Brain Research, 566 (1991) 61-69 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006.8993/911503.50
61
BRES 17204
Neuropeptide Y/angiotensin II interactions in central cardiovascular regulation of the rat J.A. Aguirre 1, K. Fuxe 1, P. Hedlund ~, J.A. Narv,Sez ~, A. Cintra 1, L. Ros6n a and L.F. Agnati 2 SDepartment of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden) and 2Department of Human Physiology, University of Modena, Modena (Italy)
(Accepted 16 July 1991) Key words: Angiotensin II; Neuropeptide Y receptor; Intracisternal injection; Autoradiography; Medulla oblongata; Blood pressure; Heart rate; Rat; Respiratory rate
Neuropeptide Y (NPY)/angiotensin II (ANG II) interactions have been studied in the central nervous system of the rat in view of their co-distribution and their opposing role in central cardiovascular control using quantitative receptor autoradiography and measurements of mean arterial blood pressure (MAP), heart rate and respiratory rate. The receptor autoradiographical experiments show that incubation with ANG II (10 nM) produces an increase in porcine (p) iodinated NPY-(1-36) [nSI]pNPY-(1-36) binding within the dorsal strip (ds) of the nucleus tractus solitarius (nTS). Immunocytochemicai analysis of intracisternally injected ANG II indicated that it could reach this area, in addition to the periventricular gray of the medulla oblongata, the pons and the periventricular part of the dorsal thalamus and dentate gyrus. Furthermore, a threshold dose of ANG II given intracisternally (3 nmol/rat) together with a dose (75 pmol/rat) of pNPY-(1-36) close to its EDso value for reducing MAP, not only counteracts the vasodepressor action of pNPY.(1-36) but also leads to a marked increase of MAP. Also the bradycardic and bradypneic actions of pNPY-(1-36) are counteracted by this dose of ANG II. In contrast, a threshold dose of pNPY-(I-36) does not counteract the prcssor action of an EDso dose of ANG II (10 nmol/rat) but even enhances the peak pressor action of ANG II. These results may be explained on the basis that central ANG II receptor activation leads to an uncoupling of the NPY Y~ receptor, which mediate the vasodepressor action of pNPY-(1-36) and which is preferentially labeled by [nSl]pNPY-(1-36). This alteration may take place in certain cardiovascular areas such as the ds of the nTS. In this way an increase in the [12"~I]pNPY-(I-36)binding takes place and the development of vasopressor responses may be due to dominance of NPY Y2 receptor mediated responses to pNPY-(1-36) which mediates vasopressor responses and is unopposed by ANG II receptor activation.
INTRODUCTION There is evidence that many of the adrenaline (A)/ neuropeptide Y (NPY) co-storing neurons of the medulla oblongata, including those of the dorsal subnuclei of the nucleus tractus solitarius (nTS) 23'26-28 (dorsal strip (ds) region and dorsal parasolitary region (dPSR)) represent vasodepressor systems t3'17. Thus, previous work has demonstrated that central administration of porcine NPY-(1-36) (pNPY-(1-36)) produces marked vasodepressor and bradycardic actions in the a.chloraloseanaesthetized and in the awake male rat, probably via activation of NPY Yt recep tOrs6'tt'lz'tT'2°'22 which only respond to the full molecule NPY-(1-36) 3s'36. The NPY Y2 receptors, which also respond to long C-terminal amino acid fragments of NPY such as NPY-(13-36), instead mediate vasopressor actions upon central administration s . High densities of angiotensin II (ANG II) immunore-
active (IR) nerve terminal networks co.distribute with high densities of noradrenaline (NA), A and NPY nerve terminal networks within central cardiovascular regions, such as the hypothalamus, the nTS and the sympathetic lateral column of the spinal cord t4'ts'29'3°. There is also evidence for the co.distribution of NPY-IR and ANG II-IR in cell bodies and terminals within the brainstem cardiovascular and respiratory areas t'2 and for the existence of ANG II-IR in subpopulations of tyrosine hydroxylase (TH)-IR neurons in the A1 and C1 cardiorespiratory areas of the ventral medulla of the male rat 7. Finally, high affinity [t2SI]ANG II and [t2Sl]pNPY-(136) binding sites inter alia co.distribute in the nTS t6'2t' 31. In the present paper previously communicated in abstract form 4 a NPY/ANG II interaction has been demonstrated in central cardiovascular regulation in a combined functional and receptor autoradiographical study.
Correspondence: K. Fuxe, Department of Histology and Neurobiology, Karolinska Institute, Box 60400, S-I04 01 Stockholm (Sweden).
62
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.e
~ - =
'"=I-NPY bindi
'=SI-NPY binding (0.5 •
. :i~"..
•=
i-0
~;~_~,-.~.
.-= -~-=_
. --~ ~ : = -
~
- ~ ~.~_
II(10 aM) .
-
~,-~,,.,~
~ ~
~
.
.~ .~ .%: . ~ , ' ~ o ~ ; .
~a - 1 3 . e mm
Fig, 1, Autoradiographi¢ localization of [t:~I]pNPY.(I-36) (0,5 nM) binding in the dorsal medulla in the absence (A) or in the presence (B) of ANG !! (10 aM) is shown in a 14./,m coronal section of the medulla oblongata using a tritium.sensitive film, Bregma level = -13,8 mm (obex =0,3 ram), The solid squares indicat~ the area illustrated in Fig, 2A, The highest binding was observed in the ds and in the AP, ANG !! increased significantly the [t=51]pNPY-(l=36) binding in the ds (B) (see Fig, 2), C and D: non-specific binding of [t=Sl]pNPY-(l=36) in the presence of 1 / , M cold pNPY.(I=36) and in the absence (C) or presence (D) of AN@ 11 (10 nM) at the same level as A and B, respectively,
TABLE
I
Cardiovascular and r,,.voiratoryeffectsof i.c. administration of various doses of AN(7 II in the a.chloralose.anaesthetized male rat Effects of i,c, injections of ANG II on MAP, HR and RR in the a-chloralose-anaesthetized male rat, The area values formed by the curves below (-) or above the base line are expressed as mean ± S,E,M, in arbitrary units, The maximal peak effects are expressed in ~, change from the respective mean basal value, JTt - The Jonekheere-Terpstra test for ordered alternatives to evaluate the dose-response action of the drug, n - 4-6 rats in each group, i.e.
MAP
HR
Basal vah~e Increases
from H~)
mCSF (10/d) ANG Ii 1 nmol ANG I! 3 nmol ANGll5nmol ANG II 10 nmoi ANG II 30 nmol
115±5 117±! 112±2 !17-+! 117-+3 115±3
Basal value
..........................................(beats/rain)
RR Decreases
Basal value
Peak
Area
Peak
Area
(%)
(arbitrary units)
(%)
(arbitrary units)
-13±5 -5±3 -7±2 -8--. 1 -8--.2 -154-6
-400±200 -818±634 -846-+575 -873±224 -278 .4-108 -1611±1109
7± 1 150±41 447±31 0.1 ± 1 66±6 397± 12 7±1 186±56 391±15 10± 1 209±63 426±8 24±4* 660±42* 391± 15 40__.7* 1016--.275' 454±10 (JTt: P < 0.001)(JTt: P < 0,001)
*P < 0,05 acrord~ng to the test: 'treatment vs control' (non-parametric procedure),
(breaths/mix)
70±3 91±37 83±5 92--.7 86±3 70+-1 (Yl't: P
Decreases Peak
Area
(%)
(arbitrary units)
-16±3 -202±27 -22±5 -6374"224 -10--.2 -1184-40 -6--.3 -145-4"92 -104-1 -318--.83 -5---3 --48±26 < 0,002)(JTt: P < 0,05)
63 TABLE II
Time course of the effects on HR by separate or simultaneous i.c. administration of pNPY-(I-36) (75 pmol/raO and ANG H (3 nmol/raO in the a-chloralose-anaesthetized male rat Means ± S.E.M. in % of the mean basal value (above (+) and below (-) the base line), are shown, pNPY-(1-36) was administered in a dose of 75 pmol/rat (close to the EDso value for its central vasodepressor, bradycardic and bradypneic action19). The interactions with a threshold dose of ANG II (3 nmol) are also shown. From the same experiment as shown in Figs. 4A, 5A, 6A and 7, where the statistics are shown, n - 4-6.
Time (rain)
mCSF (10 #l)
pNPY-(1-36) (75 pmol)
ANG 11 (3 nmol)
pNPY (75 pmol) + ANG H (3 nmol)
0 (basal value, beat/min)
447 ± 31
392.4- 10
391 __ 15
450 -+ 2
+5 +10 +15 +20 +30 +40 +50 +60
+3--. 1 +2 .4- 1 -1.5 ± 2 -1,8 ± 2 -0.6 -- 1 +3 .4- 1 +2--- 1 +1--2
-5 ±2 -15 --. 3 -12 -- 2 - 5 "- I -5 - 1 - 5 -+ 1 -10± 1 -5+2
-5 ± 1 - 8 -- 1 -2 - 1 -3 - 2 - I +- 4 -1 ± 4 -3 ±5 +2±3
-2- 1 +5 +- 1 +6 .+- 1 +6 -+ 1 - 2 -+ 1 +4 --. 1 +4- 1 +6±3
MATERIALS AND METHODS
Animals Male specific pathogen free Sprague-Dawley rats (200-250 g b. wh ALAB, Stockholm, Sweden) were used. The animals were kept under regular lighting conditions (lights on at 06.00 h and off at 20.00 h) and had free access to food pellets and tap water. The rats arrived at the animal quarters at least I week before starting the experiments,
Quantitative receptor autoradiography The rats (n -- 6) Underwent transcardiac perfusion with ice-cold sodium chloride (0.9% w/v) under methohexital natrium (30 mg/kg i,p,) (Brletal, Lilly, IN, U,S,A,) anaesthesia, The brains were thereafter dissected and frozen with CO2 gas. Coronal sections (14 pm thick) were made in a Leitz cryostat at various rostrocaudal levels of the medulla oblongata. The sections were thaw-mounted on polyethylenimine 0,1% (v/v) (Sigma Chemical Co., St. Louis,
MO, U.S.A.) coated slides to avoid binding of the radioligand to the glass. Iodinated pNPY-(1-36) ([12SI]pNPY-(l-36), Amersham Radiochemical Center, Amersham, Bucks, U.K.; spec. act. 2000 Ci/ mmol) was used and the incubation conditions were similar to those described by Goldstein et al. 19 for iodinated compounds in biochemical experiments. Briefly, a 50 mM Tris-HC! buffer (pH 7.6) containing 5 mM MgCI2, 5 mM CaCI2, 0.065 TIU/ml aprotinin and 0.1 mM bacitracin was used. The incubation concentration of [nSl]pNPY-(1-36) was 0.5 nM, which is close to its KD value 34. In this way a high signal-to-noise ratio was obtained in the receptor autoradiograms. The binding was performed under equilibrium conditions (90 rain at room temperature, 21 °C). After the incubation the sections were washed 3 times for 5 rain in 50 mM Tris.HC! buffer (pH 7.6) and finally rinsed briefly in distilled water before drying under a stream of cold dry air. Non-specific binding was defined as the binding in the presence of I pM pNPY-(I-36) (Peninsula Laboratories, Belmont, CA, U.S.A.). The influence of ANG
TABLE III
Time course of the effects on HR by separate or simultaneous i.c. administration of pNPY.(l-36) (7.5 pmollrat) and ANG !! (10 nmol/rat) in the a.chloralose.anaesthetized male rat Means ± S.E.M. in % of the mean basal value (above (+) and below (-) the base line), are shown. A subthreshold dose (7.5 pmoil see ref. 19) of pNPY-(1-36) and a dose of ANG II (10 nmol) that is equal to the EDso value for the peak and overall vasopressor effect (see Table I) were used. From the same experiment as shown in Figs. 4B, 5B and 6B, where the statistics arc shown, n = 4-6.
Time (min)
mCSF (10 pl)
pNPY.(I-36) (7.5 pmol)
ANG ll (10 nmol)
pNPY (7.S pmol) + ANG I! (10 nmol)
0 basal value, beats/min
457 ± 30
445 ± 16
393 ± 17
442 +-. 6
+5 +10 +15 +20 +30 +40 +50 +60
+3.5 -,- 2 -3-,-3
+2 +-. 1 +8- 3
-7 - I -2± 1
-1 ± 1 -3 ± 1
-1.7 ± 1
+5 ± 1
+0.4 ± 1
-8 ± 3
- 4 --. 5 -5.8 --. 3 -0.7 ± 2 -1.8 ± 2 -2±2
+1 ± +2 -0.2 ± +2 ± +4±
1 1 1 1 1
-2 -3 -5 -6 -3
+-- 1 --. 1 ± 1 ± 1 ± 1
- 7 --. 2 -7 ± 2 - 6 +-- 1 - 5 --. 1 -11±3
64 Ii on [ml]pNPY-(I-36) binding was evaluated by incubating adjacent sections also with ANG I! (10 aM; Peninsula Laboratories), the procedure otherwise beit;g the same as above. After drying the sections were exposed to a tritium-sensitive film (3H-Ultrofilm, LKB Bromma, Sweden). The exi.'osure time ranged from 16 to 48 h. The quantitative receptor autoradiographical analysis was performed by the use of the autoradiographical programs in the IBAS image analyzer (Zeiss Kontron, F.R.G.) equipped with a Bosch videocamera. The cytoarchitecture of the labelled regions was demonstrated by staining the sections with Cresyl violet, which allowed the identification of neuroanatomical landmarks and nuclei. The atlases of Paxinos and Watson32 and Kalia et al. 26 were used. The area postrema (AP), and the dorsal strip (ds) and the medial subnucleus (maTS) of the nTS were delineated (Fig. 2A-B) and the mean grey values were measured. Bilateral measurements were
made on two or 3 sections at each level of every rat. Sections containing grey matter (striatum) were applied onto dried drops of [125I]pNPY.(1-36) as previously described5'21. A standard curve consisting of 7-10 dried drops of different concentrations of [t2SI]pNPY-(1-36) wg~ present in all experiments. Using the standard curve the grey values could be recalculated to fmol/mg protein.
ANG H immunocytochemistry In order to analyze the diffusion of intracisternai ANG I1 into the brain tissue two rats received 10 nmol of ANG II/10 pl mock cerebrospinal fluid (mCSF) in the cisterna magna as in the cardiovascular experiments (see below). After 5 min the animals were perfused with fixative (4% parafo~maldehyde, 0.1% picric acid in 0.1 M phosphate). The brains were dissected out, postfixed (2 h) and washed out with buffered 10% sucrose solution (pH 7.4). Coronal cryostat sections, 14 pm thick, were made at several levels of the brain. The sections were incubated with a mouse monoclonal antibody against ANG II (1:500) tT. As controls, adjacent sections were incubated with either the antibody preabsorbed with 50 ,ug/ml ANG II or preimmune rabbit serum. The avidin-biotin-peroxidase technique (Vectastain, Vector, Burlingame, CA, U.S.A.) was employed, using 3-4-diaminobenzidine as a chromogen. Cardiovascular experiments The experiments were made in 46 a-chloralose (100 mg/kg, via the lingual vein) anaesthetized rats as described previouslytt. ANG II (1-30 nmol) (Peninsula Lab., Belm¢~, CA, U.S.A.) dissolved in 10 ld of mCSF, or mCSF alone as a control, was administered (10 pV1 miit) into the cisterna magna (intracisternaily: i.e.) of the a-chloralose anaesthetized rats. Five different doses of ANG II were injected in order to evaluate a possible dose-dependent effect and the effects were compared with the mCSF group. In separate experiments pNPY-(I-36) (Peninsula Lab., Belmont, CA, U.S.A.) was administered as above in a dose of 75 pmol/rat (close to the EDso value for its central vasodepressor, bradycardic and bradypneic action) or in a dose of 7.5 pmol/rat (a threshold dose) 23. ANG !I dissolved in 10 ld of mCSF was administered in a dose of l0 nmol/rat (a pressor dose) or in a dose of 3 nmol/rat (a subpre, sor dose) (see Table I), In another experimental group, the subpressor dose of ANG I! (3 nmol) wa~ injected together with pNPY-(I-36) (75 pmol). It was also tested if a threshold dose of pNPY-(l-36) (7.5 pmol) could modify the cardiovascular and respiratory actions of a dose of ANG ll (10 nmol) close to the EDso value for its central cardiovascular action (see 'gable !). The composition of the mCSF solution was as follows (in raM),
.~
4o
~
3O
i
2O
|
J
]
vnTS
Fig. 2. Schematic line drawings at the two levels of the dorsal medulla oblongata of the rat, where the microdensitometric measurements were performed. A corresponds to the level of Fig. 1, The various measuring fields (striped areas) are shown in the nTS region (ds and mnTS) as well as the measuring field within the AP (at both right and left sides). AP, area postrema; ncom, nucle,s commisuralis; dmnX. dorsal motor nucleus of the vagus; nXIl, nucleus hypoglossus; ds, dorsal strip; dPSR, dorsal parasolitary region; hi, intermediate nucleus; ni, interstitial nucleus; maTS, medial; dnTS, dorsal; dinTS, dorsolateral, vaTS, ventral; vlnTS, ventrolateral subnuclei of the nTS; "IS, tractus solitarius; IV. fourth ventricle.
~
I"! ml.pNPY (1-3e) rJ lsSl.pNP¥(1-,36)~NGII (10 aM)
' N
ranTS
ds Bregma -13.8 (obex -0,3)
AP
mnTS Bregma -13.3 (obex +0.2)
Fig. 3. Microdensitometric determination of [l~Sl]pNPY-(l-36) bindin&in various regions of the dorsomedial medulla oblongata of the male rat in the absence or presence of ANG II (10 nM). Means -- S,E,M, are shown; n = 6 rats; 0,5 nM of [nSl]pNPY-(1-36) was used. Size of areas measured: ranTS = 0.03 ± 0,007 mm2; ds = 0.03 ± 0,002 ram2; AP = 0,09 ± 0,008 mm 2. The Student's t-test was used (**P < 0,01; t = trend, 0,05 < P < 0.1),
65 NaC! 120, NaH2CO3 20, KC! 2, KH2PO4 0.5, CaC!2 1.2, MgCI2 1.8, Na2SO 4 0.5, D-glucose 5.8. Mean arterial blood pressure (MAP) and heart rate (HR) were measured by means of a heparinized (Heparin, 50 IEU/ml 0.9% NaC! w/v) plastic catheter (PE-50, Clay Adams, NY, U.S.A.) inserted into the common carotid artery, connected to a Statham PC23 DC transducer (Statham Co., Puerto Rico) and linked to a Grass polygraph (model 7; Grass Instruments, MA, U.S.A.). The implantation of the catheter was made under a-chloralose anaes-
Mean arterial blOOd pressure
A
----m---
ml~.RI¢
+ANG II (3 nmol)
|
,
thesia on the day of the cardiovascular experiments, The rats were allowed to recover until blood rl'essurc was stabilized. Registrations were performed as described earlier2°'22. Basal values were registered every 5 rain during a period of 15 rain before the i.c. injection. Respiratory rate (RR) was recorded via a cannula in the oesophagus, also connected to the Grass polygraph via a pressure transducer. The body temperature was monitored during the experiment and kept constant at 37.5 °C by means of a thermostatic device. Measurements of MAP, HR and RR were made during the following 1 h time interval, and the area created by the curve was calculated for each parameter and for each animal using an IBM-XT computer and a program developed by Guna Consult, Stockholm, Sweden "°. The area values (overall effects) were expressed as absolute values in arbitrary units reflecting the duration of the effect under 60 rain, and the peak effects (maximal responses) as per cent changes from the respective mean basal values. EDso values were calculated using iterative, non-linear curve fitting procedures s. The possible ANG II dose-response relationship was tested with the Jonckheere-Terpstra test and the non-parametric 'treatments versus control' test to compare peaks and overall effects between ex-
|
| Mean arterial blood pressure
-I
3 #
.| o
B I
to
2o
so 4o Time (rain)
40- * ~ * (b) ]1( so - p~
.. ,,.. : 4,
2o• /
~
~
so
so
A i
mCSF pNPV(1.~s) (7.s pmot) ~ It(10 nm¢) pNPY(1.80)(7.5pmol)+ANOtl (10nmot)
500
IIIIUlIIIIIIIIIIIIIIIIIIIIIIIIIIIII lll~i~~lllllllll IIIIIIIIIIIIIIIIIIIIII IIII 0 Ill ,i////,~~ , l , l l l l l l l ~ l i l l l l l l ,
. . Re. . .a0 .
10
40
s0
eo
~
Ill
g •500
•1000
210
I
B
800
(n ¢: :l
600
~/,~,/","~'~
~ /t * --.'k pNPY (1-36) ANG II (3 nmol) pNPY(1-36)(75pmol) (75 pmol) + ANG II (3 nmol)
Time (m~n) Fig. 4, Time course of the percent effects on MAP (m~n Hg) by separate or simultaneous i.e. administration of pNPY-(I-36) and AHG II in the a-chloralose (100 mg/kg, via the hngual vein) anaesthetized male rat. A: pNPY-(1-36) was administered in a dose of 75 pmol/rat (close to the EDso value for its central vasodepressor, bradycardic and bradypneic action19). The interactions with a threshold dose of ANG II (3 nmol/rat; see Table I) is shown. B, the interactions of i.e. administered pNPY-(1-36) in a threshold dose (7.5 pmol) are shown with a dose of ANG il (10 nmol) that is the EDs0 value for the vasopressor peak and vasopressor area (see text). MAP basal values (ram Hg) are for each group in A: mCSF = 114 -. 5; pNPY-(1-36) (75 pmol) - 110 - 6; ANt, II (3 nmol) - 112 - 2; pNPY-(I-36) (75 pmol) + ANG II (3 nmol) 110 - 5. MAP basal values (mm Hf) in B are: mCSF = 114 -+ 5; pNPY-(1-36) (7.5 pmol) - 110 -. J;; ANG II (10 nmol) - 107 3; pNPY-(1-36) (7.5 pmol) + ANG II (10 nmol) -- 108 ¢ 5. Means ± S.E.M. are shown as percent change from the respective mean basal value; n - 4-6. The Dunn's test was used to compare different groups (*P < 0.05; ***P < 0.005); a, comparisons with the peak effects of the other experimental groups; b, comparisons with the mCSF and pNPY-(1-36) (7.5 pmol) group peak values; c, comparison of the pressor peak of ANG II (10 nmol) vs the other experimental groups except for the pNPY-(1-36) (7.5 pmol) + ANG II (10 nmol) group.
s..
400
qee ,i
,,o Im ,4
200
pNPY (1-36) ANG II (10 nmol) pNPY (1-36)(7.5pmol) (7.5 pmol) + ANG II (10 nmol) Fig. 5. O~'erall effects (area values) on MAP of separate or simultaneous i.c. administration of pNPY-(1-36) and ANG II in the a.chloralose-anaesthetized male rat. The effects shown in A and B, respectively, have been obtained in the same experiments as illustrated in Fig. 4A and B where the time course of MAP and the basal values for each group are given. The hatched areas indicate the mean - S.E.M. of the effects of mCSF alone. Means -+ S.E.M. (arbitrary units) are shown; n - 4-6. The Dunn's test wa~ used to compare different groups (*P < 0.05; ***P < 0.005: the stars in the columns indicate significance with respect to the mCSF area value).
66 perimental groaps and control. For comparison~ between different experimental gt'oups the Dunn's test was used2s.
the nucleus tractus solitarius (mnTS) was unaffected by ANG lI (10 nM).
FESULTS
ANG H immunocytochemistry
Receptor autoradiographical analysis In vitro incubation with ANG II (10 nM) led to a significant increase in the [i~I]pNPY-(1-36) binding at a concentration of 0.5 nM wkhin the ds (dorsal cardiovascular region of nTS) and a trend for an increase of the [l~I]pNPY.(l-36) binding in the AP (Figs. 1-3). The [nSl]pNPY.(l-36) binding in the medial subnucleus of
As seen ia Fig. 8 intracisternal injection of ANG II (10 nmol) leads after 5 min to the appearance of a 200• 300 ~m wide zone of ANG II IR with a somewhat dotted appearance in the floor of the 4th ventricle involving
A g i
Heart rate
A O =i
e
61
BRES 17204
Neuropeptide Y/angiotensin II interactions in central cardiovascular regulation of the rat J.A. Aguirre 1, K. Fuxe 1, P. Hedlund ~, J.A. Narv,Sez ~, A. Cintra 1, L. Ros6n a and L.F. Agnati 2 SDepartment of Histology and Neurobiology, Karolinska Institute, Stockholm (Sweden) and 2Department of Human Physiology, University of Modena, Modena (Italy)
(Accepted 16 July 1991) Key words: Angiotensin II; Neuropeptide Y receptor; Intracisternal injection; Autoradiography; Medulla oblongata; Blood pressure; Heart rate; Rat; Respiratory rate
Neuropeptide Y (NPY)/angiotensin II (ANG II) interactions have been studied in the central nervous system of the rat in view of their co-distribution and their opposing role in central cardiovascular control using quantitative receptor autoradiography and measurements of mean arterial blood pressure (MAP), heart rate and respiratory rate. The receptor autoradiographical experiments show that incubation with ANG II (10 nM) produces an increase in porcine (p) iodinated NPY-(1-36) [nSI]pNPY-(1-36) binding within the dorsal strip (ds) of the nucleus tractus solitarius (nTS). Immunocytochemicai analysis of intracisternally injected ANG II indicated that it could reach this area, in addition to the periventricular gray of the medulla oblongata, the pons and the periventricular part of the dorsal thalamus and dentate gyrus. Furthermore, a threshold dose of ANG II given intracisternally (3 nmol/rat) together with a dose (75 pmol/rat) of pNPY-(1-36) close to its EDso value for reducing MAP, not only counteracts the vasodepressor action of pNPY.(1-36) but also leads to a marked increase of MAP. Also the bradycardic and bradypneic actions of pNPY-(1-36) are counteracted by this dose of ANG II. In contrast, a threshold dose of pNPY-(I-36) does not counteract the prcssor action of an EDso dose of ANG II (10 nmol/rat) but even enhances the peak pressor action of ANG II. These results may be explained on the basis that central ANG II receptor activation leads to an uncoupling of the NPY Y~ receptor, which mediate the vasodepressor action of pNPY-(1-36) and which is preferentially labeled by [nSl]pNPY-(1-36). This alteration may take place in certain cardiovascular areas such as the ds of the nTS. In this way an increase in the [12"~I]pNPY-(I-36)binding takes place and the development of vasopressor responses may be due to dominance of NPY Y2 receptor mediated responses to pNPY-(1-36) which mediates vasopressor responses and is unopposed by ANG II receptor activation.
INTRODUCTION There is evidence that many of the adrenaline (A)/ neuropeptide Y (NPY) co-storing neurons of the medulla oblongata, including those of the dorsal subnuclei of the nucleus tractus solitarius (nTS) 23'26-28 (dorsal strip (ds) region and dorsal parasolitary region (dPSR)) represent vasodepressor systems t3'17. Thus, previous work has demonstrated that central administration of porcine NPY-(1-36) (pNPY-(1-36)) produces marked vasodepressor and bradycardic actions in the a.chloraloseanaesthetized and in the awake male rat, probably via activation of NPY Yt recep tOrs6'tt'lz'tT'2°'22 which only respond to the full molecule NPY-(1-36) 3s'36. The NPY Y2 receptors, which also respond to long C-terminal amino acid fragments of NPY such as NPY-(13-36), instead mediate vasopressor actions upon central administration s . High densities of angiotensin II (ANG II) immunore-
active (IR) nerve terminal networks co.distribute with high densities of noradrenaline (NA), A and NPY nerve terminal networks within central cardiovascular regions, such as the hypothalamus, the nTS and the sympathetic lateral column of the spinal cord t4'ts'29'3°. There is also evidence for the co.distribution of NPY-IR and ANG II-IR in cell bodies and terminals within the brainstem cardiovascular and respiratory areas t'2 and for the existence of ANG II-IR in subpopulations of tyrosine hydroxylase (TH)-IR neurons in the A1 and C1 cardiorespiratory areas of the ventral medulla of the male rat 7. Finally, high affinity [t2SI]ANG II and [t2Sl]pNPY-(136) binding sites inter alia co.distribute in the nTS t6'2t' 31. In the present paper previously communicated in abstract form 4 a NPY/ANG II interaction has been demonstrated in central cardiovascular regulation in a combined functional and receptor autoradiographical study.
Correspondence: K. Fuxe, Department of Histology and Neurobiology, Karolinska Institute, Box 60400, S-I04 01 Stockholm (Sweden).
62
I-
.e
~ - =
'"=I-NPY bindi
'=SI-NPY binding (0.5 •
. :i~"..
•=
i-0
~;~_~,-.~.
.-= -~-=_
. --~ ~ : = -
~
- ~ ~.~_
II(10 aM) .
-
~,-~,,.,~
~ ~
~
.
.~ .~ .%: . ~ , ' ~ o ~ ; .
~a - 1 3 . e mm
Fig, 1, Autoradiographi¢ localization of [t:~I]pNPY.(I-36) (0,5 nM) binding in the dorsal medulla in the absence (A) or in the presence (B) of ANG !! (10 aM) is shown in a 14./,m coronal section of the medulla oblongata using a tritium.sensitive film, Bregma level = -13,8 mm (obex =0,3 ram), The solid squares indicat~ the area illustrated in Fig, 2A, The highest binding was observed in the ds and in the AP, ANG !! increased significantly the [t=51]pNPY-(l=36) binding in the ds (B) (see Fig, 2), C and D: non-specific binding of [t=Sl]pNPY-(l=36) in the presence of 1 / , M cold pNPY.(I=36) and in the absence (C) or presence (D) of AN@ 11 (10 nM) at the same level as A and B, respectively,
TABLE
I
Cardiovascular and r,,.voiratoryeffectsof i.c. administration of various doses of AN(7 II in the a.chloralose.anaesthetized male rat Effects of i,c, injections of ANG II on MAP, HR and RR in the a-chloralose-anaesthetized male rat, The area values formed by the curves below (-) or above the base line are expressed as mean ± S,E,M, in arbitrary units, The maximal peak effects are expressed in ~, change from the respective mean basal value, JTt - The Jonekheere-Terpstra test for ordered alternatives to evaluate the dose-response action of the drug, n - 4-6 rats in each group, i.e.
MAP
HR
Basal vah~e Increases
from H~)
mCSF (10/d) ANG Ii 1 nmol ANG I! 3 nmol ANGll5nmol ANG II 10 nmoi ANG II 30 nmol
115±5 117±! 112±2 !17-+! 117-+3 115±3
Basal value
..........................................(beats/rain)
RR Decreases
Basal value
Peak
Area
Peak
Area
(%)
(arbitrary units)
(%)
(arbitrary units)
-13±5 -5±3 -7±2 -8--. 1 -8--.2 -154-6
-400±200 -818±634 -846-+575 -873±224 -278 .4-108 -1611±1109
7± 1 150±41 447±31 0.1 ± 1 66±6 397± 12 7±1 186±56 391±15 10± 1 209±63 426±8 24±4* 660±42* 391± 15 40__.7* 1016--.275' 454±10 (JTt: P < 0.001)(JTt: P < 0,001)
*P < 0,05 acrord~ng to the test: 'treatment vs control' (non-parametric procedure),
(breaths/mix)
70±3 91±37 83±5 92--.7 86±3 70+-1 (Yl't: P
Decreases Peak
Area
(%)
(arbitrary units)
-16±3 -202±27 -22±5 -6374"224 -10--.2 -1184-40 -6--.3 -145-4"92 -104-1 -318--.83 -5---3 --48±26 < 0,002)(JTt: P < 0,05)
63 TABLE II
Time course of the effects on HR by separate or simultaneous i.c. administration of pNPY-(I-36) (75 pmol/raO and ANG H (3 nmol/raO in the a-chloralose-anaesthetized male rat Means ± S.E.M. in % of the mean basal value (above (+) and below (-) the base line), are shown, pNPY-(1-36) was administered in a dose of 75 pmol/rat (close to the EDso value for its central vasodepressor, bradycardic and bradypneic action19). The interactions with a threshold dose of ANG II (3 nmol) are also shown. From the same experiment as shown in Figs. 4A, 5A, 6A and 7, where the statistics are shown, n - 4-6.
Time (rain)
mCSF (10 #l)
pNPY-(1-36) (75 pmol)
ANG 11 (3 nmol)
pNPY (75 pmol) + ANG H (3 nmol)
0 (basal value, beat/min)
447 ± 31
392.4- 10
391 __ 15
450 -+ 2
+5 +10 +15 +20 +30 +40 +50 +60
+3--. 1 +2 .4- 1 -1.5 ± 2 -1,8 ± 2 -0.6 -- 1 +3 .4- 1 +2--- 1 +1--2
-5 ±2 -15 --. 3 -12 -- 2 - 5 "- I -5 - 1 - 5 -+ 1 -10± 1 -5+2
-5 ± 1 - 8 -- 1 -2 - 1 -3 - 2 - I +- 4 -1 ± 4 -3 ±5 +2±3
-2- 1 +5 +- 1 +6 .+- 1 +6 -+ 1 - 2 -+ 1 +4 --. 1 +4- 1 +6±3
MATERIALS AND METHODS
Animals Male specific pathogen free Sprague-Dawley rats (200-250 g b. wh ALAB, Stockholm, Sweden) were used. The animals were kept under regular lighting conditions (lights on at 06.00 h and off at 20.00 h) and had free access to food pellets and tap water. The rats arrived at the animal quarters at least I week before starting the experiments,
Quantitative receptor autoradiography The rats (n -- 6) Underwent transcardiac perfusion with ice-cold sodium chloride (0.9% w/v) under methohexital natrium (30 mg/kg i,p,) (Brletal, Lilly, IN, U,S,A,) anaesthesia, The brains were thereafter dissected and frozen with CO2 gas. Coronal sections (14 pm thick) were made in a Leitz cryostat at various rostrocaudal levels of the medulla oblongata. The sections were thaw-mounted on polyethylenimine 0,1% (v/v) (Sigma Chemical Co., St. Louis,
MO, U.S.A.) coated slides to avoid binding of the radioligand to the glass. Iodinated pNPY-(1-36) ([12SI]pNPY-(l-36), Amersham Radiochemical Center, Amersham, Bucks, U.K.; spec. act. 2000 Ci/ mmol) was used and the incubation conditions were similar to those described by Goldstein et al. 19 for iodinated compounds in biochemical experiments. Briefly, a 50 mM Tris-HC! buffer (pH 7.6) containing 5 mM MgCI2, 5 mM CaCI2, 0.065 TIU/ml aprotinin and 0.1 mM bacitracin was used. The incubation concentration of [nSl]pNPY-(1-36) was 0.5 nM, which is close to its KD value 34. In this way a high signal-to-noise ratio was obtained in the receptor autoradiograms. The binding was performed under equilibrium conditions (90 rain at room temperature, 21 °C). After the incubation the sections were washed 3 times for 5 rain in 50 mM Tris.HC! buffer (pH 7.6) and finally rinsed briefly in distilled water before drying under a stream of cold dry air. Non-specific binding was defined as the binding in the presence of I pM pNPY-(I-36) (Peninsula Laboratories, Belmont, CA, U.S.A.). The influence of ANG
TABLE III
Time course of the effects on HR by separate or simultaneous i.c. administration of pNPY.(l-36) (7.5 pmollrat) and ANG !! (10 nmol/rat) in the a.chloralose.anaesthetized male rat Means ± S.E.M. in % of the mean basal value (above (+) and below (-) the base line), are shown. A subthreshold dose (7.5 pmoil see ref. 19) of pNPY-(1-36) and a dose of ANG II (10 nmol) that is equal to the EDso value for the peak and overall vasopressor effect (see Table I) were used. From the same experiment as shown in Figs. 4B, 5B and 6B, where the statistics arc shown, n = 4-6.
Time (min)
mCSF (10 pl)
pNPY.(I-36) (7.5 pmol)
ANG ll (10 nmol)
pNPY (7.S pmol) + ANG I! (10 nmol)
0 basal value, beats/min
457 ± 30
445 ± 16
393 ± 17
442 +-. 6
+5 +10 +15 +20 +30 +40 +50 +60
+3.5 -,- 2 -3-,-3
+2 +-. 1 +8- 3
-7 - I -2± 1
-1 ± 1 -3 ± 1
-1.7 ± 1
+5 ± 1
+0.4 ± 1
-8 ± 3
- 4 --. 5 -5.8 --. 3 -0.7 ± 2 -1.8 ± 2 -2±2
+1 ± +2 -0.2 ± +2 ± +4±
1 1 1 1 1
-2 -3 -5 -6 -3
+-- 1 --. 1 ± 1 ± 1 ± 1
- 7 --. 2 -7 ± 2 - 6 +-- 1 - 5 --. 1 -11±3
64 Ii on [ml]pNPY-(I-36) binding was evaluated by incubating adjacent sections also with ANG I! (10 aM; Peninsula Laboratories), the procedure otherwise beit;g the same as above. After drying the sections were exposed to a tritium-sensitive film (3H-Ultrofilm, LKB Bromma, Sweden). The exi.'osure time ranged from 16 to 48 h. The quantitative receptor autoradiographical analysis was performed by the use of the autoradiographical programs in the IBAS image analyzer (Zeiss Kontron, F.R.G.) equipped with a Bosch videocamera. The cytoarchitecture of the labelled regions was demonstrated by staining the sections with Cresyl violet, which allowed the identification of neuroanatomical landmarks and nuclei. The atlases of Paxinos and Watson32 and Kalia et al. 26 were used. The area postrema (AP), and the dorsal strip (ds) and the medial subnucleus (maTS) of the nTS were delineated (Fig. 2A-B) and the mean grey values were measured. Bilateral measurements were
made on two or 3 sections at each level of every rat. Sections containing grey matter (striatum) were applied onto dried drops of [125I]pNPY.(1-36) as previously described5'21. A standard curve consisting of 7-10 dried drops of different concentrations of [t2SI]pNPY-(1-36) wg~ present in all experiments. Using the standard curve the grey values could be recalculated to fmol/mg protein.
ANG H immunocytochemistry In order to analyze the diffusion of intracisternai ANG I1 into the brain tissue two rats received 10 nmol of ANG II/10 pl mock cerebrospinal fluid (mCSF) in the cisterna magna as in the cardiovascular experiments (see below). After 5 min the animals were perfused with fixative (4% parafo~maldehyde, 0.1% picric acid in 0.1 M phosphate). The brains were dissected out, postfixed (2 h) and washed out with buffered 10% sucrose solution (pH 7.4). Coronal cryostat sections, 14 pm thick, were made at several levels of the brain. The sections were incubated with a mouse monoclonal antibody against ANG II (1:500) tT. As controls, adjacent sections were incubated with either the antibody preabsorbed with 50 ,ug/ml ANG II or preimmune rabbit serum. The avidin-biotin-peroxidase technique (Vectastain, Vector, Burlingame, CA, U.S.A.) was employed, using 3-4-diaminobenzidine as a chromogen. Cardiovascular experiments The experiments were made in 46 a-chloralose (100 mg/kg, via the lingual vein) anaesthetized rats as described previouslytt. ANG II (1-30 nmol) (Peninsula Lab., Belm¢~, CA, U.S.A.) dissolved in 10 ld of mCSF, or mCSF alone as a control, was administered (10 pV1 miit) into the cisterna magna (intracisternaily: i.e.) of the a-chloralose anaesthetized rats. Five different doses of ANG II were injected in order to evaluate a possible dose-dependent effect and the effects were compared with the mCSF group. In separate experiments pNPY-(I-36) (Peninsula Lab., Belmont, CA, U.S.A.) was administered as above in a dose of 75 pmol/rat (close to the EDso value for its central vasodepressor, bradycardic and bradypneic action) or in a dose of 7.5 pmol/rat (a threshold dose) 23. ANG !I dissolved in 10 ld of mCSF was administered in a dose of l0 nmol/rat (a pressor dose) or in a dose of 3 nmol/rat (a subpre, sor dose) (see Table I), In another experimental group, the subpressor dose of ANG I! (3 nmol) wa~ injected together with pNPY-(I-36) (75 pmol). It was also tested if a threshold dose of pNPY-(l-36) (7.5 pmol) could modify the cardiovascular and respiratory actions of a dose of ANG ll (10 nmol) close to the EDso value for its central cardiovascular action (see 'gable !). The composition of the mCSF solution was as follows (in raM),
.~
4o
~
3O
i
2O
|
J
]
vnTS
Fig. 2. Schematic line drawings at the two levels of the dorsal medulla oblongata of the rat, where the microdensitometric measurements were performed. A corresponds to the level of Fig. 1, The various measuring fields (striped areas) are shown in the nTS region (ds and mnTS) as well as the measuring field within the AP (at both right and left sides). AP, area postrema; ncom, nucle,s commisuralis; dmnX. dorsal motor nucleus of the vagus; nXIl, nucleus hypoglossus; ds, dorsal strip; dPSR, dorsal parasolitary region; hi, intermediate nucleus; ni, interstitial nucleus; maTS, medial; dnTS, dorsal; dinTS, dorsolateral, vaTS, ventral; vlnTS, ventrolateral subnuclei of the nTS; "IS, tractus solitarius; IV. fourth ventricle.
~
I"! ml.pNPY (1-3e) rJ lsSl.pNP¥(1-,36)~NGII (10 aM)
' N
ranTS
ds Bregma -13.8 (obex -0,3)
AP
mnTS Bregma -13.3 (obex +0.2)
Fig. 3. Microdensitometric determination of [l~Sl]pNPY-(l-36) bindin&in various regions of the dorsomedial medulla oblongata of the male rat in the absence or presence of ANG II (10 nM). Means -- S,E,M, are shown; n = 6 rats; 0,5 nM of [nSl]pNPY-(1-36) was used. Size of areas measured: ranTS = 0.03 ± 0,007 mm2; ds = 0.03 ± 0,002 ram2; AP = 0,09 ± 0,008 mm 2. The Student's t-test was used (**P < 0,01; t = trend, 0,05 < P < 0.1),
65 NaC! 120, NaH2CO3 20, KC! 2, KH2PO4 0.5, CaC!2 1.2, MgCI2 1.8, Na2SO 4 0.5, D-glucose 5.8. Mean arterial blood pressure (MAP) and heart rate (HR) were measured by means of a heparinized (Heparin, 50 IEU/ml 0.9% NaC! w/v) plastic catheter (PE-50, Clay Adams, NY, U.S.A.) inserted into the common carotid artery, connected to a Statham PC23 DC transducer (Statham Co., Puerto Rico) and linked to a Grass polygraph (model 7; Grass Instruments, MA, U.S.A.). The implantation of the catheter was made under a-chloralose anaes-
Mean arterial blOOd pressure
A
----m---
ml~.RI¢
+ANG II (3 nmol)
|
,
thesia on the day of the cardiovascular experiments, The rats were allowed to recover until blood rl'essurc was stabilized. Registrations were performed as described earlier2°'22. Basal values were registered every 5 rain during a period of 15 rain before the i.c. injection. Respiratory rate (RR) was recorded via a cannula in the oesophagus, also connected to the Grass polygraph via a pressure transducer. The body temperature was monitored during the experiment and kept constant at 37.5 °C by means of a thermostatic device. Measurements of MAP, HR and RR were made during the following 1 h time interval, and the area created by the curve was calculated for each parameter and for each animal using an IBM-XT computer and a program developed by Guna Consult, Stockholm, Sweden "°. The area values (overall effects) were expressed as absolute values in arbitrary units reflecting the duration of the effect under 60 rain, and the peak effects (maximal responses) as per cent changes from the respective mean basal values. EDso values were calculated using iterative, non-linear curve fitting procedures s. The possible ANG II dose-response relationship was tested with the Jonckheere-Terpstra test and the non-parametric 'treatments versus control' test to compare peaks and overall effects between ex-
|
| Mean arterial blood pressure
-I
3 #
.| o
B I
to
2o
so 4o Time (rain)
40- * ~ * (b) ]1( so - p~
.. ,,.. : 4,
2o• /
~
~
so
so
A i
mCSF pNPV(1.~s) (7.s pmot) ~ It(10 nm¢) pNPY(1.80)(7.5pmol)+ANOtl (10nmot)
500
IIIIUlIIIIIIIIIIIIIIIIIIIIIIIIIIIII lll~i~~lllllllll IIIIIIIIIIIIIIIIIIIIII IIII 0 Ill ,i////,~~ , l , l l l l l l l ~ l i l l l l l l ,
. . Re. . .a0 .
10
40
s0
eo
~
Ill
g •500
•1000
210
I
B
800
(n ¢: :l
600
~/,~,/","~'~
~ /t * --.'k pNPY (1-36) ANG II (3 nmol) pNPY(1-36)(75pmol) (75 pmol) + ANG II (3 nmol)
Time (m~n) Fig. 4, Time course of the percent effects on MAP (m~n Hg) by separate or simultaneous i.e. administration of pNPY-(I-36) and AHG II in the a-chloralose (100 mg/kg, via the hngual vein) anaesthetized male rat. A: pNPY-(1-36) was administered in a dose of 75 pmol/rat (close to the EDso value for its central vasodepressor, bradycardic and bradypneic action19). The interactions with a threshold dose of ANG II (3 nmol/rat; see Table I) is shown. B, the interactions of i.e. administered pNPY-(1-36) in a threshold dose (7.5 pmol) are shown with a dose of ANG il (10 nmol) that is the EDs0 value for the vasopressor peak and vasopressor area (see text). MAP basal values (ram Hg) are for each group in A: mCSF = 114 -. 5; pNPY-(1-36) (75 pmol) - 110 - 6; ANt, II (3 nmol) - 112 - 2; pNPY-(I-36) (75 pmol) + ANG II (3 nmol) 110 - 5. MAP basal values (mm Hf) in B are: mCSF = 114 -+ 5; pNPY-(1-36) (7.5 pmol) - 110 -. J;; ANG II (10 nmol) - 107 3; pNPY-(1-36) (7.5 pmol) + ANG II (10 nmol) -- 108 ¢ 5. Means ± S.E.M. are shown as percent change from the respective mean basal value; n - 4-6. The Dunn's test was used to compare different groups (*P < 0.05; ***P < 0.005); a, comparisons with the peak effects of the other experimental groups; b, comparisons with the mCSF and pNPY-(1-36) (7.5 pmol) group peak values; c, comparison of the pressor peak of ANG II (10 nmol) vs the other experimental groups except for the pNPY-(1-36) (7.5 pmol) + ANG II (10 nmol) group.
s..
400
qee ,i
,,o Im ,4
200
pNPY (1-36) ANG II (10 nmol) pNPY (1-36)(7.5pmol) (7.5 pmol) + ANG II (10 nmol) Fig. 5. O~'erall effects (area values) on MAP of separate or simultaneous i.c. administration of pNPY-(1-36) and ANG II in the a.chloralose-anaesthetized male rat. The effects shown in A and B, respectively, have been obtained in the same experiments as illustrated in Fig. 4A and B where the time course of MAP and the basal values for each group are given. The hatched areas indicate the mean - S.E.M. of the effects of mCSF alone. Means -+ S.E.M. (arbitrary units) are shown; n - 4-6. The Dunn's test wa~ used to compare different groups (*P < 0.05; ***P < 0.005: the stars in the columns indicate significance with respect to the mCSF area value).
66 perimental groaps and control. For comparison~ between different experimental gt'oups the Dunn's test was used2s.
the nucleus tractus solitarius (mnTS) was unaffected by ANG lI (10 nM).
FESULTS
ANG H immunocytochemistry
Receptor autoradiographical analysis In vitro incubation with ANG II (10 nM) led to a significant increase in the [i~I]pNPY-(1-36) binding at a concentration of 0.5 nM wkhin the ds (dorsal cardiovascular region of nTS) and a trend for an increase of the [l~I]pNPY.(l-36) binding in the AP (Figs. 1-3). The [nSl]pNPY.(l-36) binding in the medial subnucleus of
As seen ia Fig. 8 intracisternal injection of ANG II (10 nmol) leads after 5 min to the appearance of a 200• 300 ~m wide zone of ANG II IR with a somewhat dotted appearance in the floor of the 4th ventricle involving
A g i
Heart rate
A O =i
e
