Acta Physiol Scand 1992, 146, 185-196

Vasoconstrictor reactions in spontaneously hypertensive rats versus Wistar Kyoto can be increased or decreased depending on the conditions of perfusion I. M. RODIONOV, 0. S. T A R A S O V A , T. P. V A K U L I N A , V. B. KOSHELEV, V. G. P I N E L I S and Ch. M. MARKOV Department of H u m a n and Animal Physiology, Faculty of Biology, Moscow State University and Laboratory of Pathophysiology, Research Institute of Pediatrics, Academy of Medical Sciences, Moscow, Russia

RODIONOV, I. M., TARASOVA, 0. S., VAKULINA, T . P., KOSHELEV, V. B., PINELIS, V. G. & MARKOV, CH. M. 1992. Vasoconstrictor reactions in SHR versus WKY can be

increased or decreased depending on the conditions of perfusion. Acta Physiol Scund 146, 185-196. Received 19 July 1991, accepted 9 March 1992. ISSN 0001-6772. Department of Human and Animal Physiology, Faculty of Biology, Moscow State University, Russia. The reactions of resistance vessels in SHR and WKY hindquarters were compared during saline or blood perfusion. During saline constant-flow perfusion at all initial pressures (80-200 mmHg) sympathetic vasoconstrictor effects were greater in SHR than those in WKY. During perfusion at constant and equal pressure vasoconstrictor responses were greater in SHR vs. WKY only at high pressure - 200 mmHg. On the other hand, under constant pressure conditions at lower pressures (80 and 120 mmHg) sympathetic stimulation induced weaker responses in SHR than in WKY, which at, for example, 80 mmHg was the case at every frequency of sympathetic stimulation used (2-20 Hz). Also, the responses to exogenous noradrenaline and vasopressin occurred during perfusion at low (80 mmHg) and for both equal constant-pressure conditions lower in SHR than in WKY. Comparison of sympathetic effects in SHR and WKY during blood hindquarter perfusion revealed similar results. Also, when SHR and WKY responses were compared at their ordinary levels of constant-pressure, sympathetic vasoconstrictor effects in SHR were lower than those in WKY.

Key mords : constant flow perfusion, constant pressure perfusion, noradrenaline or vasopressin vasoconstriction, sympathetic stimulation, hypertensive resistance vessels.

T h e vascular bed of hypertensive animals and humans is characterized by a greater resistance at maximal vasodilatation (Folkow 1956, 1982, Sivertsson 1970). Further, experiments both in rats (Folkow et al. 1970) and in human vascular beds in vivo (Sivertsson 1970) show exaggerated resistance responses in hypertensives at largely unchanged sensitivity to, for example, noradrenCorrespondence: Ivan M. Rodionov, Department of Human and Animal Physiology, Faculty of Biology, Moscow State University, Moscow, 119899 Russia.

aline. However, when expressed as regional flow reductions, these were approximately the same in normotensives and hypertensives, despite 40-45 yohigher perfusion pressures in the latter. Folkow hypothesized that these facts can be explained by assuming that the quantity of smooth muscle in the resistance vessels is increased, with the resulting thicker media enroaching on the lumen. These changes in morphology represent an adaptive response to increased loading caused by an elevated blood pressure, whereas the narrowed lumen implies

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an increased minimal resistance and the thickened muscle la!-er results in exaggerated resistance responses for a given smooth muscle shortening (Folkoit- 1982). During plasma substitute constant-flow perfusion of rat hindquarter vessels, vasoconstrictor responses to exogenous noradrenaline in S H R were more pronounced than those in 11-KY (Folkow et a/.1970, Hallback et a l . 1976, Folkow & Karlstrom 1984). Likewise, a t a constant floii the constrictor responses of mesenteric vessels to sympathetic stimulation were greater in SHK than those in WKE- (Tsuda rt a / . 1984, Longhurst et N / . 1986). Similar data were obtained from 111 i.irr.o investigations on isolated, isometrically contracting mesenteric small arteries from SHK and \\.!&I’ (lumen of 150-200 p m ) (3Iulvany cf 01. 1978). T h e SHR vessels had a lio, smallt-r diameter and a .iOo, thicker media than the KKl- ones, and the wall tension increase in response to noradrenaline was increased in S H R in proportion to the media thickening. €Iowever, when the periarterial vasoconstrictor nerves were stimulated in SHR and \$-KY, similar differences were seen (Nilsson & Folkow 1982). Such observations led to the concept that responsiveness to vasoconstrictor influences in SHR is higher than in If’Kk’. Hoxeever, experimental protocols based on constant flow perfusion or isometric contraction of a vessel segment, differ markedly from the iv rizo conditions of vasomotor responses, like the results mentioned in man (Sivertsson 197O‘i, where the vasoconstrictor responses led to corresponding flow reductions. Lutz & H e n r i c i (1973) noted significant differences in vasomotor reactions as a function of the perfusion protocol. .\lathematical analysis of constrictor responses of 1-essels with different u-all stiffness suggest that under different perfusion conditions the responses may v a n , not onl!- quantitatir-el!, but also qualitatively (Nikitin & Khayutin 1962). Thus, in experimental conditions other than constant-flow perfusion vasoconstrictor responses in S H R are often not greater than in LVKE’. For example, during autoperfusion of rat hindquarter vessels at their prevailing pressures, i.e. under largely constant pressure conditions, responsiveness to intra-arterialll- administered noradrenaline did not differ in SHR and WKY (1Verber &. Fink 1985), while sympathetic vasoconstrictor effects were lower in S H R than in II’Kk-. Furthermore, at similar experimental

conditions renal vasoconstriction to both noradrenaline and sympathetic stimulation was lower in SHR (Fink & Brody 1979). Moreover, on intravenous adminstration of noradrenaline to conscious rats (after hexamethonium blockade of refles circulatory modulations) Doppler recordings of hindquarter blood flow showed less pronounced resistance increases in S H R than in \ i K Y (Touw e t ( I / . 1980). I t is therefore possible that SHR vessels may be less responsive during e.g. constant-pressure conditions, than that which appears to be the case in human hypertensive vessels, to judge e.g. from Sivertsson’s study (1970). T h i s implies that vascular responsiveness in hypertension still remains inadequate]!- understood, where species differences as well as differences in wall distensibility, extent of muscle shortening, muscle pre-stretch and, sensitivity to tissue vasodilator factors ma) all he of importance. T h e purpose of the present investigation was to stud!-, on a comparative basis, Yascular responses to constrictor effects in SHR and b’KY during constant-pressure and constantflow conditions, using: (a) saline; or (b) blood perfusion.

M E T H 0D S Experiments were performed on spontaneously hypertensive rats (SHR), aged 4-7 months, using normotensire W s t a r Kyoto (LVKY) rats as controls. Prior to the experiments, blood pressure (BP) in conscious animals was measured by the tail-cuff method. In all SHRs BP was higher than 150 mmHg. Salrtir prtjiision e.vper.inrnts Proi.rJurr. The rats were anaesthetized with nembutal (40 mg kg i.p.). Abdominal aorta and caudal cai-a1vein were exposed by midline laparotomy and cannulated into the distal direction 0.5 a n below the bifurcation of the left renal artery. The intestine n-as excluded from perfusion using t\i o tight ligatures around the rectum and duodenum. Arteries and veins supplying the proximal hindquarter parts were also ligated. The hindquarter vessels were perfused m-ith Tyode’s solution containing (in mhi) : KaCl 136.9, KCI 2.68, CaCI, 1.80, MgCI, 1.05, NaHCO, 11.90, XaH,PO, 0.42, and D-glucose 5 . 5 5 . The perfusate was warmed to 3’7 “C, pH was 7.4. Cotistatit pressuue prrfusion was performed, using a container in which the uerfusate was pressurized bv air. Perfusate outflow was measured from the caudal

Vascular reactivity in SHR versus W K Y caval vein by a photo-electric drop recordings, writing on a polygraph. Calibration was obtained by plotting outflow volume against drop frequency. Constant jow perfusion was performed by means of a peristaltic pump (LKB, Sweden). Perfusion pressure was measured at the inlet to the arterial cannula, using a Statham P23AA pressure transducer and recorded on a polygraph (Statham Instr. Inc., Oxnard, CA,

5r

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USA). Experiments with Tyrode's solution as perfusate could be carried out within 25-30 min, which excluded significant edema formation. Vascular responsiveness was investigated after blood was washed out completely. Constrictor responses were induced either by sympathetic stimulation or by vasoactive drugs added to the perfusion solution. Both lumbar sympathetic nerves were centrally blocked by a ligature at the L,-L, level and the distal ends were placed on bipolar silver electrodes at the L,-L, level. During the experiment the nerves were kept moist. Stimulation of 10 s in duration with a supramaximal voltage of 6 V, 0.5 ms stimulus duration and different frequencies (2-20 Hz) was used. T h e intervals between stimulations were 3-4 min. Noradrenaline (Noradrenaline bitartrate, Serva, Germany) and arginine-vasopressin (Serva, Germany) were used as vasoconstrictor agents. Calculations of data. T h e vasoconstrictor responses were evaluated as follows: RR;' = PP;' ( in the case of constant-flow perfusion) or RRil = QQ-' (in the case of constant-pressure perfusion), where R,, Po and Q are the initial values of resistance, perfusion pressure and flow rate, while R, P and Q represent the same parameters at the peak vasoconstriction. Protocol. I n the first experimental series the sympathetic nerve effects on S H R and WKY vessels were investigated at a constant, and for both equal, pressure of 80 mmHg and at different levels of initial tone. A 'standard' stimulation frequency of 20 Hz was used, firstly when the vessels were maximally dilated and then at increased levels of noradrenaline constriction. Increasing doses of noradrenaline, g ml-l, were for this ranging from 2 x lo-' to 1 x purpose added to the perfusate and each subsequent sympathetic stimulation was performed at a raised level of initial tone (i.e. at a reduced flow rate). Previous experiments in normotensive rats have shown that when the initial vascular tone was elevated in a stepwise fashion, the sympathetic effects are at first increased but then decreased (Rodionov et al. 1981). Maximal vasoconstrictor response occurred when the initial flow was reduced to half of that during maximal dilatation, also observed in the present study in both SHR and WKY (Fig. 1) However, such a tone called for a higher noradrenaline concentration in SHR than in WKY (4 x lo-' vs. 2 x lo-' g ml-'), flow being always lower in SHR for equal pressures. During saline perfusion vessels are fully dilated, at 7

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"

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Flow r a t e (ml min-' 1OOg-' ) Fig. 1. Constrictor responses of hindquarter vessel elicited by lumbar sympathetic nerves stimulation (6 V, 0.5 ms, 20 Hz for 10 s) in S H R (+---) and WKY (-----) during saline perfusion at constant pressure of 80mmHg as a function of perfusate flow rate prior to stimulation. Responses were evaluated as peak resistance during the stimulation (R)in relation to initial resistance (R,). Righthand points of both curves indicates the sympathetic effects at maximal vasodilatation. Subsequent stimulations were performed after the addition of graded g ml-l). doses of noradrenaline ( 2 x 10-'-1 x Vertical and horizontal bars show SEM. Constrictor responses were most pronounced at noradrenaline g ml-' and 4 x lo-' g ml-' in WKY doses of 2 x and SHR respectively.

least at lower pressure levels (Folkow et al. 1970, Clark et al. 1978) while in vivo they have an intrinsic tone. Therefore, the present study of sympathetic nerve effects towards a background of artificially raised initial tone was intended to mimic more effectively the in vivo situation. I n the second series, sympathetic stimulation with different frequencies was performed at a constant and for both SHR and WKY equal pressure of 80 mmHg. T o induce an initial, and for both largely equal, vascular tone, the perfusate was enriched with noradrenaline ( 2 x lo-' g ml-' for WKY and 4 x lo-' g ml-' for SHR) or vasopressin (1 x lo-' g ml-l for WKY and 4 x lo-' g ml-' in SHR). These concentrations reduced initial flow to about half, in which state sympathetic effects were most pronounced in both SHR and WKY. Also vasopressin was used because its action on sympathetic neurotransmission is different from that of noradrenaline (Langer 1980, Pate1 & Schmid 1988). Ten-s periods of sympathetic stimulation were used at frequencies of 2, 5 , 10, 15 or 20 Hz. T o determine whether the greater initial resistance (R,) in S H R could account for the lower response value (RR;'), initial vascular resistance was elevated in some WKY to the SHR level by partial plugging of ACT 146

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precapillary \-essels by i.,i. injection of 1W0/tm microspheres (Superfine Sephadex G-100). Then n-as gi\-en, reducing noradrenaline, 2 x lo-' g mi f l o to ~ about half, aftcr vrhich the responses to graded s!-mpathetic stimulation could be recorded. In the third series, vasoconstrictor responses to graded doses of noradrenaline or vasopressin \\ere recorded during constant pressure perfusion a t 80 mml-Ig in both the SHR and \ W Y hindquarter Lessels. Mter 5. min perfusion with 'Tyrode's solution, the J asoconstrictor effects of noradrenaline (dose range Z x lo-' to 1 x 10 g nil.') or vasopressin (dosc range 4 x 10 "' to 1 x 10-.* g mi-') were recorded to provide the cumulative dose-effect relationships. In the f o u r t h series, SHK and \l-KT hindquarter vessels were alternatingly perfused a t either constantpressure or consrant-flow, allot\ ing an adequate comparison of the t\vo perfusion patterns concerning sympathetic constrictor responses in the same pair of SHR and i\.KY vascular beds. 'I'o obtain optimal responses (see Fig. 1) initial tone \vas raised b!- nora.drenaline ( 2 x 10.' g ml-' for l\-Kl- and 4 x lo-' 5; nil ' for SHR). 1-asoconstrictor responses wert. elicited b! 'standard ' sympathetic stimulation a t c frequenc! of 20 Hz, first during constant-pressure (80 mniHg) perfusion and then, by means of tht. pump, at constant-flon perfusion, setting out frorr 80 mmHg. SubsequentlJ- vasoconstrictor responses nere studied also a t perfusion pressures of 120, 160 and 200 nimHg. first during constant-pressure and then during constant-flow conditions. Bled ptv7'i/3rot7 r \perrme~rt.z.Esperimental animals mere anaesthetized nith nenibutal (50 mg lig-' i.p.). To pre\ent clotting, heparin was injected (1000 L kg i.m.). Rectal temperature \\as maintained ;It -- c . I / C: s i t h a heated pacl. .hteriai pressure n a 5 measured through a catheter (PEIO) in the left femoral artery with a DD.1-2 (Russia) pressure transducer. After midline laparotom! the \-iscera were ciellectcd and covered with 1%arm saline-soaked gauze 10 minimize cleh!dration. The right hindlinih was perfused 117 c r t i r mith an extracorporeal flow circuit and a peristaltic pump. T h e left common carotid arter!and the right common iiiac arter?. were inserted nith J poiyethylene catheter (PE.iO) and a cannula (of lonintrinsic resistance), respective1:-, \\ hich \\ ere in turn connected to an extracorporeal circuit. T h e internal iliac arter! was ligated, and hence, perfusion involt-ed onl! the vascular bed supplied by the external iliac a r t c n , I he wlunie of cstracorporeal circuit \\-as 'ibout 2 nil, and it \\-as prior to the stud! filled nith heparinized ( 5 0 U m l ~') isotonic saline. During r'o,istat2t-prESsuI'P perfusion, pressure n a s stabilized b y a feedback system consisting ofa Statham P23.4.1 pressure transducer, a PP-111 peristaltic pump, and an electronic system to control the pump so as to keep a constant perfusion pressure. l'ascular resi5tanc.e \\-a\ derii-cd from the blood flow rate ? .

measured by a 1 mm ID electromagnetic flowmeter probe, Statham P 2022, attached to the common iliac arter!- just distal to the cannula. Both lumbar sympathetic ner\-es were prepared as described above and vasoconstrictor responses were elicited by sympathetic stimulation (6 \;, 0.5 ms, duration for 10 s) at three constant pressure levels, 80, 120 and 160 mmHg. .it 120 and 160 mmHg nerves were stimulated only at 20 Hz, hut at 80 mmHg at 2, 5, 10 and 20 Hz. In another group of rats, sympathetic effects were investigated during constant-flow perfusion when the control s!-stern was switched off and the pump output kept constant. T h e floa- was so adjusted as to produce a perfusion pressure prior to stimulation of 80, 120 and 160 mmHg. Sympathetic stimulation w a s here performed only at the frequency of 20Hz. The Iasoconstrictor responses were evaluated as RR;' during both constant-pressure and constant-flow conditions. Starrstics. .Ill data are expressed as mean values SEM. Single group comparisons between SHR and WKY were performed using Student's unpaired t-test.

R E S U L'T S Saliiie p c r j i i s i o ~

-4st h e efficacy of sympathetic influences partly depends on prevailing vascular tone (Bohlen & Lobach 1978, Rodionov rt nl. 1981), the wsoconstrictor responses to sympathetic stimulation were in this series measured as a function of the initial tone in 10 SHR (UP = 165.1 k .i.OmmIIg) and 13 WKY (BP = 112.2+ 7.0 mmElg) rats, aged 6.5-7 months. A t maximal u s o d d a t a t i o n flow was 3900 lower in SHR t h a n in K K Y (Fig. l), reflecting t h e increase of the structurally raised minimal resistance in S H R . D u r i n g 80 mmHg constant-pressure perfusion in both SHR and WKY sympathetic influences were in SHR if anything lower t h a n in WKY. I t should be emphasized that t h e sympathetic effect reached a maximum, when the initial flow was reduced t o about half in both WKY a n d SHR. This occurred a t a noradrenaline concentration of 2 x 1 0 ' g m1-l in WKY b u t in SHR a t 4 x lo-' g rn1-l. X'hen t h e vessels were maximally dilated, the vasoconstrictor responses in SHR a n d WKY were roughly similar. When, howel-er, the constrictor responses were maximal (at f l o w of 38.0+2.9 and 25.0k3.8 ml min-' 100 p-l for IVKY a n d SI-IR respectively) the s!-mpathetic effccts were significantly lower in SfIR than in WKY.

Vascular reactivity in SHR versus W K Y

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Fig. 2. Constrictor responses of hindquarter vessels in SHR WKY (--- 0---)) and WKY with vascular bed being rarified by microspheres administration to elevate resistance at maximal dilatation to the level of SHR (dashed line, filled symbols) elicited by sympathetic stimulation (6 V, 0.5 ms for 10 s) with frequencies of 2, 5 , 10, 15 and 20 Hz during saline perfusion at constant pressure of 80 mmHg. T o study the most pronounced sympathetic effects (See Fig. 1) noradrenaline (a) or vasopressin (b) was added in doses required to reduce flow rate two-fold as compared to maximal vasodilatation. Flow rates in SHR and WKY at maximal dilatation and prior to stimulation are listed in Table 1. Responses were evaluated as in Figure 1. Vertical bars represent SEM where this exceeds the size of symbol. Irrespective of the tone-inducing agent sympathetic effects in SHR were lower than in WKY and ' WKY-rarified' at every frequency of stimulation used. * P < 0.05, ** P < 0.01 vs. WKY and WKY-rarified.

T h e lower nervous vasoconstrictor effects in S H R than in WKY, when constant-pressure perfusion was performed at equal pressures (see above), occurred also at various stimulation frequencies and independently of the agent inducing the initial tone, as shown in Figures 2(a) and 2(b). I n Figure 2(a) the perfusate contained noradrenaline (2 x g ml-' for W K Y and 4 x 10-7gml-1 for S H R ) and in Figure 2(b) vasopressin (1 x lo-' g ml-I for W K Y and 4 x lo-' g ml-' for SHR), which in both W K Y and S H R reduced initial flow by about SO%, all flow values being listed in Table 1. Also here sympathetic effects were most pronounced in both WKY and SHR when initial conductance was reduced to half (See Fig. 1). It should be noted further that the structural component of resistance in S H R versus W K Y was smaller here than in series AI, perhaps because these rats were younger (4-4.5 vs.

6.5-7 month). However, irrespective of the toneinducing agent, the sympathetic vasoconstrictor responses were always smaller in S H R than in

WKY. Another experimental group included the W K Y subgroup where the vascular bed had been ' rarified ' by microsphere injections to reach about the same minimal resistance as in S H R (Table 1). Graded microplugging of vessels was used also in the study of Hallback et al. (1967) with the only exception that microspheres were then 50pm in diameter. After noradrenaline addition, 2 x lo-' g ml-l, the initial flow rate in these rats was similar to S H R (Table 1). Regardless of identical Q, (i.e. R,,), Figure 2 a shows that the responses to graded sympathetic stimulation (RR;') remained stronger in 'WKYrarified' rats than in S H R . Figure 3 shows how also the vasoconstrictor responses to noradrenaline and vasopressin were 7-2

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Table 1. Perfusate f l o ~rates in SHR, WKT and WKY v;ith vascular bed being rarified by microsphere (' N'KY-rarified ') during saline perfusion of hindquarter vessels a t constant pressure of 80 mmHg. Values for maximal vasodilation (perfusate without \-asoconstrictor agents) and prior to sympathetic stimulation (after noradrenaline or vasopressin addition) are given. Values are mean k SEhl

Flow rates (ml min-' 100 g-*) At maximal

Doses of drugs (g m1-l)

Alnimal groups

(a) Experiments with noradrenaline-induced tone 4 x lo-; SHR ( n = .i) iVKY (n = 11) 2 x lo-; 2 x lo-; LVKT-rarified ( n = 10) (b) Experiments xith vasopressin-induced tone .Ix 10-9 SHR ( 1 2 = 5 ) IVKY ?? = 12) 1x

vasodilatation

Prior to stimulation

84.4k3.6" 94.8 k 2.3 81.8 k 3 . 2 t t

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Fig. 3. Log cumulative concentration-response curx-es for noradrenaline (a) and vasopressin (b) in SHR and N-KY (---O----) rats during saline hindquarter perfusion at constant pressure of 80 mmHg. Responses were evaluated as in Figure 1. ,4t maximal dilatation flow rates in SHR and N'KY were respectively 61.7k2.9 ml min-' 100 g-' and 90.4+5.3 ml min-' 100 g-' in (a), jl.ik2.0 mi min-' 100 g-' and 91.4k6.9 ml min-' 100 g-' in (b). Vertical bars show SEM where this exceeds the size of the symbol. The curves show that for both constrictor agents responses in SHR are lower as compared to the ones in WKY. * P < 0.05, # * P < 0.01 vs. WKY.

(pa----)

weaker in SHR than in WKY, when perfused at equal constant pressure (80 mmHg). T h u s , the dose-resistance response curves for SHR are less steep than those for WKY concerning both

noradrenaline and vasopressin. These rats were 7 months old, probably explaining the greater differences in minimal resistance between SHR and FVKY than in ,411 (see legend to Fig. 3).

Vascular reactivity in SHR versus W K Y Table 2. Perfusate flow rates at various perfusion pressures in SHR and WKY during saline perfusion of hindquarter vessels

191

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R

Flow rates (ml min-' 100 g-') Perfusion pressure (mmHg)

WKY (n = 17)

SHR (n = 11)

At maximal vasodilatation 80 94.3k2.8 77.6f2.5 After noradrenaline addition 80 49.5k3.1 38.0f4.0 120 61.0k4.6 49.9f4.8 160 74.3k2.7 60.li3.0 200 88.6k4.1 72.9k6.2

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Intermittent constant pressure/constant flow perfusion of the same vascular beds was performed. For constant flow conditions values of pressure prior to stimulation are given. In individual rats at every level of pressure flow rates at constant flow and constant pressure conditions were similar. Doses of noradrenaline added were 2 x lOW and 4 x lo-' gml-' in WKY and SHR respectively. Values show mean f SEM. P-Values show significance of differences between SHR and WKY values. ns indicates P > 0.05. Table 3. Blood flow rates at various levels of perfusion pressure in SHR and WKY rats during constant-pressure perfusion of hindlimb vessels. Values given as meaniSEM. P-Values show significance of differences between SHR and WKY values. ns indicates P > 0.05 Blood flow rates (ml min-' 100 g-l) Perfusion pressure (mmHg)

WKY (n = 16)

SHR (n = 8)

P

80 120 160

6.5 k-0.8 12.9+ 1.8 26.6k2.6

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In this series, the resistance increases to standardized (20 Hz) sympathetic stimulation were compared in 5-month-old SHR and W K Y at both constant-pressure and constant-flow perfusions. Initial vascular tone was, as described above, modestly elevated by noradrenaline to attain optimal vasoconstrictor responses to nerve stimulation. These responses were compared at

80

120

160

Perfusion pressure ( m m Hg) Fig. 4. Constrictor responses of hindquarter vessels elicited by lumbar sympathetic nerves stimulation (6 V, 0.5 ms, 20 Hz for 10 s) in SHR ( 0 )and WKY (0) rats during blood constant pressure (-) and constant flow (----) perfusion. Constant flow and constant pressure experiments involved different rats. Pressure was stabilized at 80, 120 and 160mmHg. Blood flow rates prior to stimulation in SHR and WKY are listed in Table 3. At constant flow pressures prior to stimulation being as above. Flow rates in these rats were roughly similar to the ones presented in Table 3. Responses were evaluated as in Figure 1. Vertical bars show SEM where this exceeds the size of the symbol. Sympathetic effects in SHR as compared to WKY were greater at constant flow but lower at constant pressure of 80 mmHg. 'P < 0.05, *+*P< 0.001 vs. WKY.

four constant pressure values, viz. 80, 120, 160 and 200 mmHg and a t four constant flow rates which were so selected as to maintain initial perfusion pressures at about the same four levels of 80, 120, 160 or 200mmHg, which implied about 25% higher flow levels in WKY than in SHR (Table 2). At these fairly high initial pressures Figure 6 shows that the sympathetic effects were greater at constant-pressure perfusion in both SHR and WKY, although the constant-pressure responses declined at higher pressures, particularly in WKY. Thus, while the sympathetic effects were greater in W K Y than in SHR at 80 and 1 2 0 m m H g constant-pressure perfusion, they were equal at 160 m m H g but at 2 0 0 m m H g greater in SHR than in WKY. However, during constant flow perfusion sympathetic effects were greater in SHR a t every

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Frequency (Hz) Fig. 5 . \-ascumstrictor responses as a function of the fricquenc!- of sxmpathetic nerve stimulation in SHK ([email protected]) and \VKI- (----O---) rats during hlood perfusion at constant pressure of 80 mmHg. For blood tl0\1 rates prior to stirnulation in SHR and \i’K\- see Table 3 . Responses were e\-aluated as in Figure 1. Ycrtical bars shon. SEXI. * P < 0.05 \ii. \VKY

initial pressure level. It follows that the relationship between sympathetic effects a t constant-pressure and constant-flow perfusions can change eren in opposite directions in the same SHR and U’KY depending on the perfusion conditions. Blood /wr/iisioti

Qualitativel!- similar results were obtained during blood perfusion of hindlimb 1-essels. Comtarit ptrssiiw perfiisioti ini-olved eight SHR (BP =158.3k2.8 mmHg) and 16 WKY (BP == 115.4+_1.9 mmHg) rats, the animals being 5 months old. .After anaesthesia and laparotomy blood pressure fell to 117.4 i2.4 mmHg in SHR and 83.0k3.8 mmHg in K K Y ( P < 0.001). Regional hindlimb resistance was significantlv higher in SHR than WKY at all pressure levels studied (80, 120 and 160 mmHg) as shown by 30--40°,, lower flow rates in S H R (Table 3). Figure 1 shows the relationship between sympathetic resistance effects at 20 Hz frequencjand the pressure level. ,4s during saline perfusion, the sympathetic constrictor responses were more pronounced in WKY than in SHR when both

were perfused a t 80 mmHg. they were about equal a t 120 mmHg but tended to be greater in SHR a t 160 mmHg. When different frequencies of stimulation were used during 80 mmHg constant-pressure perfusion the vasoconstrictor efTects were lower in SHR than in WKY (Fig. 5) throughout. \$-hen, however, the hindlimb vessels were blood perfused at a constantftow in five SHR and 10 WKY (BP = 167.2 k 1.4 mmHg and 118.5 It 2 . 2 nimHg, respectively), and ‘standard’ sympathetic stimulation of 20 Hz was used, the S H R rcsponses were clearly stronger than in WKY at e\-ery initial pressure level (Fig. 4). Since the initial pressure levels were so high (80, 120 and 160 mmHg) the resistance increases during constant-flow perfusion were limited in extent by the high pressures reached during constriction. It can be deduced from Figure 4 that ar (for example) 120 mmHg initial pressure the SHR vasoconstriction reached about 300 mmHg versus about 220 mmHg in WKY.

DISCUSSION The above observations allow the conclusion that the pattern of perfusion as well as the initial levels of pressure and flow, must all be taken into consideration, when comparing vasoconstrictor responses in SIIR and WKY rats. During constant-flow perfusion vascular responsiveness in SHR is higher than that in WKY. Elevated vasoconstrictor effects in S H R during perfusion .at a constant flow were described previously (Folkow rt a / . 1970, Longhurst et al. 1986). Enhanced vasoconstriction can he regarded as the result of an increase in the wall thickness : inner diameter ratio (Folkow 1982). Enhanced resistance responses have been observed also in rice when hand blood flow was compared in human hypertensive and normotensive subjects, though initial flow levels were then equal while perfusion and transmural pressures were some 400,; higher in the hypertensives (Sivertsson 1970, Folkow 1990). rilso the present experiments revealed that during constant-pressure conditions the relationship between constrictor responses in SHR and WKY varies depending on perfusion pressure. During both saline and blood perfusion a t low and both equal constant-pressure conditions (80 and 120 mmHg) vascular responsiveness was lower in SHR than in WKY but with a clear

V m d w reactinity in S H R versus W K Y T

6 -

7

5 -

0

?

4 -

CG

3 -

2 -

lt

-

0 80 120 160 200 Perfusion pressure (mm Hg)

Fig. 6. Constrictor responses of hindquarter vessels elicited by sympathetic stimulation (6 V, 0.5 ms, 20 Hz for 10 s) in SHR ( 0 )and WKY (0) rats during and intermittent saline constant pressure (-) constant flow (---) perfusion. Perfusion pressure was stabilized at the levels of 80, 120, 160 and 200 mmHg. At constant flow pressures prior to stimulation being as above. T o investigate most pronounced sympathetic effects (See Fig. 1) noradrenaline was added to perfusate in the doses of 2 x g ml-' and 4x g ml-l for WKY and SHR respectively. Flow rates prior to stimulation in SHR and WKY are listed in Table 2. Responses were evaluated as in Figure 1. Vertical bars show SEM where this exceeds the size of the symbol. Sympathetic effects were greater in SHR as compared to those in WKY at constant flow and at constant pressure of 200 mmHg but lower at constant pressures of 80 and 120 mmHg. " P < 0.05, ""P < 0.01 vs. WKY. trend towards reversed relationships at higher, though still for both equal pressures (160 and 200 mmHg) coinciding with typically more pronounced responses of SHR than WKY at constant-flow perfusion (Fig. 6) Thus, at low and for both SHR and WKY equal levels of constant pressure (80 mmHg) the reactivity of the SHR vessels was lower than that of WKY, not only in response to sympathetic stimulation, but also to exogenous noradrenaline and vasopressin (Fig. 3). This indicates that the weaker SHR responses are determined by morpho-functional features of the resistance vessels per se, rather than by the sympathetic neurotransmission. With respect to the present perfusion conditions, perfusion with Tyrode's solution was used

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in most experiments, which has several advantages but also has drawbacks. For example, artificial media are hardly able to provide adequate tissue oxygenation, and to establish some initial vascular tone exogenous noradrenaline had to be added. Next about twice the amount of exogenous noradrenaline had to be given to SHR compared with WKY to raise initial resistance twofold that at maximal vasodilatation (Table 1). This indicates per se that several of the above mentioned factors are influencing the situation. However, this method was used for the following reasons. It is at these concentrations of both noradrenaline and vasopressin and consequent levels of initial tone that sympathetic vasoconstrictor responses have been shown to be most pronounced in SHR and WKY. Moreover, it is known that, in hypertension, basal resistance is elevated largely proportionally to that of maximal vasodilatation (Sivertsson 1970, Folkow 1982). Since numerous vasoactive agents affect the release of endogenous noradrenaline on sympathetic stimulation, it appeared necessary to use at least two agents to elicit an initial vascular tone. It is known that noradrenaline inhibits the adrenergic transmitter release (Langer 1980). On the contrary, vasopressin potentiates sympathetic vascular effects (Pate1 & Schmid 1988). Presumably, this explains why in both SHR and WKY sympathetic nerve effects were somewhat greater in the presence of vasopressin than of noradrenaline (Fig. 2). However, during constant-pressure conditions the sympathetic effects were lower in SHR than in WKY irrespective of the tone-inducing agent. T o investigate vascular responsiveness at natural tone, produced by arteriolar myogenic activity (Johansson 1989) and, to some extent, by blood-borne constrictor agents blood perfusion experiments were performed. Their results were qualitatively similar to the ones during saline perfusion. Vasoconstrictor effects at constantflow perfusion were greater in SHR than in WKY, while the reverse was true at constant pressure perfusion, at least at the fairly low level of 80 mmHg (Figs 4 & 5 ) . There are here several factors to consider, besides aspects like differences in inner radius, wall/lumen ratio and in wall stiffness, as studied by Folkow & Karlstrom (1984). For example, at equal and low pressures the medial smooth muscle pre-stretch must be lower in the stiffer

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I . .21. Rodtonor et al.

Sl IK resistance vessels, which all other factors being equal should reduce muscle responsiveness. Furthermore, due to the higher initial resistance in SHK, regional flon. lel-els are lo\ver a t equal pressures, n-ith less wash-out of tissue \-asodilator Factors. -4 h e r degree of pre-stretch concentration and higher local concentrations of tissue vasodilator influences for the SHR resistance vessels are eliminated during constantflow conditions which, however. introduce other o of deviations from ordinar!. 111 r ~ conditions resistance \ ascular function. On the other hand, such experimentally \-ariable conditions for Iaccular performance are needed to analyse the highi!- coniplek interactions of the many influences determining the function of norrnotensi.b-e and h! pertensiw resistance \-essels. In the end the! arc‘ adapted to function a t different ler-els of perfusion and transmural pressures, which ucuall! implies that the!- are equally distended at the same time as prevailing blood flom are the same. Apart from such aspects of 1-ascular perfimnance p r r se, the question arises a-hether the efficiency of the vasoconstrictor fibre control of thc SHR vessels is about the same as in K K Y . This calls for a discussion of some of the present findings during blood perfusion. In this situation the blood pressure values were llT.4 t_ 2.4 nimlig and S 3 . 0 i 3.8 mmHg in SHR artd \\XI-%respectivelj-, which approximates 120 and 80 rnmHg. In S € f K a t 120 mmHg and in \\.K\. a t 80 mmI-fg initial blood flow \\’ere roughl! similar (Table 3 ) . Standardized s p i pathctic stimulation induced weaker responses in SILK at 120 mmIIg than in lVKY at 80 mniIIg. In the intact SI-iK and K K l - blood pressures nere about 163 and 120 mmIIg, and also a t these ‘constant-pressure levels ’ Figure j shous relativel!. more pronounced constrictor effwts in Li-KY than in SHR. ‘Ihus, at the rcspcctir c in :.im pressure lel-els for which the respecti\ e resistance \-essels are structuralljadapted, the sympathetic nerve responses in SHR Mere weaker than those in KKJ-. This shous that the abow mentioned factors cannot he the sole reason for weaker 1-asoconstrictor responses in SHR sersus M’KY during constant-

constant. ;\long with that, the results of Sivertsson’s (1970) experiments on man revealed exaggerated resistance responses in hypertension to noradrenaline, though the effects of graded sympathetic actij-ations could not be tested. Furthermore, there may of course be differences concerning the extent of altered vascular reacti\it! in essential hypertension in man as compared with spontaneous hypertension in rat, and also concerning the efficiency of sympathetic neri-ous influences. Kh!- does the level of vasoconstrictor effects in SHR, when compared to WKY, depend on perfusion conditions? As mentioned above, the x s s e l deformation and physical condition is clearl!- different, e.g. in terms of the Laplace relationships between pressure, radius and wall thickness and also in terms of thc local chemical environment, when various methods are used to record vascular smooth muscle activity. For example, at constant-flow perfusion tissue chemical environment is constant, but the rise of vascular transmural pressure offers an increasing distension force that counteracts and finally prevents further smooth muscle shortening, though less so the thicker and stronger the media, which also increases wall stiffness (Folkow 8( Karlstrom 1981). At constant-pressure perfusion, on the other hand, the distal decline of transmural pressure at an increasing wall/lumen ratio favours vascular narrowing, though an increasing accumulation of tissue vasodilator factors at the same time attenuates smooth muscle activity. T h e present experiments show that \-ascular smooth muscle in SHR have the capacit!. to develop a greater strength if the rising pressure counteracts vasoconstriction and therefore, deformation becomes limited. This takes place during perfusion at a constant flow xhen initial pressures are fairly high, but also at ver!- high pressures (e.g. 200 mmHg) during constant-pressured conditions. Greater active tension was reported for isolated resistance vessels of S H R when compared with WKY, and largely in proportion to the thicker media (llulvany rt 01. 1978). At the same time, the net ‘isotonic’ shortening ability of vascular smooth muscle during the present conditions of low and for both equal constant pressure conditions (80 ilar data were obtained b!~Fink 8: Broci!- and 120 mmHg) proves to be lower in SHR than (1070) as \sell as U.erber & Fink (1983) with the in \YKl-. exception that these authors did not use ar.? I t is possible that in SHR the strength of spccial equipment to keep perfusion pressure contraction, which in S H R is initially greater ~

Vascular reacticity iz SHR zjersus W K Y than in WKY, may decline more rapidly as the muscle cells shorten. The mentioned changes in contractility may occur if individual smooth muscle cells in SHR become thicker and shorter than in WKY. Simultaneous structural thickening and shortening of smooth muscle cells in arterial vessels has been reported in DOCA-salt hypertensive rats (Friedman et ul. 1971). However, at least in some small arteries of SHR the thicker media seems to reflect muscle hyperplasia rather than hypertrophy (Mulvany et al. 1978, Lee & Smeda 1985, Bund et al. 1990). The shape of individual muscle cells in the resistance vessels was not different in SHR and WKY (Mulvany et al. 1985, Miller et al. 1987). Nevertheless, depressed shortening ability has been reported for caudal arterial smooth muscle from SHR compared with WKY (Packer et ul. 1986). Also, the experiments on isobarically pressurized segments of carotid and caudal arteries revealed that in SHR vasoconstriction is lower than that in WKY (Cox 1981). Another explanation of the above observations, which does not rule out the previous one, can be discussed. It is related to the mechanical properties of the vascular wall, which becomes stiffer along with hypertension (Folkow & Karlstrom 1984). Greater stiffness results in lower compliance and perhaps also a reduced wall deformability during contraction at least in some situations. It is therefore possible that the relatively reduced vasoconstriction in SHR during lower levels of constant pressure perfusion (80 and 120 mmHg), is at least partly a consequence of altered mechanical properties of the thicker vascular walls, though factors like lower pre-stretch and higher local vasodilator concentrations probably contribute. Folkow & Karlstrom (1984), by diagrammatical-mathematical transformation of constant-flow responses into constant-pressure responses, have shown that in SHR vasoconstriction is greater than in WKY not only at constant-flow but, at least in some situations, also at constant-pressure conditions. Therefore these data, at least at first sight, are not in conformity with the present findings. It should be mentioned, however, that the wide-ranging, S-shaped constant-pressure resistance curves in the study by Folkow & Karlstrom (1984) were mathematically derived from the pressure intercepts with the constant-flow curves, which implies that e.g. the counter-regulatory influence

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of tissue vasodilator factors was thereby eliminated. This was intentionally done to reveal the potential power of un-opposed constant-pressure constriction, but normally such factors oppose the vasoconstrictor responses, as in the present study. It is, however, also known that yery intense vasoconstrictor influences during constant-pressure perfusions can, at least transiently, induce even distal vascular closure M ith exceedingly high resistance values (e.g. Burton & Stinson 1960). It2 conclusaon, apart from differences in vascular geometric design, the outcome of vascular responses in SHR versus WKY may vary widely, and even in apparently opposite directions, depending on the perfusion arrangements, prevailing pressure level, the potential power of flow-dependent local vasodilator influences and the intensity of the vasoconstrictor stimuli. The present study demonstrates that vascular reactivity to constrictor influences in SHR as compared to WKY is increased during constantflow conditions but can be decreased during constant-pressure conditions. It is discussed how a variety of local factors in complex interactions, contribute to these apparently opposite patterns of reaction, which may have several consequences. The great strength of contraction, which SHR resistance vessels can develop, facilitates efficient flow regulation at elevated pressure. On the other hand, the attenuated responsiveness to constrictor influences a t low constant pressure in SHR lowers the probability of a drastic flow reduction during e.g. reflex vasoconstriction in connection with blood pressure falls. REFERENCES BOHLEN, G.H. & LOBACH, D. 1978. In cico study of microvascular wall characteristics and resting control in young and mature spontaneouslq. hypertensive rats. Blood Vessels 15, 322-330. BUND,S.J., WEST,K. & HEAGERTY, A.M. 1990. The effect of pressure attenuation upon medial growth in resistance arteries from SH and WKY rats. 3 Hyperteizs 8 (Suppl. 3), S120 (Abstr). BURTON, A.C. & STINSON, R.H. 1960. The measurement of tension in vascular smooth muscle. 3 Physiol, 153, 290-305. CLARK, D.W., JONES, D.H., PHELAN, F.L. & DEVINE, C.E. 1978. Blood pressure and vascular resistance in genetically hypertensive rats treated at birth w-it11 6-hydroxydopamine. Circ Res 43, 293-300.

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-4.P. 1987. Cell and wall morphology of intestinal Cox, R.H. 1981. Hasis for the altered arterial x i 1 1 arterioles from 4- to 6- and 17- to 19-week old mechanics in the spontaneously h!-pertensive rat. if-istar-Kyoto and spontaneously hypertensive rats. H ~ ~ p r r ~ r n s i3,o t 38.5--495. i Hyperrension 9, 59-68. FINK, G. & BRDDY,11.1979. Renal \ascular resistance I-. HAUSEN, P.K. & AALKJAER, C. 1978. and reactivitj- in the spontaneousll- h!-pertensi\-e ~ ~ L - L v . ~ Nhl.J., Direct evidence that the greater contractility of rat. .4tn f Ph,)asio/ 237, F128-Fl38. resistance vessels in spontaneously hypertensive l o ~ . k o a . B. , 19.56. Structural, m!-ogenic, humoral and rats is associated with a narrowed lumen, a thickened nervous factors, controlling peripheral resistance. media and an increased number of smooth muscle I n : Harrington (ed.j fl~;Dotetzsi;.e drugs, pp. cells. Cirr Res 43, 8.5-864. 163--174. Pergamon Press, London. \ ~ L - L v . ~ N I -$1. , J,, BAANDRUP, U. 8i GCNDERSEN, H .J.G. FOLKOW, B. 1982. Physiological aspects of primal!1985. E l idence for hyperplasia in mesenteric h!-pertension. Ph)’siol Rrr 62, 347-504. resistance vessels of spontaneously hypertensive FOLKON-, B. 1990. “Structural factor” in primar!. and rats using a three-dimensional disector. Circ Res 57, secondar!. hypertension. fl)~prrtrtisroti16, 89-101. 794-800. E‘OLKOIV, B., HALLB.ACK, \I., LLNDGRES,I.’ &- ~VEISS, XVIKITIY, L.1.. & KHAYUTIN,V.M. 1962. The theory I,. 1970. Background of increased flw resistance of measurement blood vessels resistance during and \-ascular reactivity in spontaneously hypervasomotor reactions. Fiziol Zhurn. S S S R 48, tensi\ e rats. . ~ L Y U Pli.):srol S