Differences Between the In Vitro Vasoconstrictor Responses of the Tail Artery to Potassium and Norepinephrine Between Spontaneously Hypertensive, Renovascular Hypertensive, and Various Strains of Normotensive Rats

ABDEL-KADERFOUDA, CHRISTINECAPDEVILLE,DANIEL HENRION, NATHALIETHORINTRESCASES,ERIC THORIN, AND JEFFREY ATKINSON

Isolated tail arteries removed from spontaneously hypertensive, renovascular hypertensive, or various strains of normotensive rats were perfused/superfused with norepinephrine or potassium, or subjected to electrical field stimulation. Responses in spontaneously hypertensive and outbred normotensive rat tail artery preparations were similar. Tail artery segments from renovascular hypertensive or normotensive rats of the inbred Wistar-Kyoto strain showed smaller responses to all three stimuli. Thus, in certain in vitro arterial preparations, the apparent increase in vascular reactivity observed when comparing spontaneously hypertensive rats with inbred Wistar-Kyoto rats may be due to a decrease in vascular reactivity in the Wistar-Kyoto rat strain. Rat tail artery; Norepinephrine;

Key Words: SHR; WKY

Potassium; Electrical stimulation;

INTRODUCTION Although the inbred spontaneously hypertensive rat (SHR) has a blood pressure that is higher than that of inbred or outbred normotensive rat strains, this does not imply that peripheral

resistance,

in blood pressure regulation, all normotensive rat strains. the SHR is shared

with

cardiac output,

and the other parameters

involved

will be consistently different in the SHR compared It is possible that a given cardiovascular parameter

some normotensive

rat strains and not with others.

to of

Results

obtained in SHR are often compared with those of the inbred normotensive WistarKyoto (WKY) rat strain (Mulvany, 1983), derived from the same parent stock as the SHR. Genetic traits of the SHR, which are not shared with WKY, may be related or unrelated to hypertension. Clineschmidt et al. (1970) state that “. . . studies utilizing the SHR to analyze of normotensive

From the Laboratoire Biologiques, Address

UniversitC reprint

mechanisms

involved

rats in order

to insure

de Pharmacologic de Nancy

requests

I, Nancy,

to: J. Atkinson,

in hypertension adequate

Cardio-vasculaire,

Facult6

April 1990;

revised

and accepted

Some

des Sciences

strains

authors

have

Pharmaceutiques

et

France. Laboratoire

de Pharmacologic

SciencesPharmaceutiqueset Biologiques, UniversitCde Nancy I, Received

must use multiple

controls.”

Cardio-vasculaire,

5 rue Albert

Lebrun,

Facult6

5400 Nancy,

des

France.

July 1990.

61 Journal of Pharmacological

Methods

0 1991 Elsevier Science Publishing

25, 61-68 (1991) Co., Inc., 655 Avenue of the Americas, New York, NY lWl0

62

A.-K. Fouda et al.

used outbred rat strains (Folkow, 1978) and others have pooled results from inbred (WKY) and outbred Wistar normotensive rats (Webb et al., 1981), as controls. Using the perfused/superfused tail artery as our in vitro model, we studied differences in vasoreactivity between the SHR and various inbred and outbred normotensive strains. We also compared results obtained in SHR with those of renovascular hypertensive, WKY rats (2 kidney, 1 clip model) with a blood pressure similar to that of SHR. As the possibility that hypertension represents an accelerated form of vascular aging has often been raised (Soltis, 1988), changes in vascular reactivity with age were also investigated in one of the normotensive rat strains (Sprague-Dawley). We also describe experiments on the determination of the optimal flow rate and longitudinal tension in the tail artery preparation. MATERIALS AND METHODS Animals Three-mo-old male rats of the following strains inbred SHR, inbred WKY, outbred Wistar (ICo:WI), and outbred Sprague-Dawley rats (ICo: OFA/SD), were purchased from Iffa-Credo SA, L’Abresle, France. Renovascular hypertension was produced in a separate group of 2-mo-old, inbred WKY rats by placing a solid silver clip (gap 0.2 mm) on the left renal artery under ether anesthesia. The right, contralateral kidney was left untouched. Hypertension developed during the following month and tail arteries were removed when the rats were 3 mo old. Tail arteries from a separate group of 12-mo-old, Sprague-Dawley rats (Ice: OFA/SD) were also used. All animals received food and water ad libitum and were subjected to a standard day/night cycle (lights off: 1900-0700). Systolic Arterial Pressure and Heart Rate Measurements Before removal of tail arteries, systolic arterial pressure (SAP, mmHg), and heart rate (HR, bpm) were measured under ether anesthesia using an inflatable cuff around the tail and a microphonic pulse detector. Three recordings were made on two separate occasions with 4-day intervals between the two. Rats were then allowed 4 days to recover. Tail Artery Preparation Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). The proximal 6-cm portion of the tail artery was dissected free, removed from the animal and immediately placed in oxygenated Krebs bicarbonate solution at room temperature. This 6-cm length was then cut into 3 segments of 2 cm, each of which was gently rubbed to remove any coagulated blood sticking to the outer or inner surface. Both ends of the 2-cm segment were cannulated with l-cm lengths of polyethylene tubing of 0.4-mm internal diameter and 0.8-mm external diameter. The proximal end of the segment was connected to a constant flow peristaltic pump system for perfusion of the segment (Fouda et al., 1987). The perfusate issued freely from the open end of the cannula inserted into the distal extremity of the segment and then flowed, under the effect of gravity, over the outer surface of the artery. There were no

SHR: In Vitro Vasoreactivity

significant differences in vasoreactivity to the various stimuli used between the proximal and the distal segments of the 6-cm portion removed. Vasoconstriction was estimated from the rise in perfusion pressure in the constant flow system using a strain gauge pressure transducer (Statham P23D6, Statham Inst., Puerta Rica) connected to a polygraph and placed between the peristaltic pump and the arterial segment. The pressure generated by the basal resistance of the polyethylene tubing plus the arterial segment was subtracted from the increase in perfusion pressure produced by the various stimuli. A weight (0.5 g) was attached to the cannula at the distal end. The artery was maintained in a vertical position, under a 0.5 g longitudinal tension throughout. This passive tension restored the length of the segment to that measured in vivo. The artery was perfused in a proximal to distal direction with oxygenated (95% O2 plus 5% COr?), Krebs bicarbonate containing (in mM) NaCl, 118; KCI, 4.7; MgClz, 1.2; NaH2P04, 1; CaC&, 2.6; NaHC03, 25; and glucose, 11 .I. Propranolol (2 FM) was added to block beta adrenoceptors (Hicks et al., 1984). Cocaine (4 PM) was added to block neuronal reuptake of norepinephrine (Henrion et al., 1989). Preliminary experiments showed that this concentration produced the largest Ieftward shift in the response curve to electrical stimulation in tail arteries of normotensive (ICo:WI) rats. Higher concentrations attenuated responses to electrical stimulation. lndomethacin (2.5 FM) was added to block prostanoid synthesis (Greenberg, 1978; Su et al., 1978). The pH of the perfusate was 7.4 -+ 0.2. The perfusate flowed out of the distal cannula and then superfused the tail artery segment. A period of 20 min elapsed from the beginning of the dissection to the start of the perfusion of the tail artery segment. During the first 15 min, the perfusion rate was 1 mUmin. This was increased in 1 mUmin steps to 4 mUmin over 30 min, and then held constant at this rate for the duration of the experiment (1-2 h). Preliminary experiments in outbred Wistar flCo:Wf, n = IO) rats showed that responses to repeated exposure (every IO min) to a fixed concentration of norepinephrine producing an increase in perfusion pressure of 100 mmHg, remained constant for at least 2 hr. Vasoconstriction was estimated from the peak increase in perfusion pressure (mmHg). Results are given as 1) maximal reactivity = maximal increase in perfusion pressure (mmHg), 2) sensitivity = EDloo mmHg= concentration required to produce a IOO-mmHg increase in perfusion pressure. The EDloo ,,.,r,+rgvalues were used in preference to EDs0 or pDz values because the use of the latter as an indication of sensitivity to a given agonist is theoretically possible only if there are no differences between the maximal responses. This was not the case in our experiments (see Results). As slopes relating vasoconstrictor responses to the log,* [dose] were between 140 to 160 mm Hg, and maximal responses were between 190 and 280 mm Hg, EDso values were within +5-7% of EDloo mmHgvalues. As the minimal (percentage) difference between hypertensive and normotensive rats, which was statistically significant, was 20%‘ the use of either EDs0 or EDloo ,,,,,, ng values does not alter the conclusions regarding the comparisons between hypertensive and normotensive rats, Each of the three separate tail artery segments removed from each rat, were sub-

63

64

A.-K. Fouda et al. jetted

to one of the following

pramaximal

voltage,

electrodes between

0.1-30

placed around the electrodes

carbonate

solution.

three

stimuli.

Electrical

stimulation

Hz for 15 set) was applied

the artery with 4 mm between and the arterial

As reserpine

segment

pretreatment

via two

(0.3 msec,

circular

the rings. Electrical

was by the superfusate

(2.5 mg/kg,

su-

platinum contact Krebs bi-

ip; 18 hr previously)

re-

duces tissue norepinephrine level or high performance liquid chromatography coupled to electrochemical detection (HPLC-EC) to an undetectable level and abolishes responses

of the normotensive

et al., 1987), responses endogenous solved

in

norepinephrine Krebs

fused/superfused centrations

rat tail artery to electrical

to electrical

release.

bicarbonate for 2 min.

of potassium

stimulation Exogenous

containing

Finally,

chloride

10e4

M

(Atkinson

reflect vasoreactivity

norepinephrine

Krebs bicarbonate

(IO-100

field stimulation

presumably

(10 nM-1

ascorbic

acid)

containing

mM, for 2 min, tonicity

to

PM diswas

per-

increasing

con-

kept constant

by

removal of NaCI) was perfused/superfused for 2 min. In preliminary experiments in normotensive rats pretreated with reserpine (see above), the maximal response of the tail artery to potassium

was halved (unpublished

results).

A similar

result was

obtained following perfusion of normotensive rat tail arteries with the alpha adrenoceptor blocker, phentolamine (1 ~.LM) (X. X, unpublished results). Thus, in this preparation, dent

perfusion

mechanisms:

of the smooth curves were

with potassium

liberation

muscle membrane. constructed

produces

of endogenous

vasoconstriction

norepinephrine,

For all three stimuli noncumulative

with a 5-IO-min

quiescent

period

In outbred Wistar (Ico:WI) rats (n = IO), a protocol used excepting that longitudinal tension was induced (n = IO arterial

segments

per weight).

Maximal

dose-response

between

Preliminary Experiment I: Effect of Changes in longitudinal Responses to Electrical Stimulation

0.5, and 1 g were 222 & 13,223

via two indepenand depolarization each stimulus.

Tension on

similar to the one above was by weights of 0.2, 0.5, or 1 g

vasoconstriction

2 14, and 230 ? 17 mmHg,

at weights

respectively.

values were 2.1 ? 0.1, 3.0 + 0.6, and 2.2 + 0.2 Hz, respectively.

of 0.2,

EDjo mmHg

Thus a change

in

the longitudinal tension applied to the artery, produced by varying the weight between 0.2 and 1 g, did not change the vasoconstrictor responses induced by electrical stimulation. As 0.5 g restored the arterial segment to its in vivo length, this weight

was used.

Preliminary Experiment II: Effect of Changes in Flow-rate on Responses to Electrical Stimulation In outbred Wistar (Ico:WI) rats (n = IO), a protocol similar to the one above was used excepting that the flow rate was 2, 4, or 6 mL/min (n = 10 arterial segments per flow-rate). Maximal vasoconstriction at flow-rates of 2, 4, and 6 mL/min were 231 & 20 (range: 42-270, variability 27%), 256 * 24 (range: 65-290, variability 30%), and 233 ? 39 (range: 20-308, variability 53%) mmHg, respectively. Thus a change in flow-rate did not significantly alter the maximal response produced by electrical stimulation. Variability, however, did increase with flow-rate, approximately doubling when increasing from 2 to 6 mUmin. EDloo mmHg values were 6.9 t 1.5, 4.6

SHR: In Vitro Vasoreactivity + 1.0, and 3.1 + 0.4 (p < 0.05 with 2 mL/min) electrical

stimulation

produces

significantly

an intermediate

increased

value

Hz, respectively.

with flow-rate.

for sensitivity

with

Thus sensitivity

A flow-rate

maximal

to

of 4 mUmin

responses

of lower

variability.

Preliminary Experiment III: Effect of Changes in Flow-Rate on Responses to Norepinephrine In outbred

Wistar

(Ico:WI)

sponses to norepinephrine

rats (n = IO), basal pressure

were studied at flow-rates

slightly different

to the one described

rate of 1 mL/min,

basal perfusion

above.

pressure

were vasoconstricted

with norepinephrine

increase

pressure

in perfusion

for 5 min after which norepinephrine Orthogonal

polynomial,

the data obtained;

15 min perfusion

at a flow-

and then arterial

segments

(bolus, lop4 M, 0.1 mL), and the maximal Flow was then increased at flow-rates

and exponential

the best fit was found

to 2 mUmin

and the vasoconstrictor

This was repeated

logarithmic,

re-

using a protocol

Following

pressure

were again recorded.

and vasoconstrictor mUmin

was measured

was recorded.

basal perfusion

of l-9

with

equations

a second

order

response

to

of 3-9 mUmin. were

applied

orthogonal

to

poly-

nomial. The relationship between basal perfusion pressure (y) and flow-rate (x) was y = 30.3 - 1.5x + 0.5x2 (r = 0.99, p < 0.05), and between the maximal increase in perfusion + 12.3x

pressure

following

norepinephrine

responses

(y) at flow-rates

(x) of 4-5

mL/min,

Preliminary Experiment IV: Attenuation Induced Contraction In outbred

Wistar

(Ico:WI)

a flow-rate

mM) or Krebs bicarbonate

predict

of 4 mL/min

values for

was chosen.

similar to the one above was

was perfused at a fixed concentration of 1 ~.LM After 2 min an additional perfusion of carbachol was added at a rate of 0.1 mL/min.

later this additional perfusion was stopped and norepinephrine continued for another 2 min. At the end of the experiment stained

(x) was y = 77.2 plateau

by Carbachol of Norepinephrine-

rats (n = IO), a protocol

used excepting that norepinephrine for 6 min at a flow-rate of 4 mUmin. (10 nM-1

(y) and flow-rate

1.1x2 (r = 0.87, p < 0.05). As both equations

-

using the silver nitrate technique

Two minutes

perfusion alone was the endothelium was

(Abrol et al., 1984). Norepinephrine

alone

produced a vasoconstrictor plateau of 160 + 4 mmHg (pooled results for the first and final 2-min periods which gave similar values). Addition of carbachol at concentrations produced

of less than 1 mM

had no effect.

a 20 + 2% fall to 128

+ 4 mmHg.

The latter, Silver

very high concentration

nitrate

staining

revealed

the

presence of small clusters of endothelial cells. Thus the experiments described below were carried out in the virtual absence of any endothelium-derived relaxant activity and so we can exclude any marked influence of endothelium-derived relaxing factor release on the final vasoconstrictor responses observed. The use of a flow-rate of 4 mUmin and/or of pentobarbital anesthesia (Gerkens, 1987) may explain the absence

of endothelium-derived

relaxant

activity

in our preparation.

Drugs Used The salts and glucose for the preparation of the Krebs bicarbonate solution were purchased from Merck AC, Darmstadt, FRG. Sodium pentobarbital, cocaine hydro-

65

66

A.-K. Fouda et al. chloride,

propranolol

bamylcholine from Sigma, Statistical

hydrochloride,

chloride),

reserpine,

St. Louis, MO.

All solutions

ascorbic

were

acid, carbachol

bitartrate

prepared

were

(car-

purchased

daily.

Methods

Results are given as means Student’s

indomethacin, and norepinephrine

t test. Significance

t

SEM. Significant

is denoted

differences

were

determined

by

*p < 0.05.

as follows:

RESULTS Systolic tensive

arterial

pressure

was higher

rat strains (Table 1). There

the normotensive

no significant

than

differences

in all normo-

in SAP amongst

rat strains. The SAP did not increase with age in Sprague-Dawley

rats. Vasoconstrictor stimulation,

in SHR and in WKY/RH

were

responses

norepinephrine,

of the caudal

and potassium)

artery were

to all three lower

stimuli

(electrical

in rats of the WKY strain

than in all other strains (Table 1). Sensitivity to all three stimuli decreased with age in Sprague-Dawley rats (p < 0.05 for comparison between ICo:OFA/SD of 3 and 12 months

of age).

DISCUSSION Our

results show that

1. vasoconstrictor responses arteries of various outbred 2. tail arteries

of Wistar-Kyoto

vasoconstrictor tensive rats; 3. tail arteries to electrical 4. increasing companied

stimuli

inbred

stimulation

hypertensive

and potassium,

age in one outbred by a decrease

using a perfusion

to those

are less reactive

of the tail

to the various

from the SHR or outbred

WKY rats show a reduced

normosensitivity

and;

normotensive

in sensitivity

in maximal

arise from 1) an increase in sensitivity (Vanhoutte,

rats (WKY)

used than tail arteries

from renovascular

but no change When

of the SHR tail artery are similar normotensive rat strains;

rat strain (Sprague-Dawley)

to all three

vasoconstrictor

is ac-

stimuli

used

responsiveness

may

vasoconstriction.

system

model,

increased

vascular

in receptor affinity and/or number leading to an increase 1980), and/or 2) a modification of postreceptor events, for

example, structural adaptation of the arterial wall, leading to increased reactivity, that is, an increase in maximal responses (Folkow, 1978). Evidence for both of these phenomena has been obtained in the SHR but often in studies where the only control used was the normotensive, inbred WKY. As we indicated in the introduction, this may not be valid. Comparisons with other normotensive rat strains do not show such an increase and these observations

(Clineschmidt et al., 1970; Hicks et al., 1985; present results) raise the possibility that the difference in maximal responses

between SHR and WKY may reflect a defect in vascular function in WKY. In WKY rats rendered hypertensive by unilateral renal artery clipping, exposure

25 23

Ice: OFA/SD 3 mo 12 mo

380-400 550-600

270-290 240-260 270-310 370-390

2 + ? 2

(BPM)

HR N

TA

383 ? 7’ 358 2 8=

10 9

2’ 408 ” 9’ 8 4’ 473 ? 7=sb 8 3= 365 5 Ila 12 4a 395 2 12b 20

147 ? 7a 143 2 6”

187 198 140 142

(MMHC)

SAP

& ? 2 f

IO’ 7a 7a 6’

245*gb 246 2 7’

251 200 198 257

(MMHC)

vc MAX SEN

& * 2 f

0.3 0.5a,b 0.5 0.4

2.4 ? 0.5 4.2 ? 7a,b

2.5 5.0 2.8 3.5

KD,~ornrn~~)

(Hz)

7 8

18 8 11 17

N

TA

262 ? 9’ 281 2 II’

2r~7?7~ 190 ? IO” 226*8” 264 2 106

(MMHC)

MAX

vc

* ? -c f

0.9 1.0 1.5 2.2 4.1 ” 1.4 11.6 5 1.7’

7.5 7.0 6.8 9.9

!Ehornrn~~)

IO-‘M

SEN

TAVC RESP TO NE

8 6

11 11 8 10

N

TA

263215’ 280 ? 15”

268 ? 12’ 210 * 5” 222 ? 7a 25028’

(MMHC)

MAX

vc

2 f ” +

1 I+ 1 1 28?1 32 2 lb

29 33 27 28

Whoornrn~~)

(MM)

SEN

TAVC RESP TO K

Abbreviations: SHR = spontaneously hypertensive rats (3-mo-old). WKY/RH = 2 kidney, 1 clip renovascular WKY rats (3-mo-old). WKY = inbred normotensive rats of the WKY (Wistar) strain (3-mo-old). Ice: WI = outbred normotensive rats of the Ice: WI (Wistar) strain (3-mo-old). Ice: OFA/ SD = outbred normotensive rats of the Ice: OFA’SD (Sprague-Dawley) strain (3- or 12-mo-old). SAP = systolic arterial pressure. HR = heart rate. TA = tail artery. VC max = maximum vasoconstriction. resp = response to. NE = norepinephrine. K = potassium. ES = electrical stimulation. a p < 0.05, t test for independent means with SHR. b Same as a, but with WKY.

37 18 31 47

fo)

N

SHR WKYIRH WKY Ice: WI

BODY

RATS

TAVC RESP TO ES

TABLE 1 Systolic Arterial Pressures, Heart Rates, and Maximal Vasoconstrictor Responses and Sensitivities of Tail Arteries to Electrical Stimulation, Exogenous Norepinephrine, and Potassium, of Hypertensive and Normotensive Rats

68

A.-K. Fouda et al.

to a higher pressure level-albeit for a relatively short period of 1 mo-does not produce any increase in in vitro maximal responses of the tail artery to the various stimuli applied. Furthermore, the sensitivity to stimuli whose vasoconstrictor effect depends wholly (electrical stimulation) or partially (potassium) on endogenous norepinephrine release is reduced. In renovascular hypertensive, outbred Wistar rats, we have previously shown (Henrion et al., 1989) that endogenous norepinephrine release (measured by HPLC-EC) following electrical stimulation of the tail artery is reduced. A similar phenomenon may explain our observations in WKY renovascular hypertensive rats. During aging a different phenomenon presumably explains the decrease in sensitivity to all three stimuli used. The present results confirm those previously reported in aging lvanos rats (Fouda and Atkinson, 1986) and suggest that the efficiency of the postsynaptic coupling mechanisms decreases with age.

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