Kiinische Wochenschrift

Klin. Wochenschr.56 (Suppl. I), 131-138 (1978)

© Springer-Verlag 1978

Sympathetic Vascular Tone in Spontaneous Hypertension of Rats A. Sch6mig, R. Dietz, W. Rascher, J.B. Lfith, J.F.E. Mann, M. Schmidt, and J. Weber Department of Cardiologyand Department of Pharmacology,Universityof Heidelberg

Sympathiseher GefiiBtonus bei der genetisehen Hypertonie der Ratte Zusammenfassung. Unterschiede des sympathischen Gef~il3tonus zwischen spontan hypertonen Ratten (spSHR) und Wistar Kyoto Ratten (WKR) wurden an Hand folgender Gr6Ben erfaBt: Die sympathische Aktivitfit wurde ermittelt durch die Bestimmung der Plasmakatecholamine (Noradrenalin, Adrenalin und Dopamin) bei gleichzeitiger Messung der neuronalen und extraneuronalen Wiederaufnahme yon Noradrenalin im isolierten Prfiparat (Langendorff Herz). Die Ansprechbarkeit glatter Gef~iBmuskulatur anf vasopressorische Substanzen wurde in der isoliert perfundierten Hinterextremit~it der Ratte gemessen. Die sympathische Aktivitfit war bei spSHR im Alter von 5, 12, 15 und 28 Wochen gesteigert, da die Konzentration von Noradrenalin im Plasma um 50% bei unver~inderter neuronaler und extraneuronaler Wiederaufnahme erh6ht war. Die Ansprechbarkeit der glatten GeffiBmuskulatur gegenfiber Noradrenalin war bei spSHR verstgrkt. Neben einer stfirkeren maximalen Vasokonstriktion nach supramaximalen Dosen von Noradrenalin (10 -3 tool/l) oder BaC12 (20 retool/l) fand sich eine spezifische Uberempfindlichkeit der einzelnen glatten Muskelzelle gegenfiber Noradrenalin bei 5 Wochen alten spontan hypertonen Ratten. W~ihrend nach Kaliumdepolarisation keine Unterschiede in der Schwellendosis oder der EDso auftraten, waren diese bei spSHR ffir die Noradrenalin-induzierten Widerstandserh6hungen um 25% vermindert. Die stimulierte sympathische Aktivit/it sowie die erh6hte Ansprechbarkeit der Widerstandsgef~Be gegenfiber Noradrenalin bei spSHR sind Ursache des gesteigerten sympathischen GeffiBtonus, der fiber eine Erh6hung des peripheren Widerstandes die Entwickoffprint requests to:

A. Sch6mig,M.D. (address see p. 138)

lung des hohen Blutdrucks bei der genetischen Hypertonic der Ratte verursacht.

Sehliisselwiirter: Sympathische Aktivit/it - Plasma Noradrenalin - Adrenalin -- Dopamin - Gef/iBreaktivit/it - Uptake Aktivitfit - Genetische Hypertonic. Smnmary. Differences in sympathetic vascular tone between Wistar Kyoto rats (WKR) and stroke prone spontaneously hypertensive rats (spSHR) were determined by comparing the following parameters: sympathetic activity was evaluated by determinations of plasma catecholamines (noradrenaline, adrenaline, dopamine) combined with the measurement of the neuronal and extraneuronal uptake of noradrenaline using an isolated rat heart preparation. The responsiveness of vascular smooth muscle to vasopressor agents wws tested in the isolated perfused hindlimb preparation. At the age of 5, 12, 15, and 28 weeks sympathetic nervous activity was significantly higher in spSHR than in WKR since plasma noradrenaline was elevated by almost 50% in the presence of an unaltered activity of the uptake mechanisms. The responsiveness of vascular smooth muscle to noradrenaline was markedly enhanced in spSHR. Besides increased maximal vasoconstriction in response to BaC12 (20 mmol/1) after potassium chloride depolarization or supramaximal doses of noradrenaline (10- 3 mol/1), a supersensitivity of vascular smooth muscle to noradrenaline could also be detected in spSHR (age 5 weeks). The threshold dose and the EDs0 were reduced by 25% in spSHR in response to noradrenaline infusions. No changes in threshold or EDso were found in response to potassium chloride depolarization. The stimulated sympathetic activity in spSHR and the increased responsiveness of resistance vessels to noradrenaline, both contribute to the rise in sympathetic vascular tone. The finding of an increased sympathetic vascular tone in very early stages of hyperten-

132

sion suggest that this factor, producing a primary increase in total peripheral resistance underlies the development of high blood pressure in spSHR. Key words: Sympathetic activity - Plasma noradrenaline - Adrenaline - D o p a m i n e - Vascular reactivity -- Uptake activity - Spontaneous hypertension.

The significance of the sympathetic nervous system, the most potent vasoconstrictor system for the development of high blood pressure remains speculative. This may be explained in part by theoretical considerations since the volume concept of hypertension [3, 11] has concentrated most of the research work in the field of hypertension on renal and volume factors during the past decade. On the other hand, there are methodological problems in evaluation of sympathetic activity and its effect on the target organs. In genetic hypertension of rats, conclusions about the role of the sympathetic nervous system have been derived from either indirect evidence, such as effects of immunosympathectomy [8] or from measurements of "equivalents" of sympathetic activity, as e.g. measurement of renal nerve activity [11] baroreceptor response to blood pressure elevation [4, 13] or catecholamine levels in tissue and plasma [10, 15, 22]. However, none of these approaches alone allows definite conclusions about the pathogenic significance of the sympathetic system for hypertensive diseases and data available so far are conflicting. The present study was designed to obtain a comprehensive view at the role of sympathetic activity and its action on resistance vessels in the development of spontaneous hypertension in rats. Therefore, investigations were not only confined to one aspect of the system-as the overflow of the neurotransmitter noradrenaline into the p l a s m a - b u t concerned the degree of vasoconstriction elicited by the sympathetic system. Hence it was necessary to obtain information not only about plasma catecholamines but also about the activites of the neuronal and extraneuronat uptake mechanisms and about the responsiveness of vascular smooth muscle to noradrenaline. These parameters were integrated in the term sympathetic vascular tone, the magnitude of which should correspond to the rise in blood pressure observed in spSHR.

Materials and Methods 1. General Procedure Male spontaneously hypertensive rats of the stroke prone strain (spSHR) [19] bred in Heidelberg for more than three years and age matched Wistar Kyoto rats (WKR) were used. Rats were

A. Sch6mig et al. : Sympathetic Vascular Tone in SHR housed individually in macrolon cages in a room automatically lighted from 6 a.m. to 6 p,m. with constant temperature (24_+ I ° C) and humidity (60 _+3 %). Demineralized water and a standard petlet chow (sniff~) containing 100 meq/kg sodium and 2t0 meq/kg potassium were offered ad libitum.

2. Blood ,Sampling Blood (0.5 ml) was collected in normotensive Wistar rats for determination of plasma catecholamines by four different methods for comparison of the effects of sampling techniques on plasma catecholamines: - from an incision in the tail in light ether anaesthesia (n: 10). - from an incision in the tail after thiobutabarbital anaesthesia (Incatin ® 50 mg/kg), administered i.p. 30 min before sampling (n: 20) .- from the trunk after decapitation (n: 8) -- from an arterial catheter implanted into the femoral artery and exteriorized subcutaneously at the neck two days before blood sampling. Blood was withdrawn from the distal end of the catheter outside the cage in which the rats could move freely, not being disturbed by the procedure of blood sampling (n: 10).

3. Determination o f Plasma Catecholamines Blood (0.5 ml) was collected from each rat into cooled microtubes and 1 mg ascorbic acid, 1 mg EGTA and 0.5 mg MgC12 were added. Immediately after the collection period, samples were centrifuged at 0° C at 5000 g for 10 min. 200 ~tl of 0.6 mol/1 perchloric acid were added to 200 gl of plasma for deproteinization. The mixture was centrifuged as before and the supernatant was frozen and stored at - 3 0 ° C in aliquots of 100 txl. In each of the aliquots the concentrations of the three catecholamines noradrenaline (NA), adrenaline (A) and dopamine (DA) were determined by a radioenzymatic method [5]. The three catecholamines were converted by Catechol-O-methyl-transferase(purification following Axelrod and Tomchick [1]) in the presence of S-adenosyl-methionin-3Hinto their O-methylated metabolites normetanephrin, metanephrin and methoxytyramine, respectively. After extraction into ether in the presence of tetraphenylborate and extraction into hydrochloric acid for purification the O-methylated metabolites were separated by thin layer chromatography. Prior to determination of radioactivity by liquid scintillation counting, the specificity was improved by Oxidation of normetanephrin and metanephrin to vanillin. The concentrations of the amines were corrected for the radioactivity found in the blanks and calculation based upon the activity of internal standards.

4. The ActiviO~ of Neuronal and Extraneuronal Uptake Mechanisms for Noradrenaline was determined in isolated perfused rat hearts (Langendorff technique) (spSHR n: 10, WKR n 8, age: 5 weeks). The hearts were perfused with an oxygenated modified Tyrode solution containing tritiated noradrenaline (10- 7 mol/1, Amersham). The concentration of radioactive noradrenaline, of its deaminated metabolites (DOPEG = 3,4-dihydroxyphenylglycol, DOMA = 3,4-dihydroxymandelic acid), and its O-methylated metabolites (NMN = normetanephrine, VMA=3-methoxy-4-hydroxymandelic acid, MOPEG=3-methoxy-4-hydroxyphenylglycol) were measured in both perfusate and effluent following a separation procedure described by Graefe [9]: In an initial step the catechol compounds were adsorbed on an AlzO 3 column at pH 8.2. The O-methylated metabolites, which were found in the effluem, could be separated by a Dowex 50W x 4 column into two fractions, one containing VMA and MOPEG

A. Sch6mig et al. : Sympathetic Vascular Tone in SHR

catecholaminealOin plasma ng/ml 5-

133

blood sampling withoutanesthesia with anesthesia thiobarbiturate arterial decapitation ether (20) catheter (8) (10) (10)

1.O-

0.50.10.05N noradreneline

Fig. 1. Concentrations of catecholamines in plasma of normotensive Wistar rats under different conditions of blood sampling and anesthesia. Note the log scale

iil iiliIiiiii ~ adrenaline

(effluent), the other containing N M N (6n HCI :ethanol eluate). The compounds absorbed on A1203 were eluated by 0.2 n acetic acid (NA and DOPEG) and 0.2 n HC1 (DOMA). NA and D O P E G were separated by adsorption of NA on a dowex 50W x 4 column. D O P E G was found in the effluent, whereas NA was eluted by 2 n HC1. The values for the fractions obtained by liquid scintillation counting were corrected for quench, for recovery (NA and all metabolites more than 90%), and for crosscontamination (only DOMA). Uptake and metabolization rate were calculated from the arteriovenous difference under steady state conditions before and after blockade of the neuronal uptake with cocaine (30 gmol/1) and after additional blockade of the extraneuronal uptake with corticosterone (100 gmol/1).

5. Reactivity o f Vascular Smooth Muscle to Noradrenaline An isolated perfused hindlimb preparation was used to assess changes in reactivity of vascular smooth muscle to noradrenaline. Weight and age-matched pairs of rats (one normotensive, one hypertensive rat) were perfused at constant flow (flow rate 10 ml/100 g tissue/rain). For details see Folkow et al. [7]. The preparation was perfused with an oxygenated modified Tyrode solution, containing 2% artificial colloid (Ficoll-70®). The increase in perfusion pressure, measured via a T-tube, was taken as an equivalent of resistance changes. Cumulative dose-response curves to noradrenatine were obtained in W K R (n: 13) and in spSHR (n: 13) (age 5 weeks) by perfusion of the agonist in concentrations from 10- 8 to 10- 3 mol/1. Vascular reactivity to potassium chloride was also determined (WKR n:7, SHR n:7, age 5 weeks). Dose-response curves were obtained by step-by-step replacement of sodium in the perfusion medium with potassium (4 mmol/l-120 mmol/t). Maximal contraction was achieved with BaC12 (20 mmol/1). The dose which elicited halfmaximal vasoconstriction (EDso) was estimated by graphical interpolation. The threshold dose for each agonist was defined as the dose required to raise the perfusion pressure 10 mmHg above the perfusion pressure at maximal vasodilatation.

Statistical Analysis All values given in text and figures are means + SEM. The significance of differences was calculated by Student's t-test: n.s. : not significant; * : p < 0.05 ; ** : p < 0,0 I.

N dopamine age weeks

5

0.5-

12

28 =k

15

*

noradrenaline ng/ml 0.4-

*

~

0.30.2O.10

adrenaline ng/ml

0.1] 0

dopamineng/ml 0.0.1 20"3 t

~

~

~

~

0

n-- 1010 1010 1515 anesthesia: thiobarbiturate ~WKR

i

1010 without

~SHR

Fig. 2. Concentrations of noradrenaline, adrenaline and dopamine in plasma of spSttR and W K R at the age of 5, 12, 28 weeks (thiobutabarbital anesthesia, blood sampling from tail artery) and at the age of 15 weeks (conscious, blood sampling via chronical arterial catheter)

Results

Influence of DifJbrent Blood Sampling Techniques on Plasma Catecholamine Concentrations Fig. 1 shows the influence of four different techniques of blood sampling on plasma catecholamines levels

134

A. Sch6mig et at. : Sympathetic Vascular Tone in SHR

noradrenaline uptake pmol/min /g tissue

T ....

;.=

~n

....

i/J

I

ther collected from arterial catheters-or from an inicision in the tail under thiobutabarbital anaesthesia.

Plasma Concentrations of Catechotamines in spSHR and WKR at Different ages

neuronal uptake

extraneurona! uptake

WKR (n:8)

~

SHR (n=lO)

Fig. 3. Noradrenaline uptake in the hearts of spSHR and WKR before and after blockade of the neuronal uptake by cocaine and additional blockade of the extraneuronaI uptake by corticosterone

I cocaine 30 pmol/I 50release of deaminated metabolites (DOPEG,DOMA) pmol/min 25/g tissue

]

[corticosterone / 100 rnol/ I

When plasma concentrations of adrenaline and dopamine are compared, no significant differences could be obtained between WKR and spSHR either during the developmental or established phase of hypertension (Fig. 2). In contrast, plasma noradrenaline in spSHR rats was nearly 50% greater than in WKR (SHR: 0.386+0,027 ng/ml, WKR: 0.265_+0.026 ng/ ml, p < 0.01) already in the early phase of hypertension (5 weeks). Increased values were also measured in spSHR at the age of 12 weeks (spSHR: 0.409+0.041 ng/ml, WKR: 0.292_+0.032 ng/ml, p