

The hearts of group 4 (Table 1) were perfused with 1 gmol/1 3H-isoprenaline for 30 rain, and 20 gmol/1 cortico- sterone was present throughout the experiment. The presence of corticosterone decreased the grain density over myocardial cells and, interestingly enough, also over the extramuscular space (Table1). As far as the small blood vessels are concerned, the question whether corticosterone reduced the

grain density, cannot be answered. On the one hand, no hearts were perfused for 30 min in the absence of corticosterone, on the other hand, the number of identified small blood vessels was too small for quantitative comparison (see Table 1).

Interestingly enough, heavily marked Purkinje cells were detected in groups 3 and 4. When 30 ~tmol/1 cocaine was also present (in addition to corticosterone; 30 min perfusion with 1 gmol/1 3H-isoprenaline; group 5), no marked Purkinje cells were detected (Table 1). The addition of cocaine had no effect on grain density over myocardial cells, but it reduced that over the extramuscular space (Table 1).

In some tissue samples large blood vessels were detected. When observed, they showed a silver grain density that was considerably lower than that over small blood vessels; indeed, their grain density was similar to that of myocardial cells.

3. Radiochemical Measurement of 3H-Isoprenaline

Table 1 also presents the amount of 3H-isoprenatine re- covered from the hearts at the end of the experiment. Since COMT was inhibited, 3H-OMI was not detected. Figure 2 compares the total number of silver grains per unit volume of cardiac tissue (Table 1, column "total/ram 2'') with the a H-isoprenaline content of the whole hearts. Figure 2 shows a highly significant correlation between the two parameters (r = 0.87; P < 0.001; n = 13).

4. The Surface~Volume Ratio of Different Cell Types

The surface/volume ratio was lowest for myocardial cells (Table 2). For all other cell types (arteriolar cells, capillary cells, fibroblasts and adrenergic nerves) the surface/volume ratio was considerably higher (Table 2).

Discussion

In any microscopic study, only a very small portion of the whole heart can be studied; on the other hand, the 3H-iso-

-6

i

E

c

15000-

I0000

5000.

Q �9

3o'o0 6600 ' p~ollg

Fig. 2. Correlation between the total number of silver grains per mm 2 of whole cardiac tissue and the amount of 3H-isoprenaline contained in the rat heart at the end of the perfusion (pmol/g). Shown are the results from all hearts in which both parameters were determined, irrespective of the group to which they belong

Table 2. Surface/volume ratio of different cell types in rat heart

S/V (pm2/~tm 3) n

Myocardial cells 0.67 _+ 0.07 45 Capillary cells 7.47 + 1.05 51 Arteriolar cells 2.61 _+ 0.15 20 Fibroblasts 9.87 + 1.51 60 Adrenergic nerves 3.73 _+ 0.63 15

Shown are means _+ SE determined for n cells See Methods for details of calculations

prenaline content of the tissue can be determined after homogenization of the whole heart (B6nisch et al. 1964). In this situation, it is very reassuring that the "total silver grain density" (per mm z of cardiac tissue) was highly significantly correlated with the 3H-isoprenaline content of the whole hearts of all five groups studied here. Thus, the tissue samples taken from the interventricular septum are representative of the whole tissue. Similar studies of the dog saphenous vein (Azevedo and Osswald 1976) and rabbit thoracic aorta (Branco et al. 1981) had also yielded highly significant correlations between the 3H-isoprenaline content of the tissue and the number of silver grains over the tissue.

While it is satisfying that a sample of ventricular myocar- dium was representative of the whole heart, it should be borne in mind that other cardiac tissues (e.g., atrial myocardium, nodal and conduction tissue) might differ considerably from ventricular myocardium with respect to the uptake and distribution of 3H-isoprenaline. Because of the small contri- bution by these cell types to the total weight of the heart, the 3H-isoprenaline content of the heart (in pmol/g) mainly reflects the distribution of the labelled amine into myocardial cells.

When rat hearts are perfused by the method of Langendorff (see Methods), the development of some minor degree of oedema is inevitable. Since we aimed at a corn-

parison of autoradiographic results (present study) with earlier determinations of compartments III and IV (B6nisch et al. 1974), the experimental conditions had to be kept identical. Moreover, the development of some oedema is immaterial, since the hearts of various species, when perfused by the Langendorff technique with various 3H-catechol- amines, maintain steady-state rates of uptake and metabolism of 3H-catecholamines for up to 120 min of perfusion (see Fig. 1 of Fiebig and Trendelenburg 1978; see Graefe 1981). Finally, as pointed out above, our "extramuscular space" cannot be equated with the extracellular space; the former clearly exceeds the latter.

The Purkinje cells observed in the ventricular myocar- dium seemed to differ from myocardial cells in at least one respect: like adrenergic nerve endings (Azevedo and Osswald ] 976), Purkinje cells exhibited high silver grain density when extraneuronal uptake was inhibited by the presence of corticosterone (group 4, Table 1), and they failed to accu- mulate 3H-isoprenaline in the additional presence of cocaine (group 5, Table 1). The Purkinje cells observed here probably represent Purkinje II cells which are larger and clearer than common myocardial cells. Although extrapolation to other special cell types (nodal, Purkinje I, Purkinje III) is im- possible, the finding of a cocaine-sensitive uptake of a catecholamine into Purkinje II cells is of interest for those studies, in which cocaine is used to identify "neuronal uptake". It should be noted that the effect of cocaine on grain density over Purkinje cells was not accompanied by any decrease of the 3H-isoprenaline content of the heart (Table 1, compare groups 4 and 5).

As pointed out in the Introduction, B6nisch et al. (1974) identified two distribution compartments for 3H-isoprenaline (compartments III and IV) after perfusion of the heart with 0.95 gmol/1 for 30 min, and the two compartments were of roughly similar size. Moreover, these authors suggested that compartment III might possibly be located in the vascular smooth muscle of the coronary bed, while compartment IV might be located in the myocardium. The present results indicate that the silver grain density over small blood vessels is considerably higher than over neighbouring myocardial cells. Table 1 shows that the silver grain density over arterioles or venules exceeded that over myocardial cells by factors varying from 2.7 to 6.3 (geometric mean factor of 4.4 for arterioles and of 4.8 for venules). However, the volumetric density (V,) of the small blood vessels is far too small to account for an absolute number of silver grains comparable to that over myocardial cells. To support the speculation of B6nisch et al. (1974), the ratio "absolute number of silver grains over myocardial cells/absolute number of silver grains over blood vessels" should have been close to unity. However, after perfusion of the heart with 3H-isoprenaline for 5, 20 and 60 min, the ratio amounted to 5.7, 8.9 and 5.7, respectively. Thus, we must conclude that the kinetically defined compart- ments III and IV cannot be identified with two different morphological structures (blood vessels and myocardial cells).

From the fact that this ratio was identical after 5 and after 60 min of perfusion with 3H_isoprenaline, it is evident that the accumulation of 3H-isoprenaline had a roughly similar time course for myocardial cells and for the small blood vessels. It should be noted that also the density of silver grains over the extramuscular space increased with increasing duration of perfusion with 3H-isoprenaline. It is likely that these silver grains reflect labelled amine taken up into various cell types,

since extracellularly dis t r ibuted 3H-isoprenaline must have diffused out of the tissue during the early steps of the prepara t ion of the tissue specimens. I t is of interest to note that the density of silver grains over the extramuscular space was reduced by corticosterone; this is another indication that, at least in some of the cell types, u p t a k e / w a s involved in the accumulation of 3H-isoprenaline in the structures of the extramuscular space.

Our negative finding resolves one problem. Lowe and Creveling (1979) carried out an immunocytochemical locali- zation of COMT in the rat heart. Myocardial cells were found to be rich in COMT activity. This finding was incompatible with the earlier suggestion of B6nisch et al. (1974) that compartment IV (which is very poor in, or devoid of, COMT activity) is localized in myocardial cells.

The present study showed that 3H-isoprenaline distrib- uted mainly into myocardial cells, but also into vascular smooth muscle cells. It is of interest to note that these two cell types have greatly differing surface/volume ratios. The about 4-fold higher surface/volume ratio of vascular smooth muscle cells may well explain why a small amount of 3H-isoprenaline leaves the rat heart with a very short half time (compartment 1I of B6nisch et al. 1974, with a half time of 3 min).

If both cell types would have very similar membrane properties (i.e., contain the same number ofuptake2 sites per mm z surface and allow the same number of 3H-isoprenaline molecules to diffuse through i mm 2 membrane surface), both cell types should reach the same steady-state t issue/medium ratio when exposed to 3H-isoprenaline, but the half-time for the "l ipophil ic diffusion" of 3H-isoprenaline should be propor t ionate ly shorter for cells with high surface/volume ratios than for those with low ones. Thus, on the one hand, the discrepancy between the relative size of compar tment III (B6nisch et al. 1974) and the relative propor t ion of grains over vascular smooth muscle (present study) forces us to abandon the speculative at t r ibut ion of compartments III and IV to vascular smooth muscle and myocardial cells, respectively (B6nisch et al. 1974). On the other hand, the 4 times higher surface/volume ratio of vascular smooth muscle than of myocardial cells tends to support the suggestion of B6nisch et al. (1974) that the half time for the efflux of aH-isoprenaline should be considerably shorter for vascular smooth muscle than for myocardial cells. Hence, the small distribution

compar tment in vascular smooth muscle may be represented by compartment II of B6nisch et al. (1974).

Acknowledgements. The support of the Dr. Robert Pfleger-Stiftung and the Instituto Nacioual de Investigacao Cientifica (FMP-1) is gratefully acknowledged. The authors are indebted to Dr. G. A. Johnson of Upjohn Comp., Kalamazoo, MI, USA, for the U-0521.

References

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Received September 3, 1982/Accepted December 6, 1982