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The role of endogenous norepinephrine release in potassium-evoked vasoconstriction of the rat tail artery
Potassium-containing solutions arc often used to study the scqucnce of cvcnts lcadinp from cscitation to vati;tronstricri~m.In dcnscly innervated vessels. such as the rat tail artery. potassium-induced vaulconstricticxn may lx mcdiatcd via srn~l~h mus~k depolarization and release of cndo~en~~~~~n~~r~pin~p~rin~. The rclativc ~~~ntr~~ut~#nof thcsc two mcchanismh - a *direct‘ dc~lariz~l~~n of the vascular srn~~b mus& 4 m~mbrao~. and an ‘indirect’ s~rn~a~ht~rnirn~t~c action - to the ~~~c~~~tr~~~~~r response r‘as studied in the present paper. Perfusion/supcrfinsion of the ra? rail artcry in vitro with prttas~ium-containing solutions had different effects depending on Ihe conccntratiun used. A change in potssium cunccntration from 47 to 31 mM had no effect on cithcr perfusion pressure or norcpincphrinc ovvcrku. From 30 to 70 mM. potassium produced inncasing amounts of norcpincphrinc overflow. Experiments with phentolaminc and rcscrpinc shutted that this norcpincphrinc ovcrfk~ contrihutcd for up to half of the vasoconstrictor response observed. A second norepincphrin~-indc~ndcnt mechanism was also involved but the latter appeared lo be incapabk of producing sustained contraption. At conc~~~rat~~~ns of prttassiumahchf %f-?U m&l. the results of experiments with ( I~I~-prop~n~~lol suggest that the norcpincphrjn~ rckascd by pr)tasGum has a @-adreooccptor-mediated vasorclaxant cffcct. Tail artcry: K l; Norcpincphrinc: Phcntolaminc; Propranolol: Rcserpinc:
1. Introduction In vivo, small. physiological increases in the concentration of potassium in the extracellular fluid cause vasodilatation (Vanhoutte et al., 1975). In vitro, however, higher con~ntrations of potassium (> 20 mM) are often used to study the sequence of events leading from excitation to vasoconstriction (Weiss, 1972). An increase in the extracellular concentration of potassium will depolarize the muscle cell membrane and induce calcium influx and/or the release of calcium from membrane or intracellular stores, and thus provoke contraction of vascular smooth muscle (Bolton, 1979). In in vitro preparations of sparsely innervated blood vessels. such as the rat aorta, this mechanism appears to be the main one involved in potassiumevoked contractions. In these preparations, depletion of catecholamines with reserpine or blockade of cy-
Conespondence to: A.-K. Fouda. lnstitut de Pharmacolog~e de Wniversiti de Lausanne. Rue du Bugnon 27. ItNIt) Lausanne. Switzerland. Tel. 33.X3.32.7h.48, fan 33.83.32.3tf.58.
Diltiazem:
(Rat)
adrenoceptors with phentolamine does not modi& the vasoconstrictor response to potassium (Shibata and Carrier, 1967; Garret and Carrier. 1971). In densely innervated vessels. such as the rat tail artery, potassium-induced vasoconstriction can also be mediated via neurogenically released norepinephrine (Polidoro and Savino, 1982; Xiao and Rand, 1991). The relative contribution of these two mechanisms - a ‘direct’ depolarization of the vascular smooth muscle cell membrane. and an ‘indirect’ sympathomimetic action - to the vasoconstrictor response is equivocal. Fouda et al. ( 1986) reported that the cr-adrenoccptor antagonist. phentolamine (4 PM), almost completely abolished the to potassium (50 mMW). vasoconstrictor response whereas Webb et al. (t981) showed that phentolamine (10 p&l) produced only a 50% inhibition of the response to potassium 175 mM). In order to gain insight into the reasons for this apparent discrepancy, we reinvestigated the effects of phentolamine on the potassium-induced vasoconstriction of the normotensive rat tail artery. We compared these results with those obtained after depIetion of norepinephrine by pretreatment with reserpine. &per-
pm__ 1, .. C-1 for aF Icast 1 \wck t-&we each expcrimcnF. Rats wcrc ancsFhetizrd with sodium pcntobarhiFat (50 w,‘kg. i.p.1 anit _;Omin later two 2-cm lengths & Fbc pmxintal pm of Fhc tail artery wcrc removed and immcdiatcty ptaccd in oxygenated Krchs hicarbonaft SC~UF~MI. Rats wcrc Fhcn kittcd with an overdose of so&ma pcntobarbitat. PrcEiminso espcriments showed no significant diffcrcncc?rin \ssorcactivity to cithcr potassium or norcpin&~-& titwccn the two scparatc scgmcnts of the Fait arte~. After gwtlr’ rubbing to remove any coagulated blood sticking to the oufer or inner surface. both ends of each I-cm sgmc‘nr were cannutated with t-cm lengths k%fpotycthytcnc tubing (0.4 mm internal diameter and 0.S mm external diamctrr). The proximal end of the Fait ancry scgmcnt was connected to a constant flow p&.sFa!Fic pump system (Fouda ct al.. 1057). VasoconsFricFor rcsponsc after potassium or norepinephrine were es:imated from the increases in perfusion pressure in the constant flow system. using a strain-gauge prcsure transducer (Statham F23DB) placed bchveen the peristaltic pump and the arterial segment. The pressure caused by the basal resistance of the potyethytene tubing plus the unstimulated arterial segment was suhtrzted from the increases in perfusion pressure produced by potassium chloride or norepinephrine. The arterial se_ementwas maintained in a vertical position by a 05 g weight attached via a pulley system to the distal cannuta of the arterial segment. The passive tension generated by the 0.5 g weight. restored the tengFh of the segment to that measured in vivo.
‘1%~ pcrfuartc used was it Krebs solution containing (mht): NaCl I IS. KCI 4.7. MgCt, 1.2. NaH,PO, 1. 24.9 and ~IUCOSCI I. I. The SOIUC’aCI , 2.2, NittICOi tion &IS osypcnatcd with 95% 0, plus 5?; CO?; the pH N;IS 7.4 + 0.Z at 37 oC. The tlow rate was incrcascd gradually o\cr I h. from I to 4 mt/min by I mt/min increments cve~y 20 min. and was then maintained constant at I mt/min for the remainder of the experimcnt. The first cxposurc to potassium or norepincphrinc ws ilfkr 3 20-min equilibration period at 4 ml/min. This tlow rate was chosen on the basis of a prctiminary cspcriment (n = IO arterial segments), in which basal perfusion pressure and increases in perfusion prcssurc after perfusion with norepinephrine (injection of 0. I ml of a solution of 0.1 mM in 3 s into the perfusion circuit). were determined after a 15min cquitibration period at increasing flow rates of 2 to 9 mt/min (sfcps of I ml/min). The equations relating haslit perfusion pressure ( = 30.3- 1.5x + 0.5x’, r = 0.99, P < O.tW and norepinephrine-induced increases in perfusion pressure ( = 77.2 + 12.3x- 1.Ix’. r = 0.87, P < 0.05) to flow rate (x) predicted that plateau responses for both parameters would be obtained at 4 to 5 mt/min. The flow rate was reduced to 1.5 mt/min for the experiments in which potassium-evoked vasoconstriction and norepinephrine overflow were measured simultaneously. as preliminary experiments showed that the amount of norepinephrine recovered was highest at this lower flow rate. Two tail artery segments were mounted in parallel. Four pairs of arterial segments were perfused each day, two in the morning and two in the afternoon. The perfusate flowed freely from the open end of the distal cannuta and in so doing superfused the tail artery segment. A period of 20 min elapsed from the beginning of the dissection to the perfusion/ superfusion of the tail artery. 2.2 Cotlceturatiolt-respo,lse
ctwes
of the tail artery to
norepirlephritte arid potassium
Non-cumulative concentration-response curves were made. Perfusion pressure was allowed to return to baseline for 5 min between each challenge. One agonist only was applied to each tail artery segment. Each concentration of potassium or norepinephrine was applied for 2 min. Potassium was applied at concentrations of IO. 20.30.3S.40,4_5, 50.60, 70,80.90. 100. 110 and 120 mM. tsotonicity was maintained by removal of NaCt. In preliminary experiments to determine which concentration of phentotamine to use (see below), concentration-response curves were made with concentrations of potassium of lo-50 mM only. Norepinephrine, dissolved in Krebs solution plus 0.1 mM ascorbic acid, was applied at concentrations of 1
nM to 3 mM. dcpcnding on whether rats had been prctrcatcd with rcserpinc or not and whether the arterial segment was co-perfused with phentolaminc or not (WC below). Control concentration-response curves for
potassium or norepinephrine were made in each series of experiments on the effects of a- or @-adrenoceptor antagonists or reserpine. Results presented are the pooled data from all such control experiments. In a preliminary experiment, in which potassium (50 mM for 2 min. at S-min intervals) was applied I4 times to tail artery segments (n = 4). responses were constant throughout. For example, at the 6th and 14th challenge responses were 107 + 3 and 102 f 10%” of those obtained during the first challenge. It should be noted that in this particular in vitro arterial preparation endothelial-derived relaxation is greatly attenuated (Atkinson et al., 1988). 2.3. Frequency-response curt’es of the tail artery to elec-
trical stimulation in the presence of phentolamine Arterial segments were mounted as described previously and, after equilibration at a tlow rate of 4 ml/min, they were electrically stimulated via two circular platinum electrodes placed 4 mm apart around the seg-
ment. Frequencies of l-256 Hz were applied at 5-min intervals for 10 s, with a pulse duration of 0.3 ms at supramaximal voltage. The protocol was repeated after perfusion with phentolamine (0.2 and 4 PM). Perfusion with phentolamine started 5 min after the final increase in flow rate from 3 to 4 ml/min and continued for the next 15 min before electrical stimulation. Perfusion was maintained up to the end of the experiment. 2.4. Effect of reserpine pretreatment on the rasoconstrictor responses of the tail artery evoked by potassium or norepinephrine
The protocol used was the same as that described above except that rats were injected with reset-pine (2.5 mg/kg, i.p.1 18 h before removal of the tail artery. Two segments of the tail artery of each rat pretreated with reserpine were mounted simultaneously with two segments from the tail artery from a rat that had not received reserpine. Potassium or norepinephrine was applied as described above. As before, each arterial segment was exposed to one agonist only. 2.5. Effect of LYor @adrenoceptor antagonists on the rasoconstrictor responses of the tail artery evoked by potassium or norepinephrine The protocol used was similar to the one described above except that rats were not pretreated with reserpine and that one of each pair of arterial segments mounted in parallel was perfused/superfused with ei-
ther phcntolaminc (0.2 and 4 PM), f F I-propranotd (1.
PM) or a combination of phent&minr (1 p (+ )_propranolol (2 FM). Perfusion with ore UT the combination of the two antagonists started S min after the final increase in flow rate from 3 to 4 ml/min. Perfusion continued for the next 15 min before co-perfusion with the first concentration of either potassim or norepinephrine and was maintained up to the cd of the experiment. The concentrations of phentolaminc used were based on the results of preliminary experimenti (rcsuits not shown), in which potassium was applied at
concentrations of IO-50 mM in the presence 0% 0.1, 0.2. 1, 4 or 10 PM phentolamine. A coneentration of 0.1 PM phentolamine had no significant effect on vasoconstrictor responses whereas 10 PM phentolamine abolished responses to potassium. Tbe intermediate concentrations of 0.2 and 4 PM phtntcrlamine were used in subsequent experiments. The concentration of ( f )_propranolol chosen (2 FM) was based on the concentration used in previous reports with this in vitro rat tail artery preparation (Medgett and Langer, 1984). The second member of each pair of arterial segments was treated as in the original protocol and was not perfused with either adrenuceptor agonist. 2.6. Vasoconstrictor responses of depo/atiTed arteries produced by CaCI, in the presence of phentolami~te (3 p M) or diltiazem (0. I p M) Rats were pretreated with reserpine, and two arterial segments removed from each rat and perfused/ superfused as described above. After equilibration for 20 min at a flow rate of 4 ml/min, one of each pair of arterial segments was perfused/ superfused with Krebs solution of the following composition (mM): NaCl 1IX, potassium chloride 4.7, MgCl,, NaH?PO, 1. NaHCO, 24.9, glucose 11.1 and ethylene glycol-bis(/3-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) I. After a 30-min exposure to this calcium-free plus chelatcr solution, the perfusate was switched to a calcium-free plus chelator depolarizing solution of the following composition (mM): potassium chloride 110. MgCI, 1.2. NaH ,PO, 1, NaHCO, 24.9, glucose I I. 1 and EGTA 1. After a further 30 min, the perfusate was switched to a calcium-free depolarizing solution (without chelator) of the following composition (mM): potassium chloride 110. MgCl? 1.2, NaH,PO, 1. NaHCO, 24.9 and glucose 11.1. After a further 30-min equilibration period, distilled water (0.1 ml in 3 s) was injected into the perfusion circuit. This produced a transient rise in perfusion pressure of 5-8 mmHg. The value of this injection artefact was subtracted from she subsequent responses to injections of CaCl,. Five minutes after the injection of distilled water, the rapid. smafl volume injection was
Tail t1Ftr‘I-y sc-gnwnts K&S
\verc pe‘rfused/ SUptXfUSd
with
(N&I
dutitEn
1IS. potassium chloride -4.7. M&J. 4.2, NaH .FU,. C&I. 2.2. NaHCO, 2-4.9 and glt.~&e I I.1 mMj_ After ;1 $min cquilihration period at ;1!tkm rxc of 1 mlfmin. the flow rate was increased to E3 ml/min and arterial preparations were stabilized at this tCnv rrrtr for 20 ruin 4xforc‘ the first concentra-
potassium was applied. Increasing concentra(40-120 mM1 were applied as descriid ahcne except that the flow rate was 1.5 ml/min. Preliminrtry experiments showed that the percentage
tion of
) x g at -4°C for 3 min. The supcrnatant was discarded and norcpincphrine was elutcd with 150 ~1 perchloric acid (0.1 MI by mixing on a vortex at low xpccd for -45 s. The suspension was then centrifuged at IStW)x !: at 1 “C for 10 min. One hundred microlitres of the supernatant were injected into the high-performance liquid chromatography (HPLC) apparatus. Tail artery norepincphrinc content was determined according to the method described by Fouda et al. ( 1987). The proximal I cm of the tail artery was washed in Krehs solution. blotted dry and homogenized in 0.5 ml of 0.1 N perchloric acid solution containing 2 mM sodium disulfitc. I mM EDTA and 3A-dihydroxybensylamine as internal standard. After centrifugation at 25W x I: at -4“C for 20 min. the supernatant was diluted and injected into the HPLC apparatus. The HPLC-EC system consisted of a single piston pump with a pulse damper (LC pump T-414. Kontron, Ziirich. Switzerland). an ODS reverse-phase column (250 mm x 4 mm I.D.. 5 pm, Machety Nagel. Diiren, Germany) iand an amperometric detector (LC 4-A, B&nalyticai systems, Lafayette, Indiana, USA) with a carbon paste electrode held at an oxidation potential of +I).6 V compared to a Ag-AgCl reference electrode. The mobile phase consisted of 0.15 M NaHPO,, 50 /IM Na, EDTA and 0.3 mM sodium octyl sulfate, pH 3.00-3.05. The flow rate was 0.6 ml/min and the column temperature 37 o C. The limit of detection was 5 pg norepinephrine. As we were unable to detect norepinephrine in the superfusate in the absence of an increase in potassium concentration, we concluded that the basal norepinephrine overflow in this preparation was < 5 pg/ml perfusate/ superfusate. 2.8. Substances used
tions of potrtsium
r~~ovzry
of
norepinrphrine
bupcrfwwre was greater
in
the
perfusate/
at a flow rate of 1.5 ml/min
T4~e perfusate/
superfusate was collected in ice-cold concyntrated perchloric acid (7W w/v. 25 PI/ml perfusate). 3.4-DihydrosTbenzylamine hydrobromide ( 100 ~4 of a solution of 2.5 ng/ml 0.1 N perchloric acid) was added to each tuhc as an internal standard together with 100 p I of a solution of 1Cc Na , EDTA plus 0.5? Na,S,O,. The pH of the solution was brought to S-25-S-35 b adding 100 ~1 of Tris buffer (2 M. pH 8.5) and 40 to 20 yl of 5 N NaOH. Norepinep,rine was then adsortard onto acid-Nfashed Al,O; (4 me/ml) by shaking for 10 min at 1 o C. The supernatant was removed and the alumina was washed 4 times with a solution of 0.1% Na,EDTA plus 0.05% Na?S,O, adjusted to pH 7 with Tris buffer (2 MI and centrifuged tubes during the challenge. The tubes contained
Diltiazem was a gift from Tanabe Seiyaku Ltd., Osaka, Japan. Reset-pine and phentolamine were gifts from Ciba-Geigy Ltd., Base& Switzerland, and (+)_ propranolol was a gift from ICI Ltd., Macclesfield. England. EGTA was purchased from Fluka AG, Buchs, Switzerland. Norepinep:.,me and 3,4-dihydroxybenzylamine hydrobromide were purchased from the Sigma Chemical Company. St. Louis, Missouri, U.S.A. 2.9. Statistical analysis
ED,,, (ED,,,rnmlIg 1 values represent the intensity of stimulus required to produce a 20-mmHg increase in perfusion pressure. They were calculated as means of individual values. each taken from linear regression analysis, following logit transformation of responses and log,,, transformation of concentration for each individual concentration-response curve. After mean responses had been calculated at each concentration used, concentration-response curve data were fitted to
the sigmoid equation: response = ~‘(1 + cxp((a x)/h)) + d. where a = log,,, ED5,,r;, b = slope, c = maximum, d = minimum and x = log),, concentration, using a computer program written by M. Ekdes, IMPC. Sofia-Antipolis, France. The affinities of phentolamine or diltiazem were estimated from -log Ku = [antagonistI/fx - 11, where x = ratio of pD, or EDX, values before and after antagonist. Results are given as means jt S.E.M. Means were compared using Students* t-test. 3. Results
40
t
I 3.1.. Vasoconrrrittion indttc ‘d by nonepittephitte
20
!I
Perfusion/superfusion with increasing concentrations of norepinephrine for 2 min produced concentration-related increases in perfusion pressure (fig. I), with a maximum of 250 + 12 mmHg at 1 FM norepinephrine.
?erfusion
60
pressure
reached
a maximum
0 0
10-7
10-s
10-5
10-a
norepinepbrin~
10-a
lo-2
CM)
Fig. I. increases in perfusion pressure tmmHg) after norepinephrine (NE) in the absence (open squares) or presence of phentolamine tP 0.2 pM, full squares. or 1 pM. full circles). flest fits to the sigmoid curve (response = c/t
NE + P 0.2 fiM +P3brM
NE +PO.2 fiM +P4/iM
1+ exti(a - x)/b)) + d).
a
b
C
d
n
- 6.8339 - 5.2881 - 4.2644
0.3515 0.4363 0.3495
261 26Y 246
-1 -Y -5
‘3 7 8
ED,,
Maximum (mmHg)
-log K,
32 f7nM l.O+OS /AM 7.6kO.6 gM
250+12 St* 10 237+ 9
8.2+0.1 g.o+o.1
LOO
1SO
200
sreondr
Fig. I. Increases in perfusion pressure (5 maximum1 after norepineph~ne (NE. open squares!. pttassium fK-, open ckcte4. K * phzs phentofam~oe (I? 0.2 FM, futf circfes) or K” in tail anet-& from reserpinized rats tfult triangiesk
NE(I pMI I;- 450 mMf K‘ fS’tmM)+ P (0.2 PM) K- (SO mMB after reserpine
0 10-a
SO
Maximum (mutHat
n
Open squares C&en circtes
254I* 12 -T-t’+ -- Y
23 -tl
Fuil circles
il6+
14
10
x
9
Full triangles
905
70-90 s after the start of the perfusion (see fig. 2 for response to 1 PM norepinephrine). At this concentration of norepinephrine the rate of increase in perfusion pressure from 0 to 70 s was approximately linear. with a slope of + 3.39 f 0.49 mmHg/s. From 80 s onwards. va~onstriction remained at a plateau value for 40 s until perfusion with norepinephrine ceased Cat fM sf, whereupon perfusion pressure decreased at a rate of -4.23 rf: 0.20 mmHg/s from 130 to 180 s. reaching baseline at 120 s. Vasoconstrictor responses at lower concentrations of norepinephrine had similar profiles with upward and downward slopes which were shallower. Phentolamine acted as a competitive antagonist of norepinephrine-evoked vasoconstriction (fig. I). with a -log K n value of 8.1, Vasoconstrictor responses to norepinephrine in the presence of phentolamine had similar profiles to those obtained in the absence of phentolamine fresuits not shownf. In tail arteries from rats pretreated with reserpine (n = 8) there was a parallel shift to the left in the norepinephrine concentration-response curve with no change in slope or rn~rn~ WO + I1 mmHgf. but a
Bt’\t tit\ to the Gpmoid curve tresponsc = c/(1 a
h
EDa,
C
+c?ip((ad
xl/h)l+d). n
Maximum (mmHg)
perfusion pressure was 137 f S mmHg, approximately half the increase at 50 mM. The profile of the vasoconstrictor response curve of potassium had a bell shape similar to that of norepinephrine (fig. 2). The initial (O-30 s) rate of rise of perfusion pressure was +5.50 + 0.47 mmHg/s (at 50 mM): the rate then slowed to + 1.78 _t 0.11 mmHg/s from 30 to 70 s. Vasoconstriction remained at a maximal plateau value from 80 to 120 s then decreased at a rate of - 5.8 +_0.25 mmHg/s from 130 to 160 s to reach baseline at 200 s. Thus the onset and termination of the potassium-evoked vasoconstriction were more rapid than those of the norepinephrine-evoked vasoconstriction. The vasocnnstrictor response to 50 mM potassium was diminished bv 52% in the presence of 0.2 PM phentolamine and by 86% in the presence of 4 ,F~M phentolamine (fig. 4). Maximal vasoconstrictor responses also decreased and were observed at higher concentrations of potassium chloride: at 90 mM potassium for 0.2 PM and at 120 mM potassium for 4 PM
Thus. in contrast to its antagonis iiorepi~e~~ri~c responses. oiaminc did not behave as a competitive anta of potassium-evoked vasoconstriction. n the presence of the a-adrenocepe~to~amine, potassium did not protractions (see fig. 2 for response to 50 mM potassium in t e presence of 0.2 p phentolamine). After an I peak at 30 s. perfusion pressure fell to 50% of eak value at 120 s. At this time ~lient~l~niine.
the vasoconstrictor response to potassium in t scnce of phentolamine was still at a maGmai plateau value (fig. 2). Vasoconstrictor responses to concentrations of potassium at 50 mM or lower were not modified by co-perfusion with ( f bproprdnolol (fig. 5). At concentrations of 60 to 120 mM potassium. the vasoconstric-
Fig. h. Increaxh in perfusion pressure (mmHg1 after K- (o,pen squares). K - m the presence of phcntc~lammr (P: 4 PM). and (k )-propranolol (Pr: 1 p M. fuR trianglrs). and K. attrr rcwrpine (full circles).
I + sxp(ta- sb/bl) - cit.
Best fits to the sigmoid cuwe (response = c/t
K+P +Pr
after rcxrpmr
1x9
- 1
13
21x
-3
142
I .h52h i .X37
IWXY! 0.1 IX4 ED,,,
Maximum (mmH@
30*2
z-E= (!
201t2 32+1
191* 8 3f)li II
Y
100
10
potassium
(zuld)
Fi_e. 5. Increases in perfusion pressure (mmH_e) after K _ (open squares) or K* (i) plus phentolamine (P: -I pM. open circles). (ii) plus (+I-propranolol (Pr: 2 PM. full squares). (iii) plus P (-t PM) and ( f )-Pr (2 FM. full trisnglrs). Best fits to the sigmoid curve (response = c/t I + esp(C;1- x)/b))+ 3
+P
I.5789 I.7912
+ Pr +P + Pr
b -XI453
C
‘12
n
d -I
41
t I.0869
!‘2
0
8
I.5638
0.054 I
245
I
Y
I .653l
NOXYI
IX’)
ED,,,
-I Maximum CmmHg)
I3
d).
tion remained elevated in the presence of ( f I-propranolol. In the presence of (+ )-propranolol (2 PM) and phentolamine (4 PM). the potassium-evoked vasoconstrictor responses were greater than those obsen;ed in the presence of phentolamine (3 PM) alone. Responses in the presence of combined LY-and P-adrenoceptor blockade were very similar to those obtained after perfusion/ superfusion with potassium of tail arteries from rats previously treated with reserpine (fig. 6). Once again. as was seen after a-adrenoceptor blockade. potassium was unable to produce a sustained vasoconstriction in tail arteries from rats pretreated with reserpine (fig. 2). 3.4. Calciwn-induced
+P + Pr +P +Pr
II
C
tw453
+P + Pr after rrserpinr
K-
n
-I
b
1.5789
K-
K-
d
a
chloride-depolarized
rasocoratriction tail arten
in the potassium
Calcium chloride induced concentration-related increases in perfusion pressure in depolarized tail arter-
200
G 1Q
0
iuoo
The ~~9re~i~ephrine content of the tail artery was 8.1 + 0.5 ng/mg tissue wet weight (n = 10). As arterial seg~l~~ts weighted 4.1 zt 0.1 ng (II = 10). the total amount of nor~p~~e~h~~~e recovered from the superfusate (during th 12 c;lallenges with 10. 20, 30, 40. 50, ho. 70. #I. %I. I 1. 110 and I20 mM potassium) was equal to “75 of the total arterial norepinephrine content.
1. Discussion
e increases in PC” Gun pressure ~rod~~ed by ass&m in this ~~~erime~t (fig. 7). in which the fusion rate was 1.~~~~~~~s~~descri fusion %w rate was 1 ml/min (fig. 41. The maximum wrf-&on pressure was atta~~~$ at a higher oride ~~~~e~tratio~: 70 vs. 50 mM. This ly arose from the large dead space k:; rcn the ~re~ar~t~o~ and the potassium chloride soIutic~ resenoir. If this is so. then the speed of the incnzase in the ~t~si~~ concentration near the ~re~~~~t~o~ evoked be a cted by the flow rate.
Perfusion/superfusion of the rat tail artery in vitro with a solution of potassium chloride has different effects depending on the concentration used. Changing the potassium c~~~e~tration from 4.7 to 20 mM had no effect; there was no increase in either perfusion pressure or norepinephrine overflow. From 30 to Xl-70 mM. potassium produced increasing amounts of nore~i~epbri~e overflow. Experiments with phento~amine and reserpine show that this norepinephrine overffow contributed for up to half of the vasoconstrictor response observed. A second norepinephrine-independent mechanism was also involved but this latter mechanism appeared to be incapable of producing sustained contraction. At concentrations of ~tassium above 5070 mM, the results of experiments with ( t_ I-propranolol suggest that the norepinephrine released by potassium exerts a /3-adrenoceptor-mediated vasorelaxant effect. An increase in the extracellular concentration of potassium wiIt provoke exocytotic release of nore~ine~hirine and at the same time inhibit neuronal uptake by depolarization (Webb et al., 1981). The observation that potassium-evoked vasoconstriction was diminished by pretreatment with reserpine suggests that potassium-evoked norepinephrine release contribiites to the vas~onstrictor response obtained. The
rcscrpinc treatment used in the present study produces a fall in the tail artery norepinephrine content below e limit of detection of the HPLC-EC system and com~~eteiy abolishes responses to electrical stimulation ~Atk~n~n ct al., 1983. As tctrodotox~n ~~cdgett and Langer, 1984) and ~nnnethidin~ fDuckles and Siherman, 19809 also abolish responses to electrical stimulation in this preparation. it can be suggested that reserpine diminishes potassium-evoked vas~onstrictor responses by dcpictin~ arterial norcpinepbrine stores. Furthermore, responses in reserpinizcd prcparatiuns were similar to those obtained after cy- and &adrenoceptor blockade. It can be assumed that the response remaining after reserpine or combined LY- and /3adrenoceptor blockade represents that produced by smooth muscle cell de~lari2ation. These responses are not only smaller but they also have a different form. In the present study. responses to the various vasoconstrictor stimuli were taken as the maximal increases in perfusion pressure. A closer examination of the responses reveals that in tail arteries from rats pretreated with reserpine. potassium cannot produce a sustained increase in perfusion pressure. The observation that phentolamine, an cY-adrenoceptor blocking agent, reduced responses to potassium appears to confirm the hypothesis that potassium induces va~onstriction of the rat tail artery - at least partially - by releasing norepinephrine. However, phentolamine antagonized responses to norepinephrine and to potassium chloride in different ways. This may suggest that, for instance, in the case of potassium, phentolamine aIso acts as a calcium entry blocker and thus blocks pot ~ssium-evoked vasoconstriction arising from smooth ,nuscIe membrane depolarization. This would appear to be unlikely as phentolaminc did not modi@ the vasoconstrictor responses of depolarized arteries to calcium chloride (whereas diltiazem did). Phentolamine blocked responses to electrical stimulation in a manner similar to its effect on potassium-evoked vasoconstriction. Rajanayagam et ai. (1990) reported that electrical stimulation of the rat tail artery produces vasoconstriction via activation of ar,-adrenoceptors (an effect partially counteracted hy vasodilatation due to activation of /3-adrenoceptors). As both electrical stimulation and elevated potassium produce vasoconstriction via ncurogenically released norepinephrine and stimulation of cu-adrenoceptors in close proximity to nerve terminals. it may be that antagonism at the level of these receptors is different from that at receptors that are more remote from the nerve terminals. The latter would presumably be stimulated after perfusion/ superfusion with norepinephrine. At high concentrations, potassium-evoked responses gradualfy diminished. This may be due to ‘fatigue’ of the segment. This appears unlikely as in a prelimina~
experiment with 50 m potaGim (vv maximum response I, consecuti:,e produced without any sign of ‘fatigue’. co~cenFr~* tions of ~tassium prime the re!eease of lar @f nor~~inephr~ne. which may st~rnu~at tars. This would explain why the blocker. i rf: I-propranolol, increased responses to ~~~~~ concenfrations of potassium. Final!*, our obseiervation that ~t~s~urn-i~d~c~d norep~nephrine overflow and ~tass~urn~~nd~ced vast+ constriction showed app~ximate~y the same co~en~ration-response relationship further strengthens ah’e argument that potassium induces vasoconstriction, at kast partly, by releasing norepinephrine. This confirms previous reports. for instance that of Xiao and Ran.d 4t991k who used a different appr~c~ but arrived at a similar conclusion. The contribution of the sympathomimetic action to the overall vasoconstrictor response to potassium dapends upon the concentration of the latter. At a potassium concentration of 50 mM, which produces maximal vas~onstriction in our preparation, the sympathomimetic action accounts for approximately 5% of the total vasoconstrictor response.
Atliinson. J., N. Boillat. A.K. Fouda. H. Guikain.
lYX7.Noradrenaline
Sonnay. electrical Alkinwt.
Gen. Pharmacol.
J.. N. Trescabrc.
Krausr. M.
stimulation.
C. Bmrdes.
M.C. Pitton. C. Rafizadeh.
Sonnay.
IYXX. Alpha-f
induce vasomnstriction vitro
by stimulation
adrenoceptors. Bolton.
T.B.,
Ducklrs.
N. Boillat. AK
S.P. and R.W.
F.
agonists
rat caudal artery in
pupulation
Arch. Pharmacol.
of action of transmittrrs
Silverman.
Fouda.
adrenoceptor
of a heterogenrom
on smooth muscle. Ph,xiol.
t+
Sautri md
J.C. De Rivaz. hl.
and alpha-2
Naunyn-Schmiedrh.
ind,xrd
18. 719.
of the normotcnsive
IY79. Mechanisms
whstancrs
M. Saute1 and M.
inhibits vawcunstrictiun
of alpha-t 33X. T2Y. and other
Rev. 59. Mlh.
1YWt. Transmural
nerve stirnub
tion of blood ~essrls in vitro: a critical examination.
Bkxod Vrs-
sels. 17. 53. Fouda.
A.K.,
A. Marazzi.
Atkinson. arteries
of spootaneously
endogcnous Fouda.
N. Boillat.
M. Sonnay. H. Guillain
and J.
19X7. Changes in the vascu!:!r reactivity of the k&ted
A.K..
and renovascular
and exogenous noradrmafinc. A.
Kaufmann
and J. Atkinson.
hb-portenske
rats to
Blond L’esxk
21. 6.:.
19%.
Potassium-in-
duced ~asoc~~nstrict~on of the isolated rat tail artr~ mediated
via liberation
of cndogrnous
noradrenaiinr.
is p~rna~i? .!. &pert.
1. 661. Garret.
R.L.
and 0.
Carrier.
cium dependence
1971. Alteration
of rntracclkkv
in vascular tissue by resrrpinr.
cab
Eur. J. Pharma-
col, 13. 3%. Mrdgett.
cle
I.C. and S.Z. Langer.
19X4. HeterogcflriW of smoorh mustail artep in sitro. J. Pha;hnacot.
alpha ~dr~~~~pto~in rat
Exp. Ther.
229. 823.
Polidoro. J.H. and E.A. Savino. 19X2. Responses of the rat tail artW to high external
potassium concentration.
Arch. Int.
Pharmaco-
dyn. 256. 36. Rajanayagam. strirtor
M.A.S..
rqxutses
I.C. bfedgett
and M.J. Rand. I‘@#. ~‘~SCGM~mpathctic tKIs‘c stimula-
of rat tail artery to
1. Introduction In vivo, small. physiological increases in the concentration of potassium in the extracellular fluid cause vasodilatation (Vanhoutte et al., 1975). In vitro, however, higher con~ntrations of potassium (> 20 mM) are often used to study the sequence of events leading from excitation to vasoconstriction (Weiss, 1972). An increase in the extracellular concentration of potassium will depolarize the muscle cell membrane and induce calcium influx and/or the release of calcium from membrane or intracellular stores, and thus provoke contraction of vascular smooth muscle (Bolton, 1979). In in vitro preparations of sparsely innervated blood vessels. such as the rat aorta, this mechanism appears to be the main one involved in potassiumevoked contractions. In these preparations, depletion of catecholamines with reserpine or blockade of cy-
Conespondence to: A.-K. Fouda. lnstitut de Pharmacolog~e de Wniversiti de Lausanne. Rue du Bugnon 27. ItNIt) Lausanne. Switzerland. Tel. 33.X3.32.7h.48, fan 33.83.32.3tf.58.
Diltiazem:
(Rat)
adrenoceptors with phentolamine does not modi& the vasoconstrictor response to potassium (Shibata and Carrier, 1967; Garret and Carrier. 1971). In densely innervated vessels. such as the rat tail artery, potassium-induced vasoconstriction can also be mediated via neurogenically released norepinephrine (Polidoro and Savino, 1982; Xiao and Rand, 1991). The relative contribution of these two mechanisms - a ‘direct’ depolarization of the vascular smooth muscle cell membrane. and an ‘indirect’ sympathomimetic action - to the vasoconstrictor response is equivocal. Fouda et al. ( 1986) reported that the cr-adrenoccptor antagonist. phentolamine (4 PM), almost completely abolished the to potassium (50 mMW). vasoconstrictor response whereas Webb et al. (t981) showed that phentolamine (10 p&l) produced only a 50% inhibition of the response to potassium 175 mM). In order to gain insight into the reasons for this apparent discrepancy, we reinvestigated the effects of phentolamine on the potassium-induced vasoconstriction of the normotensive rat tail artery. We compared these results with those obtained after depIetion of norepinephrine by pretreatment with reserpine. &per-
pm__ 1, .. C-1 for aF Icast 1 \wck t-&we each expcrimcnF. Rats wcrc ancsFhetizrd with sodium pcntobarhiFat (50 w,‘kg. i.p.1 anit _;Omin later two 2-cm lengths & Fbc pmxintal pm of Fhc tail artery wcrc removed and immcdiatcty ptaccd in oxygenated Krchs hicarbonaft SC~UF~MI. Rats wcrc Fhcn kittcd with an overdose of so&ma pcntobarbitat. PrcEiminso espcriments showed no significant diffcrcncc?rin \ssorcactivity to cithcr potassium or norcpin&~-& titwccn the two scparatc scgmcnts of the Fait arte~. After gwtlr’ rubbing to remove any coagulated blood sticking to the oufer or inner surface. both ends of each I-cm sgmc‘nr were cannutated with t-cm lengths k%fpotycthytcnc tubing (0.4 mm internal diameter and 0.S mm external diamctrr). The proximal end of the Fait ancry scgmcnt was connected to a constant flow p&.sFa!Fic pump system (Fouda ct al.. 1057). VasoconsFricFor rcsponsc after potassium or norepinephrine were es:imated from the increases in perfusion pressure in the constant flow system. using a strain-gauge prcsure transducer (Statham F23DB) placed bchveen the peristaltic pump and the arterial segment. The pressure caused by the basal resistance of the potyethytene tubing plus the unstimulated arterial segment was suhtrzted from the increases in perfusion pressure produced by potassium chloride or norepinephrine. The arterial se_ementwas maintained in a vertical position by a 05 g weight attached via a pulley system to the distal cannuta of the arterial segment. The passive tension generated by the 0.5 g weight. restored the tengFh of the segment to that measured in vivo.
‘1%~ pcrfuartc used was it Krebs solution containing (mht): NaCl I IS. KCI 4.7. MgCt, 1.2. NaH,PO, 1. 24.9 and ~IUCOSCI I. I. The SOIUC’aCI , 2.2, NittICOi tion &IS osypcnatcd with 95% 0, plus 5?; CO?; the pH N;IS 7.4 + 0.Z at 37 oC. The tlow rate was incrcascd gradually o\cr I h. from I to 4 mt/min by I mt/min increments cve~y 20 min. and was then maintained constant at I mt/min for the remainder of the experimcnt. The first cxposurc to potassium or norepincphrinc ws ilfkr 3 20-min equilibration period at 4 ml/min. This tlow rate was chosen on the basis of a prctiminary cspcriment (n = IO arterial segments), in which basal perfusion pressure and increases in perfusion prcssurc after perfusion with norepinephrine (injection of 0. I ml of a solution of 0.1 mM in 3 s into the perfusion circuit). were determined after a 15min cquitibration period at increasing flow rates of 2 to 9 mt/min (sfcps of I ml/min). The equations relating haslit perfusion pressure ( = 30.3- 1.5x + 0.5x’, r = 0.99, P < O.tW and norepinephrine-induced increases in perfusion pressure ( = 77.2 + 12.3x- 1.Ix’. r = 0.87, P < 0.05) to flow rate (x) predicted that plateau responses for both parameters would be obtained at 4 to 5 mt/min. The flow rate was reduced to 1.5 mt/min for the experiments in which potassium-evoked vasoconstriction and norepinephrine overflow were measured simultaneously. as preliminary experiments showed that the amount of norepinephrine recovered was highest at this lower flow rate. Two tail artery segments were mounted in parallel. Four pairs of arterial segments were perfused each day, two in the morning and two in the afternoon. The perfusate flowed freely from the open end of the distal cannuta and in so doing superfused the tail artery segment. A period of 20 min elapsed from the beginning of the dissection to the perfusion/ superfusion of the tail artery. 2.2 Cotlceturatiolt-respo,lse
ctwes
of the tail artery to
norepirlephritte arid potassium
Non-cumulative concentration-response curves were made. Perfusion pressure was allowed to return to baseline for 5 min between each challenge. One agonist only was applied to each tail artery segment. Each concentration of potassium or norepinephrine was applied for 2 min. Potassium was applied at concentrations of IO. 20.30.3S.40,4_5, 50.60, 70,80.90. 100. 110 and 120 mM. tsotonicity was maintained by removal of NaCt. In preliminary experiments to determine which concentration of phentotamine to use (see below), concentration-response curves were made with concentrations of potassium of lo-50 mM only. Norepinephrine, dissolved in Krebs solution plus 0.1 mM ascorbic acid, was applied at concentrations of 1
nM to 3 mM. dcpcnding on whether rats had been prctrcatcd with rcserpinc or not and whether the arterial segment was co-perfused with phentolaminc or not (WC below). Control concentration-response curves for
potassium or norepinephrine were made in each series of experiments on the effects of a- or @-adrenoceptor antagonists or reserpine. Results presented are the pooled data from all such control experiments. In a preliminary experiment, in which potassium (50 mM for 2 min. at S-min intervals) was applied I4 times to tail artery segments (n = 4). responses were constant throughout. For example, at the 6th and 14th challenge responses were 107 + 3 and 102 f 10%” of those obtained during the first challenge. It should be noted that in this particular in vitro arterial preparation endothelial-derived relaxation is greatly attenuated (Atkinson et al., 1988). 2.3. Frequency-response curt’es of the tail artery to elec-
trical stimulation in the presence of phentolamine Arterial segments were mounted as described previously and, after equilibration at a tlow rate of 4 ml/min, they were electrically stimulated via two circular platinum electrodes placed 4 mm apart around the seg-
ment. Frequencies of l-256 Hz were applied at 5-min intervals for 10 s, with a pulse duration of 0.3 ms at supramaximal voltage. The protocol was repeated after perfusion with phentolamine (0.2 and 4 PM). Perfusion with phentolamine started 5 min after the final increase in flow rate from 3 to 4 ml/min and continued for the next 15 min before electrical stimulation. Perfusion was maintained up to the end of the experiment. 2.4. Effect of reserpine pretreatment on the rasoconstrictor responses of the tail artery evoked by potassium or norepinephrine
The protocol used was the same as that described above except that rats were injected with reset-pine (2.5 mg/kg, i.p.1 18 h before removal of the tail artery. Two segments of the tail artery of each rat pretreated with reserpine were mounted simultaneously with two segments from the tail artery from a rat that had not received reserpine. Potassium or norepinephrine was applied as described above. As before, each arterial segment was exposed to one agonist only. 2.5. Effect of LYor @adrenoceptor antagonists on the rasoconstrictor responses of the tail artery evoked by potassium or norepinephrine The protocol used was similar to the one described above except that rats were not pretreated with reserpine and that one of each pair of arterial segments mounted in parallel was perfused/superfused with ei-
ther phcntolaminc (0.2 and 4 PM), f F I-propranotd (1.
PM) or a combination of phent&minr (1 p (+ )_propranolol (2 FM). Perfusion with ore UT the combination of the two antagonists started S min after the final increase in flow rate from 3 to 4 ml/min. Perfusion continued for the next 15 min before co-perfusion with the first concentration of either potassim or norepinephrine and was maintained up to the cd of the experiment. The concentrations of phentolaminc used were based on the results of preliminary experimenti (rcsuits not shown), in which potassium was applied at
concentrations of IO-50 mM in the presence 0% 0.1, 0.2. 1, 4 or 10 PM phentolamine. A coneentration of 0.1 PM phentolamine had no significant effect on vasoconstrictor responses whereas 10 PM phentolamine abolished responses to potassium. Tbe intermediate concentrations of 0.2 and 4 PM phtntcrlamine were used in subsequent experiments. The concentration of ( f )_propranolol chosen (2 FM) was based on the concentration used in previous reports with this in vitro rat tail artery preparation (Medgett and Langer, 1984). The second member of each pair of arterial segments was treated as in the original protocol and was not perfused with either adrenuceptor agonist. 2.6. Vasoconstrictor responses of depo/atiTed arteries produced by CaCI, in the presence of phentolami~te (3 p M) or diltiazem (0. I p M) Rats were pretreated with reserpine, and two arterial segments removed from each rat and perfused/ superfused as described above. After equilibration for 20 min at a flow rate of 4 ml/min, one of each pair of arterial segments was perfused/ superfused with Krebs solution of the following composition (mM): NaCl 1IX, potassium chloride 4.7, MgCl,, NaH?PO, 1. NaHCO, 24.9, glucose 11.1 and ethylene glycol-bis(/3-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) I. After a 30-min exposure to this calcium-free plus chelatcr solution, the perfusate was switched to a calcium-free plus chelator depolarizing solution of the following composition (mM): potassium chloride 110. MgCI, 1.2. NaH ,PO, 1, NaHCO, 24.9, glucose I I. 1 and EGTA 1. After a further 30 min, the perfusate was switched to a calcium-free depolarizing solution (without chelator) of the following composition (mM): potassium chloride 110. MgCl? 1.2, NaH,PO, 1. NaHCO, 24.9 and glucose 11.1. After a further 30-min equilibration period, distilled water (0.1 ml in 3 s) was injected into the perfusion circuit. This produced a transient rise in perfusion pressure of 5-8 mmHg. The value of this injection artefact was subtracted from she subsequent responses to injections of CaCl,. Five minutes after the injection of distilled water, the rapid. smafl volume injection was
Tail t1Ftr‘I-y sc-gnwnts K&S
\verc pe‘rfused/ SUptXfUSd
with
(N&I
dutitEn
1IS. potassium chloride -4.7. M&J. 4.2, NaH .FU,. C&I. 2.2. NaHCO, 2-4.9 and glt.~&e I I.1 mMj_ After ;1 $min cquilihration period at ;1!tkm rxc of 1 mlfmin. the flow rate was increased to E3 ml/min and arterial preparations were stabilized at this tCnv rrrtr for 20 ruin 4xforc‘ the first concentra-
potassium was applied. Increasing concentra(40-120 mM1 were applied as descriid ahcne except that the flow rate was 1.5 ml/min. Preliminrtry experiments showed that the percentage
tion of
) x g at -4°C for 3 min. The supcrnatant was discarded and norcpincphrine was elutcd with 150 ~1 perchloric acid (0.1 MI by mixing on a vortex at low xpccd for -45 s. The suspension was then centrifuged at IStW)x !: at 1 “C for 10 min. One hundred microlitres of the supernatant were injected into the high-performance liquid chromatography (HPLC) apparatus. Tail artery norepincphrinc content was determined according to the method described by Fouda et al. ( 1987). The proximal I cm of the tail artery was washed in Krehs solution. blotted dry and homogenized in 0.5 ml of 0.1 N perchloric acid solution containing 2 mM sodium disulfitc. I mM EDTA and 3A-dihydroxybensylamine as internal standard. After centrifugation at 25W x I: at -4“C for 20 min. the supernatant was diluted and injected into the HPLC apparatus. The HPLC-EC system consisted of a single piston pump with a pulse damper (LC pump T-414. Kontron, Ziirich. Switzerland). an ODS reverse-phase column (250 mm x 4 mm I.D.. 5 pm, Machety Nagel. Diiren, Germany) iand an amperometric detector (LC 4-A, B&nalyticai systems, Lafayette, Indiana, USA) with a carbon paste electrode held at an oxidation potential of +I).6 V compared to a Ag-AgCl reference electrode. The mobile phase consisted of 0.15 M NaHPO,, 50 /IM Na, EDTA and 0.3 mM sodium octyl sulfate, pH 3.00-3.05. The flow rate was 0.6 ml/min and the column temperature 37 o C. The limit of detection was 5 pg norepinephrine. As we were unable to detect norepinephrine in the superfusate in the absence of an increase in potassium concentration, we concluded that the basal norepinephrine overflow in this preparation was < 5 pg/ml perfusate/ superfusate. 2.8. Substances used
tions of potrtsium
r~~ovzry
of
norepinrphrine
bupcrfwwre was greater
in
the
perfusate/
at a flow rate of 1.5 ml/min
T4~e perfusate/
superfusate was collected in ice-cold concyntrated perchloric acid (7W w/v. 25 PI/ml perfusate). 3.4-DihydrosTbenzylamine hydrobromide ( 100 ~4 of a solution of 2.5 ng/ml 0.1 N perchloric acid) was added to each tuhc as an internal standard together with 100 p I of a solution of 1Cc Na , EDTA plus 0.5? Na,S,O,. The pH of the solution was brought to S-25-S-35 b adding 100 ~1 of Tris buffer (2 M. pH 8.5) and 40 to 20 yl of 5 N NaOH. Norepinep,rine was then adsortard onto acid-Nfashed Al,O; (4 me/ml) by shaking for 10 min at 1 o C. The supernatant was removed and the alumina was washed 4 times with a solution of 0.1% Na,EDTA plus 0.05% Na?S,O, adjusted to pH 7 with Tris buffer (2 MI and centrifuged tubes during the challenge. The tubes contained
Diltiazem was a gift from Tanabe Seiyaku Ltd., Osaka, Japan. Reset-pine and phentolamine were gifts from Ciba-Geigy Ltd., Base& Switzerland, and (+)_ propranolol was a gift from ICI Ltd., Macclesfield. England. EGTA was purchased from Fluka AG, Buchs, Switzerland. Norepinep:.,me and 3,4-dihydroxybenzylamine hydrobromide were purchased from the Sigma Chemical Company. St. Louis, Missouri, U.S.A. 2.9. Statistical analysis
ED,,, (ED,,,rnmlIg 1 values represent the intensity of stimulus required to produce a 20-mmHg increase in perfusion pressure. They were calculated as means of individual values. each taken from linear regression analysis, following logit transformation of responses and log,,, transformation of concentration for each individual concentration-response curve. After mean responses had been calculated at each concentration used, concentration-response curve data were fitted to
the sigmoid equation: response = ~‘(1 + cxp((a x)/h)) + d. where a = log,,, ED5,,r;, b = slope, c = maximum, d = minimum and x = log),, concentration, using a computer program written by M. Ekdes, IMPC. Sofia-Antipolis, France. The affinities of phentolamine or diltiazem were estimated from -log Ku = [antagonistI/fx - 11, where x = ratio of pD, or EDX, values before and after antagonist. Results are given as means jt S.E.M. Means were compared using Students* t-test. 3. Results
40
t
I 3.1.. Vasoconrrrittion indttc ‘d by nonepittephitte
20
!I
Perfusion/superfusion with increasing concentrations of norepinephrine for 2 min produced concentration-related increases in perfusion pressure (fig. I), with a maximum of 250 + 12 mmHg at 1 FM norepinephrine.
?erfusion
60
pressure
reached
a maximum
0 0
10-7
10-s
10-5
10-a
norepinepbrin~
10-a
lo-2
CM)
Fig. I. increases in perfusion pressure tmmHg) after norepinephrine (NE) in the absence (open squares) or presence of phentolamine tP 0.2 pM, full squares. or 1 pM. full circles). flest fits to the sigmoid curve (response = c/t
NE + P 0.2 fiM +P3brM
NE +PO.2 fiM +P4/iM
1+ exti(a - x)/b)) + d).
a
b
C
d
n
- 6.8339 - 5.2881 - 4.2644
0.3515 0.4363 0.3495
261 26Y 246
-1 -Y -5
‘3 7 8
ED,,
Maximum (mmHg)
-log K,
32 f7nM l.O+OS /AM 7.6kO.6 gM
250+12 St* 10 237+ 9
8.2+0.1 g.o+o.1
LOO
1SO
200
sreondr
Fig. I. Increases in perfusion pressure (5 maximum1 after norepineph~ne (NE. open squares!. pttassium fK-, open ckcte4. K * phzs phentofam~oe (I? 0.2 FM, futf circfes) or K” in tail anet-& from reserpinized rats tfult triangiesk
NE(I pMI I;- 450 mMf K‘ fS’tmM)+ P (0.2 PM) K- (SO mMB after reserpine
0 10-a
SO
Maximum (mutHat
n
Open squares C&en circtes
254I* 12 -T-t’+ -- Y
23 -tl
Fuil circles
il6+
14
10
x
9
Full triangles
905
70-90 s after the start of the perfusion (see fig. 2 for response to 1 PM norepinephrine). At this concentration of norepinephrine the rate of increase in perfusion pressure from 0 to 70 s was approximately linear. with a slope of + 3.39 f 0.49 mmHg/s. From 80 s onwards. va~onstriction remained at a plateau value for 40 s until perfusion with norepinephrine ceased Cat fM sf, whereupon perfusion pressure decreased at a rate of -4.23 rf: 0.20 mmHg/s from 130 to 180 s. reaching baseline at 120 s. Vasoconstrictor responses at lower concentrations of norepinephrine had similar profiles with upward and downward slopes which were shallower. Phentolamine acted as a competitive antagonist of norepinephrine-evoked vasoconstriction (fig. I). with a -log K n value of 8.1, Vasoconstrictor responses to norepinephrine in the presence of phentolamine had similar profiles to those obtained in the absence of phentolamine fresuits not shownf. In tail arteries from rats pretreated with reserpine (n = 8) there was a parallel shift to the left in the norepinephrine concentration-response curve with no change in slope or rn~rn~ WO + I1 mmHgf. but a
Bt’\t tit\ to the Gpmoid curve tresponsc = c/(1 a
h
EDa,
C
+c?ip((ad
xl/h)l+d). n
Maximum (mmHg)
perfusion pressure was 137 f S mmHg, approximately half the increase at 50 mM. The profile of the vasoconstrictor response curve of potassium had a bell shape similar to that of norepinephrine (fig. 2). The initial (O-30 s) rate of rise of perfusion pressure was +5.50 + 0.47 mmHg/s (at 50 mM): the rate then slowed to + 1.78 _t 0.11 mmHg/s from 30 to 70 s. Vasoconstriction remained at a maximal plateau value from 80 to 120 s then decreased at a rate of - 5.8 +_0.25 mmHg/s from 130 to 160 s to reach baseline at 200 s. Thus the onset and termination of the potassium-evoked vasoconstriction were more rapid than those of the norepinephrine-evoked vasoconstriction. The vasocnnstrictor response to 50 mM potassium was diminished bv 52% in the presence of 0.2 PM phentolamine and by 86% in the presence of 4 ,F~M phentolamine (fig. 4). Maximal vasoconstrictor responses also decreased and were observed at higher concentrations of potassium chloride: at 90 mM potassium for 0.2 PM and at 120 mM potassium for 4 PM
Thus. in contrast to its antagonis iiorepi~e~~ri~c responses. oiaminc did not behave as a competitive anta of potassium-evoked vasoconstriction. n the presence of the a-adrenocepe~to~amine, potassium did not protractions (see fig. 2 for response to 50 mM potassium in t e presence of 0.2 p phentolamine). After an I peak at 30 s. perfusion pressure fell to 50% of eak value at 120 s. At this time ~lient~l~niine.
the vasoconstrictor response to potassium in t scnce of phentolamine was still at a maGmai plateau value (fig. 2). Vasoconstrictor responses to concentrations of potassium at 50 mM or lower were not modified by co-perfusion with ( f bproprdnolol (fig. 5). At concentrations of 60 to 120 mM potassium. the vasoconstric-
Fig. h. Increaxh in perfusion pressure (mmHg1 after K- (o,pen squares). K - m the presence of phcntc~lammr (P: 4 PM). and (k )-propranolol (Pr: 1 p M. fuR trianglrs). and K. attrr rcwrpine (full circles).
I + sxp(ta- sb/bl) - cit.
Best fits to the sigmoid cuwe (response = c/t
K+P +Pr
after rcxrpmr
1x9
- 1
13
21x
-3
142
I .h52h i .X37
IWXY! 0.1 IX4 ED,,,
Maximum (mmH@
30*2
z-E= (!
201t2 32+1
191* 8 3f)li II
Y
100
10
potassium
(zuld)
Fi_e. 5. Increases in perfusion pressure (mmH_e) after K _ (open squares) or K* (i) plus phentolamine (P: -I pM. open circles). (ii) plus (+I-propranolol (Pr: 2 PM. full squares). (iii) plus P (-t PM) and ( f )-Pr (2 FM. full trisnglrs). Best fits to the sigmoid curve (response = c/t I + esp(C;1- x)/b))+ 3
+P
I.5789 I.7912
+ Pr +P + Pr
b -XI453
C
‘12
n
d -I
41
t I.0869
!‘2
0
8
I.5638
0.054 I
245
I
Y
I .653l
NOXYI
IX’)
ED,,,
-I Maximum CmmHg)
I3
d).
tion remained elevated in the presence of ( f I-propranolol. In the presence of (+ )-propranolol (2 PM) and phentolamine (4 PM). the potassium-evoked vasoconstrictor responses were greater than those obsen;ed in the presence of phentolamine (3 PM) alone. Responses in the presence of combined LY-and P-adrenoceptor blockade were very similar to those obtained after perfusion/ superfusion with potassium of tail arteries from rats previously treated with reserpine (fig. 6). Once again. as was seen after a-adrenoceptor blockade. potassium was unable to produce a sustained vasoconstriction in tail arteries from rats pretreated with reserpine (fig. 2). 3.4. Calciwn-induced
+P + Pr +P +Pr
II
C
tw453
+P + Pr after rrserpinr
K-
n
-I
b
1.5789
K-
K-
d
a
chloride-depolarized
rasocoratriction tail arten
in the potassium
Calcium chloride induced concentration-related increases in perfusion pressure in depolarized tail arter-
200
G 1Q
0
iuoo
The ~~9re~i~ephrine content of the tail artery was 8.1 + 0.5 ng/mg tissue wet weight (n = 10). As arterial seg~l~~ts weighted 4.1 zt 0.1 ng (II = 10). the total amount of nor~p~~e~h~~~e recovered from the superfusate (during th 12 c;lallenges with 10. 20, 30, 40. 50, ho. 70. #I. %I. I 1. 110 and I20 mM potassium) was equal to “75 of the total arterial norepinephrine content.
1. Discussion
e increases in PC” Gun pressure ~rod~~ed by ass&m in this ~~~erime~t (fig. 7). in which the fusion rate was 1.~~~~~~~s~~descri fusion %w rate was 1 ml/min (fig. 41. The maximum wrf-&on pressure was atta~~~$ at a higher oride ~~~~e~tratio~: 70 vs. 50 mM. This ly arose from the large dead space k:; rcn the ~re~ar~t~o~ and the potassium chloride soIutic~ resenoir. If this is so. then the speed of the incnzase in the ~t~si~~ concentration near the ~re~~~~t~o~ evoked be a cted by the flow rate.
Perfusion/superfusion of the rat tail artery in vitro with a solution of potassium chloride has different effects depending on the concentration used. Changing the potassium c~~~e~tration from 4.7 to 20 mM had no effect; there was no increase in either perfusion pressure or norepinephrine overflow. From 30 to Xl-70 mM. potassium produced increasing amounts of nore~i~epbri~e overflow. Experiments with phento~amine and reserpine show that this norepinephrine overffow contributed for up to half of the vasoconstrictor response observed. A second norepinephrine-independent mechanism was also involved but this latter mechanism appeared to be incapable of producing sustained contraction. At concentrations of ~tassium above 5070 mM, the results of experiments with ( t_ I-propranolol suggest that the norepinephrine released by potassium exerts a /3-adrenoceptor-mediated vasorelaxant effect. An increase in the extracellular concentration of potassium wiIt provoke exocytotic release of nore~ine~hirine and at the same time inhibit neuronal uptake by depolarization (Webb et al., 1981). The observation that potassium-evoked vasoconstriction was diminished by pretreatment with reserpine suggests that potassium-evoked norepinephrine release contribiites to the vas~onstrictor response obtained. The
rcscrpinc treatment used in the present study produces a fall in the tail artery norepinephrine content below e limit of detection of the HPLC-EC system and com~~eteiy abolishes responses to electrical stimulation ~Atk~n~n ct al., 1983. As tctrodotox~n ~~cdgett and Langer, 1984) and ~nnnethidin~ fDuckles and Siherman, 19809 also abolish responses to electrical stimulation in this preparation. it can be suggested that reserpine diminishes potassium-evoked vas~onstrictor responses by dcpictin~ arterial norcpinepbrine stores. Furthermore, responses in reserpinizcd prcparatiuns were similar to those obtained after cy- and &adrenoceptor blockade. It can be assumed that the response remaining after reserpine or combined LY- and /3adrenoceptor blockade represents that produced by smooth muscle cell de~lari2ation. These responses are not only smaller but they also have a different form. In the present study. responses to the various vasoconstrictor stimuli were taken as the maximal increases in perfusion pressure. A closer examination of the responses reveals that in tail arteries from rats pretreated with reserpine. potassium cannot produce a sustained increase in perfusion pressure. The observation that phentolamine, an cY-adrenoceptor blocking agent, reduced responses to potassium appears to confirm the hypothesis that potassium induces va~onstriction of the rat tail artery - at least partially - by releasing norepinephrine. However, phentolamine antagonized responses to norepinephrine and to potassium chloride in different ways. This may suggest that, for instance, in the case of potassium, phentolamine aIso acts as a calcium entry blocker and thus blocks pot ~ssium-evoked vasoconstriction arising from smooth ,nuscIe membrane depolarization. This would appear to be unlikely as phentolaminc did not modi@ the vasoconstrictor responses of depolarized arteries to calcium chloride (whereas diltiazem did). Phentolamine blocked responses to electrical stimulation in a manner similar to its effect on potassium-evoked vasoconstriction. Rajanayagam et ai. (1990) reported that electrical stimulation of the rat tail artery produces vasoconstriction via activation of ar,-adrenoceptors (an effect partially counteracted hy vasodilatation due to activation of /3-adrenoceptors). As both electrical stimulation and elevated potassium produce vasoconstriction via ncurogenically released norepinephrine and stimulation of cu-adrenoceptors in close proximity to nerve terminals. it may be that antagonism at the level of these receptors is different from that at receptors that are more remote from the nerve terminals. The latter would presumably be stimulated after perfusion/ superfusion with norepinephrine. At high concentrations, potassium-evoked responses gradualfy diminished. This may be due to ‘fatigue’ of the segment. This appears unlikely as in a prelimina~
experiment with 50 m potaGim (vv maximum response I, consecuti:,e produced without any sign of ‘fatigue’. co~cenFr~* tions of ~tassium prime the re!eease of lar @f nor~~inephr~ne. which may st~rnu~at tars. This would explain why the blocker. i rf: I-propranolol, increased responses to ~~~~~ concenfrations of potassium. Final!*, our obseiervation that ~t~s~urn-i~d~c~d norep~nephrine overflow and ~tass~urn~~nd~ced vast+ constriction showed app~ximate~y the same co~en~ration-response relationship further strengthens ah’e argument that potassium induces vasoconstriction, at kast partly, by releasing norepinephrine. This confirms previous reports. for instance that of Xiao and Ran.d 4t991k who used a different appr~c~ but arrived at a similar conclusion. The contribution of the sympathomimetic action to the overall vasoconstrictor response to potassium dapends upon the concentration of the latter. At a potassium concentration of 50 mM, which produces maximal vas~onstriction in our preparation, the sympathomimetic action accounts for approximately 5% of the total vasoconstrictor response.
Atliinson. J., N. Boillat. A.K. Fouda. H. Guikain.
lYX7.Noradrenaline
Sonnay. electrical Alkinwt.
Gen. Pharmacol.
J.. N. Trescabrc.
Krausr. M.
stimulation.
C. Bmrdes.
M.C. Pitton. C. Rafizadeh.
Sonnay.
IYXX. Alpha-f
induce vasomnstriction vitro
by stimulation
adrenoceptors. Bolton.
T.B.,
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N. Boillat. AK
S.P. and R.W.
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pupulation
Arch. Pharmacol.
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Silverman.
Fouda.
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18. 719.
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IY79. Mechanisms
whstancrs
M. Saute1 and M.
inhibits vawcunstrictiun
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Rev. 59. Mlh.
1YWt. Transmural
nerve stirnub
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Bkxod Vrs-
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A.K.,
A. Marazzi.
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of spootaneously
endogcnous Fouda.
N. Boillat.
M. Sonnay. H. Guillain
and J.
19X7. Changes in the vascu!:!r reactivity of the k&ted
A.K..
and renovascular
and exogenous noradrmafinc. A.
Kaufmann
and J. Atkinson.
hb-portenske
rats to
Blond L’esxk
21. 6.:.
19%.
Potassium-in-
duced ~asoc~~nstrict~on of the isolated rat tail artr~ mediated
via liberation
of cndogrnous
noradrenaiinr.
is p~rna~i? .!. &pert.
1. 661. Garret.
R.L.
and 0.
Carrier.
cium dependence
1971. Alteration
of rntracclkkv
in vascular tissue by resrrpinr.
cab
Eur. J. Pharma-
col, 13. 3%. Mrdgett.
cle
I.C. and S.Z. Langer.
19X4. HeterogcflriW of smoorh mustail artep in sitro. J. Pha;hnacot.
alpha ~dr~~~~pto~in rat
Exp. Ther.
229. 823.
Polidoro. J.H. and E.A. Savino. 19X2. Responses of the rat tail artW to high external
potassium concentration.
Arch. Int.
Pharmaco-
dyn. 256. 36. Rajanayagam. strirtor
M.A.S..
rqxutses
I.C. bfedgett
and M.J. Rand. I‘@#. ~‘~SCGM~mpathctic tKIs‘c stimula-
of rat tail artery to