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Spinal substance P transmits bradykinin but not osmotic stimuli from hepatic portal vein to hypothalamus in rat
f?eur#seience Vol. 11, No. Printed in Great Britain
4, pp. 903-912,
0~5~/84
1984
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Pergamon Press Ltd @ 1984IBRO
SPINAL SUBSTANCE P TRANSMITS BRADYKININ BUT NOT OSMOTIC STIMULI FROM HEPATIC PORTAL VEIN TO HYPOTHALAMUS IN RAT L. STOPPINI, F. BARJA, R. MATHWN and J. BAERTSCHI* Department of Animal Biology, University of Geneva, 1211 Geneva 4, Switzerland Abet-Pe~phe~l osmo- and b~dy~n-s~sitive receptors which have been previously localised within the hepa& portal vein area, activate the hypothal~o-neur~h~ophysial system through a neural pathway projecting to the lower thoracic spinal cord. In this paper we attempted to identify the spinal transmitter(s) involved and to answer the question whether osmoreceptors are in fact chemosensitive nociceptors. T’he portal vein of anesthetised rats was superfused with 0.2ml of 4% NaCl or 1 PM bradykinin, and hypothalamo-neurohypophysial responses were measured either electrophysiologically or by radioimmunoassay of arginine vasopressin. Responses to bradykinin, but not to hypertouic saline, were abolished in rats pretreated 2 wks previously with capsaicin s.c., and ~~o~t~h~st~ for substance P in these animals showed that substance P was strongly depleted both in the dorsal thoracic spinal cord and in the portal vein. The spinal injection of 8~1 0.1 mM capsaicin at T&T9 elicited a pronounced hypothalamo-neurohypophysial response, and diminished reversibly the response to bradykinin superfusion of the portal vein. Spinal capsaicin had no effect on responses to hypertonic saline. Similarly, the spinal (TS-T9) injection of 8 ,I.J~substance P antagonist, the [D-Pro”, ~-Trp~.~*‘~,Vals]substance P (4-l 1), reduced reversibly the responses to bradykinin by about 50% without affecting those to hypertonic saline. The spinal injection of 8 fig substance P, at the same site where substance P antagonist was applied, elicited within 4s a prolonged response (several min). A slightly longer delay between stimulus and neuroph~ioio~~l response was observed for spinal capsaicin and for bradykinin superfusion. Responses to hypertonic saline superfusion of the portal vein, however, occurred within l-2 s. The results show that portal vein osmoreceptors are distinct from chemo-sensitive nociceptors, and suggest that substance P may be a spinal mediator for chemo-sensitive portal vein nociceptors. The spinal transmitter for osmosensitive afferents, and the physiological importance of the portal vein area in chemosensation remain to be established.
we have reported that the hepatic portal vein area of the rat contains sodium chloride sensitive receptors that activate the hypothalamoneurohypophysial system.*’ Subsequently, these receptors were shown to be cholinergic osmoreceptors and to induce the re&ase of arginine vasopressin through a spinal pathway.4,22 During investigations on the cellular mechanisms of activation of this osmosensitive area, we applied various vasoactive substances to the hepatic portal vein to check whether the subsequent responses to osmotic stimulation could be- modified by local distension or ~n~action of portal vein tissue. Although this was not the case, we noticed that one of the substances tested, bradykinin, was able to elicit through a spinal pathway a pronounced response of the hypothalamoneurohypophysial system.*’ In view of the possible involvement of substance P as a spinal trans~tter for noxious chemical stimuli,‘2,‘6*24in particular those induced by bradykinin,23 we examined if substance P should be considered seriously as a spinal transmitter for chemically induced visceral pain, and also if the portal vein osmoreceptors were in fact identical to bradyki~n-sensitive nociceptors.
EXPERIMENTAL PROCEDURES
Previously,
*To whom correspondence should be addressed. Abbreviations: AVP, arginine vasopressin; CBP, compound action potential.
Surgicai preparation Neurophysiological experiments. Female rats of 250-350 g body wt were anaesthetised with urethane (0.4g in 2 ml 0.9% NaCl/300 g body wt, i.p.f and cannulas were inserted into the trachea and into a jugular vein. The basal hypothalamus was approached transpha~n~~ly and the medial part of the median eminence and stalk were exposed for positioning the electrodes (see below). In one half of the animals, a flexible stainless steel cannula of 0.3 mm outer diameter and 60 mm length was inserted into the spinal cord at L3 through a dorsal root and advanced u&l the tip reached the level T8-T9. The caudal end was connected bv a PPlO cannula to a Hamilton syringe of 10 ~1; total deai volume of the tubings was 10~1. Correct position of the tip was checked post-mortem by injection of methylene blue. The abdominal wall was opened by a midline incision, and intraperitoneal tissues were pushed aside with cotton wool in order to expose the hepatic portal vein for stimulating its sensory structures (see below). The abdominal incision was covered with cotton wool and ~~iniurn foil in order to prevent evaporative water loss. Studies on vasopressin release. Female rats of 250-35Og body wt were anaesthetised with pentobarbital (Nembutal, 15 mg/3OOg, i.p.). This type of anaesthesia was chosen because “basal” plasma levels of arginine vasopressin (AVP) were much lower than with urethane. Cannulas were inserted into the trachea, into a jugular vein for infusing a Ringer solution, and into a mmmon carotid artery for withdrawing blood samples (see protocols). Abdominal surgery was performed as above. Electrophysiological recordings
Antidromic compound action potentials (CAP) were recorded with a glass micropipette (tip diameter 1-2pm), 903
904
filled with 4M NaCI, within the hypothalamoneurohypophysial tract. Biphasic 0.2-0.4 ms pulses were applied once per second at constant current (0.52 mA) through a stimulating electrode positioned at the junction of the pituitary stalk with the hypophysis. A decrease of the CAP amplitude has previously been correlated with an increase of orthodromic impulse traffic and neurohypophysial hormone secretions.’ The CAP amplitude was continuously plotted on-line on the chart by an electronic peak-detector. Stable recordings could be made for several hours and this permitted us to repeatedly apply peripheral stimuli to the same animal in the presence or absence of various pretreatments and antagonists (see below). Hormone measurements Prorocols. The first blood sample was collected in ethylenediaminetetraacetate containing tubes on ice 30 min after the end of surgery by withdrawing arterial blood at the rate of 0.6ml/min over I min, and by replacing it with Ringer solution i.v. at the same rate. A peripheral stimulus (see below) was applied to the portal vein 30s later, and the collection of the second blood sample started 15-30 s thereafter. Following a I2 min rest period to allow hormone levels to return to prestimulation values, the same sequence of blood sample collection and stimulation was initiated, except that a ditTerent stimulus was applied (hypertonic NaCl if the first stimulus was bradykinin, or vice versa). The blood samples were centrifuged at 4°C. the plasma collected and frozen at -20°C pending assay. Radioimmunoassay of arginine aasopressin. The method as adapted from Dogterom ” has already been applied in a previous study on portal vein osmoreceptors.’ Briefly, vasopressin was extracted with Vycor glass powder. Recovery, as checked with Brattleboro rat blood. was 82.1 + 2.87; (mean + SEM). The AVP tracer came from New England Nuclear, the AVP standard (504 U/m@ was a gift of Dr. K. Lederis (Calgary). The AVP-antiserum, a gift from Dr. J. Diirr (Denver), was diluted 600,000 times. The antiserum does not crossreact with adrenocorticotropic hormone, endorphin, DD-AVP, oxytocin, vasotocin or the enkephalins. Sensitivity of the assay was 0.61 pg/ZOO~1 sample. SIimulation of the heparic porral cein area Osmoreceptors. We superfused the hepatic portal vein with 0.2ml of a 40/o NaCI solution; much lower concertRations also were able to activate the hypothalamoneurohypophysial system,J,21 but the 43,; NaCI, 0.2 ml stimulus was adopted for obtaining repeatedly clear-cut responses. Intervals between two osmotic stimulations were at least 7 min, and the portal vein was rinsed previously with 0.5ml of 0.45% NaCl and excess fluid was sponged with cotton wool. No habituation of the response was observed under these conditions. Bradyknin receptors. The portal vein was superfused with 0.2 ml of bradykinin, 0.9% NaCl solution (O.Ol-IOpM). Habituation of the hypothalamo-neurohypophysial response was negligible when stimulations with I PM bradykinin were separated by at least I5 min, and when the portal vein was rinsed in between each treatment. The user of higher concentrations (IOpM bradykinin) led to tachyphylaxis. Bradykinin and osmotic stimulations, when applied alternatively. did not crossreact with each other. Pretrealments,
drugs and peprides
Capsaicin prelrearmenls.
Since capsaicin pretreatments in adult rats are known to diminish the content of sensory spinal neurones in substance P and other peptides,‘2.‘s~‘Brats were injected S.C.with increasing doses of caps&in (Merck) over 2 days, 25 mg/kg on the first day, and 50mg/kg and I50 mg/kg in separate injections on the 2nd day, 15-20 days before the experiments (immunocytochemistry and hormone measurements). Control animals were injected with solvent alone. that is 10% ethanol and 10% Tween 80 (Merck) in 0.9’?; NaCl solution.
Spinal icjecrions. The possibility. that spinal substance I’ may be a link in sensory transmission from portal \,cin bradykinin receptors to the hypothalamus. was tested by injecting various compounds into the spinal cord. Tn that effect a stock solution of 0.1 M capsaicin was prepared m IO”,; Tween 80. IOY;,ethanol and 0.9y; NaCI. and the solution was diluted IOOO-foldin 0.9’!;,NaCI before injecting 8 ~1 amounts intraspinally (T8-TIO). Furthermore. peptides, substance P and somatostatin (Beckman) and a substance P antagonist (see below) were also injected m 8 111 (8 pg) amounts. The stock solution were made of I mgml peptide in 0.2Sy0 acetic acid and 0.9Ya NaCI. This solution was adjusted to pH 7.4 with NaHCO, before injection. The spinal cannula was flushed with 10 111vehicle or 0.9”,, Nac‘l. Subsrances P anragonisfs. The agonistic and antagonistic actions of various substance P antagonists have been reported in detail elsewhere.” In the present work we have used the new antagonist [D-Pro4, tl-Trp”,‘“, Valx]substance P (4-i I)” that has been manufactured by Bachem (Basel). This antagonist had the lowest agonistic action of all antagonists tested.
The direct immunofluorescence method of Coons and Kaplan’ was employed for the localization of substance P in the portal vein and the dorsal horn of the spinal cord. Animals, with or without capsaicin pretreatment, were prepared by intracardiac perfusion with lOOmI of Tyrode solution, followed by 200ml of 4% formaldehyde in 0.1 M phosphate buffer @H 7.2). The portal vein and thoracic spinal cord (T8-TIO) were removed from the animal, and immersed in the same fixative overnight. After washing for 24 h in 5”, (w/v) sucrose, spinal cords were sectioned frontally on a cryostat (IOpm) and portal veins were prepared as whole mounts (spreads). Rabbit antiserum against substance P raised in our laboratory’ was applied at a final dilution of I :400 to the spinal cord sections and I :240 to the portal veins. Following an overnight incubation at 4°C with the antiserum the tissues were rinsed in phosphatebuffered saline and then incubated with fluorescein isothiocyanatetonjugated anti-rabbit immunoglobulin G a dilution of I : 100 for 1.5 h at room temperature. After a final rinsing the tissues were mounted in buffered glycerol (I :3) and examined with a Leitz Orthoplan microscope. Sections and whole mounts. incubated with the antiserum previously preabsorbed with an excess of exogeneous substance P (IO-’ M), served as controls. RESULTS Hypothalamo-neurohypophysial vein stimuli
responses to portal
The superfusion of the hepatic portal vein with 0.2 ml of 1PM bradykinin resulted in a decrease of the antidromic compound action potentials (CAP) recorded within the hypothalamo-neurohypophysial tract. Compared to the response to 4% NaCI, the decrease of CAP amplitude was about the same, but occurred with a detay of S-6 s instead of 1-2 s (Fig. 1). The subsequent response to the 4% NaCl superfusion was not changed by the bradykinin stimulus. Infusion of the same amount of bradykinin into the jugular vein had no effect on CAP amplhudes (results not shown), although larger amounts do so as reported previously.3 The decrease in CAP amplitude induced by bradykinin was dose dependent (10-8-10 -‘M; Fig. 2). Further evidence that bradykinin acted locally and not centrally was given by
Spinal transmission for visceral afferents
I
lmin
I
Fig. 1. Response of the hypothalamo-neurohypophysial system to portal vein superfusions with 4% NaCl or 1O-6M bradykinin (volume 0.2ml). Responses were measured by the amplitude of tire antidromic compound action potential (CAP) monitored continuously within the hypotbalamoneuroh~ophysial tract. The responses were obtained in a normal rat in the sequence indicated by the figure (top to bottom) at 9min intervals (control = RIO%,amplitude before stimulation). the fact that local anaesthesia of the hepatic portal vein area with xylocaine (2%, As&a) abolished the hypothalamo-neurohypophysial responses to both bradykinin and 4% NaCl superfusions. To investigate if the NaCl superfusion did act as a nociceptive chemical stimulus rather than as an os-
905
motic stimulus, the same experiment was repeated in rats pretreated 2 wks previously with capsaicin. This pretreatment is supposed to abolish chemically induced pain.14 As shown in Fig. 3, even the large dose of 10 PM bradykinin in 0.2 ml of 0.9% NaCl was not able to elicit a detectable hypothalamoneuroh~ophysial response. In contradistinction, the responses to 4% NaCl su~rfusions were as large as in control animals. In groups of five-seven rats pretreated or not with capsaicin, CAP responses and vasopressin measurements were obtained for both the bradykinin and osmotic stimulus (Fig. 4). Both types of measurements gave identical results, as the CAP responses and the vasopressin secretions to bradykinin superfusion of the portal vein were abolished when the rats were pretreated with capsaicin. Responses to the osmotic stimulus were perfectly preserved. To check if substance P was depleted in the capsaicin treated animals, the portal vein and spinal cord at level T&T10 were examined for substance P immuno~acti~ty. In all three pretreated rats, substance P was no longer detectable in the portal vein and was also strongly diminished in the dorsal spinal cord (Fig. 8). The T8-TlO levels were studied in detail because other experiments indicated that the bradykinin-sensitive fibres enter the spinal cord at this level (see below). Hypothalamo-neurohypophysial responses in the presence of spinal capsaicin, substance P and substance P antagonist
The spinal injection of capsaicin at T8-T9 elicited a large decrease of the CAP amplitude (Fig. SC). Responses to an osmotic stimulus applied to the portal vein were not changed by previous application of spinal capsaicin (Fig. 5E), but the response to portal vein bradykinin was strongly diminished (Fig. 5D). Partial recovery of this response was achieved ____y&-.-v-----
_r,-d--
.p.,__...t.--3.
l% NaCl 100 50
*_,**t.+. ._... _~._.*~~,.~“:....~-...--*~~~.-~
i-5,
BRABYKIWN
BRADYKININkLogbl) Fig. 2. Dose--response curve for the hypothalamoneurohypophysial response to varying doses of bradykinin superfused onto the portal vein of normal rats. The maximal decrease of the CAP amplitude was normalized with respect to that observed with 10m6M brady~nin in each rat, and means f SEM from three to eight rats have been indicated.
Volume of each superfusion was 0.2ml.
Fig. 3. Response of the hypothalamo-neurohypophysial system to portal vein superfusion with 4”/, NaCl and 10e5 M bradykinin (volume 0.2ml) in capsaicin-treated rat. Responses were obtained in the same animal in the sequence indicated by the figure (top to bottom) at 9min intervals (control = lOOo/ CAP amplitude before stimulation).
L. Stopprni cl ul
E
T
Fig. 4. Response of the hypothalamo-neurohypophysial system (left) and vasopressin release (right) in normal rats and rats pretreated with capsaicin i.p. 2 wks before the experiment. The responses of CAP amplitude were normalized in each group of rats (n = 611) with respect to the response obtained with 4% Nail superfusions (= 1WA). The release of vasoprauin, studied in other rats was expressed by the increase of AVP concentration after stimulation (=AAVP) over the initial concentration (n = 6-7). Restimulation concentrations rauged from 66 to 124 p&ml in the control group and 3&98 pg/ml in the treated group and were drawn at the 0 level f SEM. The AVP for normal rats increased by 70 f 25 pg/ml (mean f SEM) in response to the 4% NaCl superfusions. in the vasopreasin group of normal rats, four had received no pretreatment (no vehicle), and two had tived the vehicle. The results from the vehicle treated group were similar to those from non-treated rats.
1 h later (Fig. 5F). Results from a group of six rats are summarized in Table 1, where the maximal decrease of CAP amplitude (mean + SEM) is shown for the same stimulus sequence as shown in Fig. 5. Thus the responses to bradykinin were, on the average, z times less pronounced when capsaicin had been injected spinally 5 min previously. Recovery after 1 h was almost complete. To check if substance P may have mediated the hypothalamo-neurohypophysial responses to portal vein bradykinin superfusions, the substance P antagonist [bPro4, ~-Trp~.~.“, Val*] substance P (4-l 1) was injected spinally at tire T8-T9 level 5 min before applying bradykinin to the portal vein. Again, the responses to bradykinin, but not to hypertonic saline, were reduced markedly by this pretreatment (Fig. 6); on the average, responses to bradykinin diminished by 49.1 + 1.9% (mean + SEM, n = 5 rats) when the antagonist was injected spinally beforehand. Responses to bradykinin were not affected when the cannula was moved up or down in the spinal cord by f 20 mm and the antagonist was injected above or
below the T8-T9 level. However, injection of xylocaine at T8-T9, or above, abolished the response to both bradykinin and hypertonic saline. Finally, substance P was injected spinally at the same level where the antagonist blocked the response to bradykinin (Fig. 7). The spinal substance P elicited a very pronounced and long lasting response of the hypothalamo-neurobypophysial systetm responses to spinal substance P were also obtained when the cannula was moved
up to T2 and down
to L2.
The delay between the stimulation (spinal or portal) and the onset of the neurophysiological response was studied in detail. This delay was 1.9 + 0.2 s (mean + SEM) for the hypertonic saline, and 6.2 f 0.5 s for the bradykinin portal superfusion (same group of 22 rats). For spinal injections at the T&T10 level, the delay was 3.8 + 0.6s (n = 6) for substance P and 5.2 +_0.4 s (n = 8, different group) for capsaicin. The delay for the response to spinal substance P was increased to 9.0 + 3.0 s (n = 3) when capsaicin was injected spinally 6-l 1 min beforehand.
DISCUSSION Separate bradykinin- and osmo-receptors hepatic portal vein area
in the
Three types of experiments show that osmotic and bradykinin stimulation of the hepatic portal vein receptors are subserved by separate sensory mechanisms. First, the delay between the superfusion of the hepatic portal vein and the onset of the hypothalamo-neurohypophysti response was consistently only l-2 s for the osmotic stimulus and 5-6 s for the bradykinin stimulus (Figs I, 6 and 7). Second, responses to bradykinin superfusion were abolished 2 wks after systemic capsaicin pretmatment, whereas superfusions with hypertonic NaCl .soWons still reauRod in byP&h&mo-aeu@#f@&&il &vation and vasops&n lccntion Qzis j aud 4). Third, spinal injection of capsaicin or substance P antagonist reversibIy diminished the response to the bra-
907
Spinal transmission for visceral aKerents
10% BRAIXKININ
LYo N&l
2
I
E
lmin
I
8 0
0
k
!s 2
100
0
. _*.‘/.f--..‘_-._..--‘._______.-~“‘..-._. _...:.....: ,:_s__---. --..--_..___..__._.._--~‘”
50[
2 3
’
m
Spinal 16’~ CAP5AICIN
lC?n BRAIYYKININ
% 0 6
El 100
50[
_._A-_‘.-.e.._..:.____.___-2._..‘.-:~--------------_-..e..._ _____Y_..__.‘:=“-‘--
.
l
4% Nca
10?1 BRAIXKININ [ RECOVERY1
Fig. 5. Responses of the hypothalamo-neurohypophysial system of a normal rat to 4% NaCl and 1O-6M bradykinin superfusions of the portal vein before (A, B) and 5-6 min after spinal capsaicin (D, E). Partial recovery to bradykinin is obtained 1h later (F). Statistical results for a group of rats are indicated in Table 1. dykinin stimulus, but had no effect on the response to osmotic stimulation (Figs 5, 6; Table 1). The long delay between the onset of the bradykinin superfusion and the onset of the hypothalamic response may have occurred either at the peripheral or at the spinal level. The induction of prostaglandin synthesis has been advanced as an intermediary step in the action of bradykinin (Ref. 19 for review). We did, however, note no significant effects of prostaglandin F,,(10-4 M in 0.6 ml 0.9% NaCl) superfusion of the portal vein, and portal pretreatments with indomethacin (IO-’ M in 0.6 ml 0.9% NaCl) did not alter the hypothalamo-neurohypophysial responses to portal bradykinin (results not shown). The slow neuronal activation by substance P, on the other hand, has often been demonstrated (e.g. Henry’3) as is also the case for the induction of scratching behavior by this peptide.
Assuming that substance P may be involved in the neurotransmission of the bradykinin stimulus (see below), the slow onset of responses may perhaps explain the long delay observed. In fact, spinal injection of substance P results only 4s later in an activation of the hypothalamo-neurohypophysial system (Fig. 7 and statistical data). It must be kept in mind, however, that a delayed ejection of substance P from the micropipette during electrophoresis, or diffusion from the tip of the cannula to the site of action may account for part of the delay. Substance P as a neurotransmitter sensitive afferents
for
bradykinin
It has been generally recognized in recent years that capsaicin pretreatment of adult rats leads to depletion of primary sensory neurones not only in substance P,” but also in somatostatin’2J* and perhaps other
908 Table
I
Response,
(n - 6; last column bradykmm
of rhe hypolhalamo-neur[)hypophyslal ,I = 4) to superfusmn
before
and
after
spmal
of rhe portdl capwan
(xx
system
of normal
ve,” wth
4”,. IriaCI
protocol
11, legend
ral~
or 10. ‘M I’I~.
5)
Sumulus. NaCI
4”.,
IOO”,, Responses
of CAP
respect
BK’
(‘apsalunt
BK:
NaC‘I
x3 +8
l47zY
3x+x
93,
amplitude
to the first
indicates
(“,, decrease)
response
6 7 min
after
Wapsaun capsaicin:
IO ’ BK
’
M. X pl:
$BK
100
capsaicm;
&NtCI
4’;,
_---_ _-__=_ :
____=-‘___
_--------5
-__..-___...
4% Noa
16; 6WSKI#ZN
1 min
_ --;:_
I
0
-
--_
‘_....‘.
. _-_____-_.~_;_‘_“-
.5-z.: .-_
____ _____/
=-_ - _:_-
.--=_-__-
--__.=--..
B
. 4% NOCI
l&4 BIwwaMN
[SP AfnmaasTl
[SP-mrl
0
8
50 [
with Table
m
8
100
rat
l
1
[
each = IW,,).
stimuli. Since substance P has been proposed as a possible spinal transmitter for chemically (bradykinin) induced nociceptive stimuli applied intraarterially to the rat hind limb,23 we tested the hypothesis that it may also be a transmitter for chemical nociceptive stimuli applied to the viscera, in particular to the portal vein area. Three findii support this hypothesis. First, capsaicin pretreatntent abolished
..a----.
-2_. ,_~_,_.__-_-._:-----
100
within
(control
5 mm after
.-.*-. .__.Q L-
_A
XI +7
(Recovery).
peptides (See Ref. 6 for review). Acute application of capsaicin to sensory neurones leads to the release of both substance P and somatostatin, the release of substance P being six times larger than that of somatostatin.” Although capsaicin treatment may not be as selective for substance P depletion as originally thought, substance P remains a prime candidate in the neurotransmission of nociceptive
50
normalized
superfusion
BK’
I5
means f SEM.
lBK = Bradykinin:
50
were
to 4” ,, NaCl
J‘ ,,b
--_-_ -_-_ _, _~____z,__,‘.__~~~-_~=_-_--~ -=-------~=_~.~___-._==----“--------, _ _- _-l
. 4% MCI
ti6n
BECOVERvl
DSi%MRYl
Fig. 6. Responses of the hypothaiamo-neuroh ysiol system of a nor+ rat to 4% NaCl and toe6 M bradykinin (vol 0.2 ml) before (A, 9, 5-10 nX@aher (C, D) and 1 h after (E, F) spinai injection (‘l9) of a substance P (SP) antagonist (8 ~8).
Fig. 8. hnnma~ for subaance P in spinal cord and portal vein (A) Et04 spinal cord (T3) P antiserum was 1:400. @I) Dorsal spinal cord cp) of * rat treated 2 wks previously with CapsPicin from the same rat a~ in (A). The caudal portal vein is shown. Dilution of antiserum was 1:240. (D) portal vein as in (C). Dilution of antiserum was I :240.
of a rat treat& 2 arks previously with vehicle S.C. diith of substance SC. same dilution of rntisrmm as in (A). (C) Spread of a portal vein Spread of the portal vein from the same rat as in (B). same region of Calibration bar is 20 jan.
911
Spinal transmission for visceral aBerents
Q 8 ***.? --‘*=**** ---*~_~‘*_*-‘t~* ~~~*~~~~~~~~~-~~~~~-.r ~**~****-‘l*_**_“‘** a 6’~
BRADYKININ
BP-ANTAGONIST]
0 *r----X--r,*_~&.*-.rr-?------
l
m
4% NaCl
lOa n BRADWIMN
[SP-ANTAGONIST1
[RECOVERYI
0 *_******-. * _**,_=-,;=I=_*-*--.~***~**~*~***--*_~.**<*=-ah_**~-~-~~~~~~.~~~*~,~~~~
so[
~***.~~~=~~=~ff****.‘~*
m SPINAL SP IQlg)
Fig. 7. Responses (A) and 4% NaCl
of the hypothalamo-neurohypophysial system of a normal rat to 10m6M bradykinin (B) superfusion of the portal vein (~010.2 ml), 3 and 10 min after spinal (T8) injection of 8 pg substance P (SP) antagonist. Response to bradykinin was obtained 1 h later (C). Spinal injection, at the same site, of 8~8 substance P elicits a pronounced and very much prolonged response (D).
hypothalamo-neurohypophysial kinin superfusion of the portal
responses to bradyvein (Figs 3 and 4),
and substance P was depleted in the portal vein as well as in the dorsal spinal cord (Fig. 8). Second, the spinal injection at T&T10 of a substance P antagonist, as well as that of capsaicin, diminished the responses to portal vein superfusions with bradykinin (Figs 5 and 6). Third, the spinal injection of substance P at the site where antagonists blocked the response, activated the hypothalamo-neurohypophysial system (Fig. 7). This response occurred within the time frame expected from the response to bradykinin superfusion. The hypothesis is also strengthened by the fact that spinal injection of somatostatin had no effect (data not shown). Another criterium, the spinal release of substance P in response to bradykinin su~rfusion of the portal vein, remains to be verified experimentally. It is still uncertain whether substance P is the only transmitter released from bradykininsensitive visceral afferents, since a permissive role of substance P in neurotransmission cannot be excluded by our studies. Possible role of portal vein chemoreceptors
From the two previous paragraphs it appears that the superfusion of the hepatic portal vein with hypertonic saline is unlikely to evoke a non-specific activation of chemosensitive nociceptors. The results confirm our previous sugg~tions,4~’ that os-
moreceptors are located within the portal vein area and probably play a role in anticipating forthcoming changes in plasma osmolality. Hypertonic saline and bradyki~n activate receptors easily from outside the portal vein, raising the question if changes in physical and chemical properties of the intra-peritoneal fluid may be involved in overall body fluid and electrolyte control. No studies have, to our knowledge, been performed to answer this question. The vasopressin released in response to bradykinin superfusion of the portal vein (Fig. 4) may possibly act on the mesenteric circulation’ or on intestinal absorption,* thus restricting blood flow and reducing the absorption of chemical irritants. In view of the potent vasodilatating properties of bradykinin, a regulatory vas~nst~cting action of vasopressin may be appropriate for local blood flow distribution within visceral organs. In this context it is interesting to note that bradykinin stimulations of skin and cornea, where capsaicin-sensitive substance P fibres have been found,” do not activate the hypothalamoneurohypophysial system (data not shown). Further experiments are clearly needed to determine the physiological importance of the hepatic portal vein area in chemosensation. Acknowledgements---Thisstudy was supported in part by the Swiss National Science Foundation, grants 3.195.82 and 3.800.80. We thank MS M. Friedli for excellent technical assistance.
REFERENCES
1. Altura B. M. (1978) Humoral, hormonal and myogenic mechanisms in microcirculatory regulation. In Microcirculation (eds Kaley G. and Altura B. M.) Vol. II, pp, 431-502. University Park Press. 2. Baertschi A. J. and D&fuss J. J. (1979) The antidromic compound potential of the hypothalamo-neurohypophysial tract, a tool for assessing posterior pituitary activity in t&o. Brain Res. 171, 437451. 3. Baertschi A. J. and Dreifuss J. J. (1981) Enkephalins, substance P, bradykinin and angiotensin II: Differential sites of action on the hypothalamo-neurohypophysial system. Brain Res. 2u), 107-l 19.
912
L. Stoppini or ul
4. Baertxhi A. J. and Vallet P. G. (1981) Osmosensitivity of the hepatic portal vein area and vasopressin release in rats. J. Physiol.. Lond. 315, 217-230. 5. Barja F. and Mathison R. D. (1982) Adrenergic and peptidergic (substance P and vasoactive intestinal polypeptide) innervation of the rat portal vein. Blood Vessels 19, 263-272. 6. Buck S.. Walsh J. H.. Yamamura H. and Buhks T. (1982) Neuropeptides in sensory neurons. f.ge Sci. 30. 1857. 1866. 7. Coons A. H. and Kaplan M. H. (1950) Localization of antigens in tissue cells. II. Improvement in a method for the detection of antigens by fluorescent antibodies. J. exp. Med. 91, I-13. 8. Dennhardt R., Lingdbach B. and Haberich F. J. (I 979) Intestinal absorption under the influence of vasopressin: Studies in unanaesthetized rats. Gut 20, 107--l 13. 9. Dobry P. J.. Piercey M. F. and Schroeder L. A. (1981) Pharmacological characterization of scratching behaviour induced by intracranial injection of substance P and somatostatin. Neurophormacology 20, 267-272. IO. Dogterom J. (1977) The release and presence of vasopressin in plasma and cerebrospinal fluid as measured by radioimmunoassay. Studies on vasopressin as a mediator of memory processes in the rat. Thesis, Utrecht. I I. Gamse R., Lackner D.. Gamse G. and Leeman S. E. (1981) Effect ofcapsaicin pretreatment on capsaicin-evoked release of immunoreactive somatostatin and substance P from primary sensory neurones. Vuunyn-Schmiedebergs Arch. Pharmuc.. 316, 38-41. 12. Gamse R., Leeman, S. E., Holzer P. and Lembek F. (1981) Differential effects of capsaicin on the content of somatostatin. substance P and neurotensin in the nervous system of the rat. Nuunyn-Schmiedebergs Arch Phurmuc. 317, 140-148. 13. Henry J. L. (1976) Effects of substance P on functionally identified units in cat spinal cord. Bruin Res. 114, 439-451. 14. Jancso N. (1966) Desensitization with capsaicin and related acylamides as a tool for studying the function of pain receptors. In Pharmacology oj’ pain, 3rd in!. Phurmuc. Meeting,. pp. 33-35. Pergamon Pr&, Oxford.
IS. Jesse1 Y. M.. lversen L. L. and Cuello A. C. (1978) Caosaicin-induced deDletion of substance P from mimarv . . s sensorv neurones. B&in Res. 152, 183-188. 16. Lundberg J. M. and Saria A. (1983) Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature 302, 251-253. 17. Mathison R. D., Stofer P., Baja F. and lmmer H. (1983) Development of a new tachykinin antagonist. Experientiu. 18. Nagy J. I., Vincent S. R., Stain= W. M. A., Fibiger, H. C., Reisine T. D. and Yamamura H. 1. (1980) Neurotoxic action of capsaicin on spinal substance P neurones. Bruin Res. 186, 4354. 19. Regoli D. and Barabe J. (1980) Pharmacology of bradykinin and related kinins. Phormuc. Rev. 32, l-46. 20. Stoppini L., Baertschi A. J., Mathison R. D. and Baja F. (1983) Neural actions of several substance P antagonists in the rat spinal cord. Neurosci. Lprrs. 21. Vallet P. and Baertschi A. J. (1980) Sodium-chloride sensitive receptors located in hepatic portal vein of the rat. Neurosci. lerts 17, 283-288. 22. Vallet, P. and Baertschi A. J. (1982) Spinal afferents for peripheral osmoreceptors
in the rat. Bruin res. 239, 271-274. 23. Wright D. M. and Roberts M. H. T. (1980) Responses of spinal neurones to a substance P analogue. noxious pinch and bradykinin. Eur. J. of Phurmuc. 64, 165-167. 24. Yaksh T. L., Farb D. H., Leeman S. E. and Jesscll T. M. (1979) lntrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia. Science, N.Y. 206, 481483 (Accepted 5 October 1983)
4, pp. 903-912,
0~5~/84
1984
$3.00 + 0.00
Pergamon Press Ltd @ 1984IBRO
SPINAL SUBSTANCE P TRANSMITS BRADYKININ BUT NOT OSMOTIC STIMULI FROM HEPATIC PORTAL VEIN TO HYPOTHALAMUS IN RAT L. STOPPINI, F. BARJA, R. MATHWN and J. BAERTSCHI* Department of Animal Biology, University of Geneva, 1211 Geneva 4, Switzerland Abet-Pe~phe~l osmo- and b~dy~n-s~sitive receptors which have been previously localised within the hepa& portal vein area, activate the hypothal~o-neur~h~ophysial system through a neural pathway projecting to the lower thoracic spinal cord. In this paper we attempted to identify the spinal transmitter(s) involved and to answer the question whether osmoreceptors are in fact chemosensitive nociceptors. T’he portal vein of anesthetised rats was superfused with 0.2ml of 4% NaCl or 1 PM bradykinin, and hypothalamo-neurohypophysial responses were measured either electrophysiologically or by radioimmunoassay of arginine vasopressin. Responses to bradykinin, but not to hypertouic saline, were abolished in rats pretreated 2 wks previously with capsaicin s.c., and ~~o~t~h~st~ for substance P in these animals showed that substance P was strongly depleted both in the dorsal thoracic spinal cord and in the portal vein. The spinal injection of 8~1 0.1 mM capsaicin at T&T9 elicited a pronounced hypothalamo-neurohypophysial response, and diminished reversibly the response to bradykinin superfusion of the portal vein. Spinal capsaicin had no effect on responses to hypertonic saline. Similarly, the spinal (TS-T9) injection of 8 ,I.J~substance P antagonist, the [D-Pro”, ~-Trp~.~*‘~,Vals]substance P (4-l 1), reduced reversibly the responses to bradykinin by about 50% without affecting those to hypertonic saline. The spinal injection of 8 fig substance P, at the same site where substance P antagonist was applied, elicited within 4s a prolonged response (several min). A slightly longer delay between stimulus and neuroph~ioio~~l response was observed for spinal capsaicin and for bradykinin superfusion. Responses to hypertonic saline superfusion of the portal vein, however, occurred within l-2 s. The results show that portal vein osmoreceptors are distinct from chemo-sensitive nociceptors, and suggest that substance P may be a spinal mediator for chemo-sensitive portal vein nociceptors. The spinal transmitter for osmosensitive afferents, and the physiological importance of the portal vein area in chemosensation remain to be established.
we have reported that the hepatic portal vein area of the rat contains sodium chloride sensitive receptors that activate the hypothalamoneurohypophysial system.*’ Subsequently, these receptors were shown to be cholinergic osmoreceptors and to induce the re&ase of arginine vasopressin through a spinal pathway.4,22 During investigations on the cellular mechanisms of activation of this osmosensitive area, we applied various vasoactive substances to the hepatic portal vein to check whether the subsequent responses to osmotic stimulation could be- modified by local distension or ~n~action of portal vein tissue. Although this was not the case, we noticed that one of the substances tested, bradykinin, was able to elicit through a spinal pathway a pronounced response of the hypothalamoneurohypophysial system.*’ In view of the possible involvement of substance P as a spinal trans~tter for noxious chemical stimuli,‘2,‘6*24in particular those induced by bradykinin,23 we examined if substance P should be considered seriously as a spinal transmitter for chemically induced visceral pain, and also if the portal vein osmoreceptors were in fact identical to bradyki~n-sensitive nociceptors.
EXPERIMENTAL PROCEDURES
Previously,
*To whom correspondence should be addressed. Abbreviations: AVP, arginine vasopressin; CBP, compound action potential.
Surgicai preparation Neurophysiological experiments. Female rats of 250-350 g body wt were anaesthetised with urethane (0.4g in 2 ml 0.9% NaCl/300 g body wt, i.p.f and cannulas were inserted into the trachea and into a jugular vein. The basal hypothalamus was approached transpha~n~~ly and the medial part of the median eminence and stalk were exposed for positioning the electrodes (see below). In one half of the animals, a flexible stainless steel cannula of 0.3 mm outer diameter and 60 mm length was inserted into the spinal cord at L3 through a dorsal root and advanced u&l the tip reached the level T8-T9. The caudal end was connected bv a PPlO cannula to a Hamilton syringe of 10 ~1; total deai volume of the tubings was 10~1. Correct position of the tip was checked post-mortem by injection of methylene blue. The abdominal wall was opened by a midline incision, and intraperitoneal tissues were pushed aside with cotton wool in order to expose the hepatic portal vein for stimulating its sensory structures (see below). The abdominal incision was covered with cotton wool and ~~iniurn foil in order to prevent evaporative water loss. Studies on vasopressin release. Female rats of 250-35Og body wt were anaesthetised with pentobarbital (Nembutal, 15 mg/3OOg, i.p.). This type of anaesthesia was chosen because “basal” plasma levels of arginine vasopressin (AVP) were much lower than with urethane. Cannulas were inserted into the trachea, into a jugular vein for infusing a Ringer solution, and into a mmmon carotid artery for withdrawing blood samples (see protocols). Abdominal surgery was performed as above. Electrophysiological recordings
Antidromic compound action potentials (CAP) were recorded with a glass micropipette (tip diameter 1-2pm), 903
904
filled with 4M NaCI, within the hypothalamoneurohypophysial tract. Biphasic 0.2-0.4 ms pulses were applied once per second at constant current (0.52 mA) through a stimulating electrode positioned at the junction of the pituitary stalk with the hypophysis. A decrease of the CAP amplitude has previously been correlated with an increase of orthodromic impulse traffic and neurohypophysial hormone secretions.’ The CAP amplitude was continuously plotted on-line on the chart by an electronic peak-detector. Stable recordings could be made for several hours and this permitted us to repeatedly apply peripheral stimuli to the same animal in the presence or absence of various pretreatments and antagonists (see below). Hormone measurements Prorocols. The first blood sample was collected in ethylenediaminetetraacetate containing tubes on ice 30 min after the end of surgery by withdrawing arterial blood at the rate of 0.6ml/min over I min, and by replacing it with Ringer solution i.v. at the same rate. A peripheral stimulus (see below) was applied to the portal vein 30s later, and the collection of the second blood sample started 15-30 s thereafter. Following a I2 min rest period to allow hormone levels to return to prestimulation values, the same sequence of blood sample collection and stimulation was initiated, except that a ditTerent stimulus was applied (hypertonic NaCl if the first stimulus was bradykinin, or vice versa). The blood samples were centrifuged at 4°C. the plasma collected and frozen at -20°C pending assay. Radioimmunoassay of arginine aasopressin. The method as adapted from Dogterom ” has already been applied in a previous study on portal vein osmoreceptors.’ Briefly, vasopressin was extracted with Vycor glass powder. Recovery, as checked with Brattleboro rat blood. was 82.1 + 2.87; (mean + SEM). The AVP tracer came from New England Nuclear, the AVP standard (504 U/m@ was a gift of Dr. K. Lederis (Calgary). The AVP-antiserum, a gift from Dr. J. Diirr (Denver), was diluted 600,000 times. The antiserum does not crossreact with adrenocorticotropic hormone, endorphin, DD-AVP, oxytocin, vasotocin or the enkephalins. Sensitivity of the assay was 0.61 pg/ZOO~1 sample. SIimulation of the heparic porral cein area Osmoreceptors. We superfused the hepatic portal vein with 0.2ml of a 40/o NaCI solution; much lower concertRations also were able to activate the hypothalamoneurohypophysial system,J,21 but the 43,; NaCI, 0.2 ml stimulus was adopted for obtaining repeatedly clear-cut responses. Intervals between two osmotic stimulations were at least 7 min, and the portal vein was rinsed previously with 0.5ml of 0.45% NaCl and excess fluid was sponged with cotton wool. No habituation of the response was observed under these conditions. Bradyknin receptors. The portal vein was superfused with 0.2 ml of bradykinin, 0.9% NaCl solution (O.Ol-IOpM). Habituation of the hypothalamo-neurohypophysial response was negligible when stimulations with I PM bradykinin were separated by at least I5 min, and when the portal vein was rinsed in between each treatment. The user of higher concentrations (IOpM bradykinin) led to tachyphylaxis. Bradykinin and osmotic stimulations, when applied alternatively. did not crossreact with each other. Pretrealments,
drugs and peprides
Capsaicin prelrearmenls.
Since capsaicin pretreatments in adult rats are known to diminish the content of sensory spinal neurones in substance P and other peptides,‘2.‘s~‘Brats were injected S.C.with increasing doses of caps&in (Merck) over 2 days, 25 mg/kg on the first day, and 50mg/kg and I50 mg/kg in separate injections on the 2nd day, 15-20 days before the experiments (immunocytochemistry and hormone measurements). Control animals were injected with solvent alone. that is 10% ethanol and 10% Tween 80 (Merck) in 0.9’?; NaCl solution.
Spinal icjecrions. The possibility. that spinal substance I’ may be a link in sensory transmission from portal \,cin bradykinin receptors to the hypothalamus. was tested by injecting various compounds into the spinal cord. Tn that effect a stock solution of 0.1 M capsaicin was prepared m IO”,; Tween 80. IOY;,ethanol and 0.9y; NaCI. and the solution was diluted IOOO-foldin 0.9’!;,NaCI before injecting 8 ~1 amounts intraspinally (T8-TIO). Furthermore. peptides, substance P and somatostatin (Beckman) and a substance P antagonist (see below) were also injected m 8 111 (8 pg) amounts. The stock solution were made of I mgml peptide in 0.2Sy0 acetic acid and 0.9Ya NaCI. This solution was adjusted to pH 7.4 with NaHCO, before injection. The spinal cannula was flushed with 10 111vehicle or 0.9”,, Nac‘l. Subsrances P anragonisfs. The agonistic and antagonistic actions of various substance P antagonists have been reported in detail elsewhere.” In the present work we have used the new antagonist [D-Pro4, tl-Trp”,‘“, Valx]substance P (4-i I)” that has been manufactured by Bachem (Basel). This antagonist had the lowest agonistic action of all antagonists tested.
The direct immunofluorescence method of Coons and Kaplan’ was employed for the localization of substance P in the portal vein and the dorsal horn of the spinal cord. Animals, with or without capsaicin pretreatment, were prepared by intracardiac perfusion with lOOmI of Tyrode solution, followed by 200ml of 4% formaldehyde in 0.1 M phosphate buffer @H 7.2). The portal vein and thoracic spinal cord (T8-TIO) were removed from the animal, and immersed in the same fixative overnight. After washing for 24 h in 5”, (w/v) sucrose, spinal cords were sectioned frontally on a cryostat (IOpm) and portal veins were prepared as whole mounts (spreads). Rabbit antiserum against substance P raised in our laboratory’ was applied at a final dilution of I :400 to the spinal cord sections and I :240 to the portal veins. Following an overnight incubation at 4°C with the antiserum the tissues were rinsed in phosphatebuffered saline and then incubated with fluorescein isothiocyanatetonjugated anti-rabbit immunoglobulin G a dilution of I : 100 for 1.5 h at room temperature. After a final rinsing the tissues were mounted in buffered glycerol (I :3) and examined with a Leitz Orthoplan microscope. Sections and whole mounts. incubated with the antiserum previously preabsorbed with an excess of exogeneous substance P (IO-’ M), served as controls. RESULTS Hypothalamo-neurohypophysial vein stimuli
responses to portal
The superfusion of the hepatic portal vein with 0.2 ml of 1PM bradykinin resulted in a decrease of the antidromic compound action potentials (CAP) recorded within the hypothalamo-neurohypophysial tract. Compared to the response to 4% NaCI, the decrease of CAP amplitude was about the same, but occurred with a detay of S-6 s instead of 1-2 s (Fig. 1). The subsequent response to the 4% NaCl superfusion was not changed by the bradykinin stimulus. Infusion of the same amount of bradykinin into the jugular vein had no effect on CAP amplhudes (results not shown), although larger amounts do so as reported previously.3 The decrease in CAP amplitude induced by bradykinin was dose dependent (10-8-10 -‘M; Fig. 2). Further evidence that bradykinin acted locally and not centrally was given by
Spinal transmission for visceral afferents
I
lmin
I
Fig. 1. Response of the hypothalamo-neurohypophysial system to portal vein superfusions with 4% NaCl or 1O-6M bradykinin (volume 0.2ml). Responses were measured by the amplitude of tire antidromic compound action potential (CAP) monitored continuously within the hypotbalamoneuroh~ophysial tract. The responses were obtained in a normal rat in the sequence indicated by the figure (top to bottom) at 9min intervals (control = RIO%,amplitude before stimulation). the fact that local anaesthesia of the hepatic portal vein area with xylocaine (2%, As&a) abolished the hypothalamo-neurohypophysial responses to both bradykinin and 4% NaCl superfusions. To investigate if the NaCl superfusion did act as a nociceptive chemical stimulus rather than as an os-
905
motic stimulus, the same experiment was repeated in rats pretreated 2 wks previously with capsaicin. This pretreatment is supposed to abolish chemically induced pain.14 As shown in Fig. 3, even the large dose of 10 PM bradykinin in 0.2 ml of 0.9% NaCl was not able to elicit a detectable hypothalamoneuroh~ophysial response. In contradistinction, the responses to 4% NaCl su~rfusions were as large as in control animals. In groups of five-seven rats pretreated or not with capsaicin, CAP responses and vasopressin measurements were obtained for both the bradykinin and osmotic stimulus (Fig. 4). Both types of measurements gave identical results, as the CAP responses and the vasopressin secretions to bradykinin superfusion of the portal vein were abolished when the rats were pretreated with capsaicin. Responses to the osmotic stimulus were perfectly preserved. To check if substance P was depleted in the capsaicin treated animals, the portal vein and spinal cord at level T&T10 were examined for substance P immuno~acti~ty. In all three pretreated rats, substance P was no longer detectable in the portal vein and was also strongly diminished in the dorsal spinal cord (Fig. 8). The T8-TlO levels were studied in detail because other experiments indicated that the bradykinin-sensitive fibres enter the spinal cord at this level (see below). Hypothalamo-neurohypophysial responses in the presence of spinal capsaicin, substance P and substance P antagonist
The spinal injection of capsaicin at T8-T9 elicited a large decrease of the CAP amplitude (Fig. SC). Responses to an osmotic stimulus applied to the portal vein were not changed by previous application of spinal capsaicin (Fig. 5E), but the response to portal vein bradykinin was strongly diminished (Fig. 5D). Partial recovery of this response was achieved ____y&-.-v-----
_r,-d--
.p.,__...t.--3.
l% NaCl 100 50
*_,**t.+. ._... _~._.*~~,.~“:....~-...--*~~~.-~
i-5,
BRABYKIWN
BRADYKININkLogbl) Fig. 2. Dose--response curve for the hypothalamoneurohypophysial response to varying doses of bradykinin superfused onto the portal vein of normal rats. The maximal decrease of the CAP amplitude was normalized with respect to that observed with 10m6M brady~nin in each rat, and means f SEM from three to eight rats have been indicated.
Volume of each superfusion was 0.2ml.
Fig. 3. Response of the hypothalamo-neurohypophysial system to portal vein superfusion with 4”/, NaCl and 10e5 M bradykinin (volume 0.2ml) in capsaicin-treated rat. Responses were obtained in the same animal in the sequence indicated by the figure (top to bottom) at 9min intervals (control = lOOo/ CAP amplitude before stimulation).
L. Stopprni cl ul
E
T
Fig. 4. Response of the hypothalamo-neurohypophysial system (left) and vasopressin release (right) in normal rats and rats pretreated with capsaicin i.p. 2 wks before the experiment. The responses of CAP amplitude were normalized in each group of rats (n = 611) with respect to the response obtained with 4% Nail superfusions (= 1WA). The release of vasoprauin, studied in other rats was expressed by the increase of AVP concentration after stimulation (=AAVP) over the initial concentration (n = 6-7). Restimulation concentrations rauged from 66 to 124 p&ml in the control group and 3&98 pg/ml in the treated group and were drawn at the 0 level f SEM. The AVP for normal rats increased by 70 f 25 pg/ml (mean f SEM) in response to the 4% NaCl superfusions. in the vasopreasin group of normal rats, four had received no pretreatment (no vehicle), and two had tived the vehicle. The results from the vehicle treated group were similar to those from non-treated rats.
1 h later (Fig. 5F). Results from a group of six rats are summarized in Table 1, where the maximal decrease of CAP amplitude (mean + SEM) is shown for the same stimulus sequence as shown in Fig. 5. Thus the responses to bradykinin were, on the average, z times less pronounced when capsaicin had been injected spinally 5 min previously. Recovery after 1 h was almost complete. To check if substance P may have mediated the hypothalamo-neurohypophysial responses to portal vein bradykinin superfusions, the substance P antagonist [bPro4, ~-Trp~.~.“, Val*] substance P (4-l 1) was injected spinally at tire T8-T9 level 5 min before applying bradykinin to the portal vein. Again, the responses to bradykinin, but not to hypertonic saline, were reduced markedly by this pretreatment (Fig. 6); on the average, responses to bradykinin diminished by 49.1 + 1.9% (mean + SEM, n = 5 rats) when the antagonist was injected spinally beforehand. Responses to bradykinin were not affected when the cannula was moved up or down in the spinal cord by f 20 mm and the antagonist was injected above or
below the T8-T9 level. However, injection of xylocaine at T8-T9, or above, abolished the response to both bradykinin and hypertonic saline. Finally, substance P was injected spinally at the same level where the antagonist blocked the response to bradykinin (Fig. 7). The spinal substance P elicited a very pronounced and long lasting response of the hypothalamo-neurobypophysial systetm responses to spinal substance P were also obtained when the cannula was moved
up to T2 and down
to L2.
The delay between the stimulation (spinal or portal) and the onset of the neurophysiological response was studied in detail. This delay was 1.9 + 0.2 s (mean + SEM) for the hypertonic saline, and 6.2 f 0.5 s for the bradykinin portal superfusion (same group of 22 rats). For spinal injections at the T&T10 level, the delay was 3.8 + 0.6s (n = 6) for substance P and 5.2 +_0.4 s (n = 8, different group) for capsaicin. The delay for the response to spinal substance P was increased to 9.0 + 3.0 s (n = 3) when capsaicin was injected spinally 6-l 1 min beforehand.
DISCUSSION Separate bradykinin- and osmo-receptors hepatic portal vein area
in the
Three types of experiments show that osmotic and bradykinin stimulation of the hepatic portal vein receptors are subserved by separate sensory mechanisms. First, the delay between the superfusion of the hepatic portal vein and the onset of the hypothalamo-neurohypophysti response was consistently only l-2 s for the osmotic stimulus and 5-6 s for the bradykinin stimulus (Figs I, 6 and 7). Second, responses to bradykinin superfusion were abolished 2 wks after systemic capsaicin pretmatment, whereas superfusions with hypertonic NaCl .soWons still reauRod in byP&h&mo-aeu@#f@&&il &vation and vasops&n lccntion Qzis j aud 4). Third, spinal injection of capsaicin or substance P antagonist reversibIy diminished the response to the bra-
907
Spinal transmission for visceral aKerents
10% BRAIXKININ
LYo N&l
2
I
E
lmin
I
8 0
0
k
!s 2
100
0
. _*.‘/.f--..‘_-._..--‘._______.-~“‘..-._. _...:.....: ,:_s__---. --..--_..___..__._.._--~‘”
50[
2 3
’
m
Spinal 16’~ CAP5AICIN
lC?n BRAIYYKININ
% 0 6
El 100
50[
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.
l
4% Nca
10?1 BRAIXKININ [ RECOVERY1
Fig. 5. Responses of the hypothalamo-neurohypophysial system of a normal rat to 4% NaCl and 1O-6M bradykinin superfusions of the portal vein before (A, B) and 5-6 min after spinal capsaicin (D, E). Partial recovery to bradykinin is obtained 1h later (F). Statistical results for a group of rats are indicated in Table 1. dykinin stimulus, but had no effect on the response to osmotic stimulation (Figs 5, 6; Table 1). The long delay between the onset of the bradykinin superfusion and the onset of the hypothalamic response may have occurred either at the peripheral or at the spinal level. The induction of prostaglandin synthesis has been advanced as an intermediary step in the action of bradykinin (Ref. 19 for review). We did, however, note no significant effects of prostaglandin F,,(10-4 M in 0.6 ml 0.9% NaCl) superfusion of the portal vein, and portal pretreatments with indomethacin (IO-’ M in 0.6 ml 0.9% NaCl) did not alter the hypothalamo-neurohypophysial responses to portal bradykinin (results not shown). The slow neuronal activation by substance P, on the other hand, has often been demonstrated (e.g. Henry’3) as is also the case for the induction of scratching behavior by this peptide.
Assuming that substance P may be involved in the neurotransmission of the bradykinin stimulus (see below), the slow onset of responses may perhaps explain the long delay observed. In fact, spinal injection of substance P results only 4s later in an activation of the hypothalamo-neurohypophysial system (Fig. 7 and statistical data). It must be kept in mind, however, that a delayed ejection of substance P from the micropipette during electrophoresis, or diffusion from the tip of the cannula to the site of action may account for part of the delay. Substance P as a neurotransmitter sensitive afferents
for
bradykinin
It has been generally recognized in recent years that capsaicin pretreatment of adult rats leads to depletion of primary sensory neurones not only in substance P,” but also in somatostatin’2J* and perhaps other
908 Table
I
Response,
(n - 6; last column bradykmm
of rhe hypolhalamo-neur[)hypophyslal ,I = 4) to superfusmn
before
and
after
spmal
of rhe portdl capwan
(xx
system
of normal
ve,” wth
4”,. IriaCI
protocol
11, legend
ral~
or 10. ‘M I’I~.
5)
Sumulus. NaCI
4”.,
IOO”,, Responses
of CAP
respect
BK’
(‘apsalunt
BK:
NaC‘I
x3 +8
l47zY
3x+x
93,
amplitude
to the first
indicates
(“,, decrease)
response
6 7 min
after
Wapsaun capsaicin:
IO ’ BK
’
M. X pl:
$BK
100
capsaicm;
&NtCI
4’;,
_---_ _-__=_ :
____=-‘___
_--------5
-__..-___...
4% Noa
16; 6WSKI#ZN
1 min
_ --;:_
I
0
-
--_
‘_....‘.
. _-_____-_.~_;_‘_“-
.5-z.: .-_
____ _____/
=-_ - _:_-
.--=_-__-
--__.=--..
B
. 4% NOCI
l&4 BIwwaMN
[SP AfnmaasTl
[SP-mrl
0
8
50 [
with Table
m
8
100
rat
l
1
[
each = IW,,).
stimuli. Since substance P has been proposed as a possible spinal transmitter for chemically (bradykinin) induced nociceptive stimuli applied intraarterially to the rat hind limb,23 we tested the hypothesis that it may also be a transmitter for chemical nociceptive stimuli applied to the viscera, in particular to the portal vein area. Three findii support this hypothesis. First, capsaicin pretreatntent abolished
..a----.
-2_. ,_~_,_.__-_-._:-----
100
within
(control
5 mm after
.-.*-. .__.Q L-
_A
XI +7
(Recovery).
peptides (See Ref. 6 for review). Acute application of capsaicin to sensory neurones leads to the release of both substance P and somatostatin, the release of substance P being six times larger than that of somatostatin.” Although capsaicin treatment may not be as selective for substance P depletion as originally thought, substance P remains a prime candidate in the neurotransmission of nociceptive
50
normalized
superfusion
BK’
I5
means f SEM.
lBK = Bradykinin:
50
were
to 4” ,, NaCl
J‘ ,,b
--_-_ -_-_ _, _~____z,__,‘.__~~~-_~=_-_--~ -=-------~=_~.~___-._==----“--------, _ _- _-l
. 4% MCI
ti6n
BECOVERvl
DSi%MRYl
Fig. 6. Responses of the hypothaiamo-neuroh ysiol system of a nor+ rat to 4% NaCl and toe6 M bradykinin (vol 0.2 ml) before (A, 9, 5-10 nX@aher (C, D) and 1 h after (E, F) spinai injection (‘l9) of a substance P (SP) antagonist (8 ~8).
Fig. 8. hnnma~ for subaance P in spinal cord and portal vein (A) Et04 spinal cord (T3) P antiserum was 1:400. @I) Dorsal spinal cord cp) of * rat treated 2 wks previously with CapsPicin from the same rat a~ in (A). The caudal portal vein is shown. Dilution of antiserum was 1:240. (D) portal vein as in (C). Dilution of antiserum was I :240.
of a rat treat& 2 arks previously with vehicle S.C. diith of substance SC. same dilution of rntisrmm as in (A). (C) Spread of a portal vein Spread of the portal vein from the same rat as in (B). same region of Calibration bar is 20 jan.
911
Spinal transmission for visceral aBerents
Q 8 ***.? --‘*=**** ---*~_~‘*_*-‘t~* ~~~*~~~~~~~~~-~~~~~-.r ~**~****-‘l*_**_“‘** a 6’~
BRADYKININ
BP-ANTAGONIST]
0 *r----X--r,*_~&.*-.rr-?------
l
m
4% NaCl
lOa n BRADWIMN
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Fig. 7. Responses (A) and 4% NaCl
of the hypothalamo-neurohypophysial system of a normal rat to 10m6M bradykinin (B) superfusion of the portal vein (~010.2 ml), 3 and 10 min after spinal (T8) injection of 8 pg substance P (SP) antagonist. Response to bradykinin was obtained 1 h later (C). Spinal injection, at the same site, of 8~8 substance P elicits a pronounced and very much prolonged response (D).
hypothalamo-neurohypophysial kinin superfusion of the portal
responses to bradyvein (Figs 3 and 4),
and substance P was depleted in the portal vein as well as in the dorsal spinal cord (Fig. 8). Second, the spinal injection at T&T10 of a substance P antagonist, as well as that of capsaicin, diminished the responses to portal vein superfusions with bradykinin (Figs 5 and 6). Third, the spinal injection of substance P at the site where antagonists blocked the response, activated the hypothalamo-neurohypophysial system (Fig. 7). This response occurred within the time frame expected from the response to bradykinin superfusion. The hypothesis is also strengthened by the fact that spinal injection of somatostatin had no effect (data not shown). Another criterium, the spinal release of substance P in response to bradykinin su~rfusion of the portal vein, remains to be verified experimentally. It is still uncertain whether substance P is the only transmitter released from bradykininsensitive visceral afferents, since a permissive role of substance P in neurotransmission cannot be excluded by our studies. Possible role of portal vein chemoreceptors
From the two previous paragraphs it appears that the superfusion of the hepatic portal vein with hypertonic saline is unlikely to evoke a non-specific activation of chemosensitive nociceptors. The results confirm our previous sugg~tions,4~’ that os-
moreceptors are located within the portal vein area and probably play a role in anticipating forthcoming changes in plasma osmolality. Hypertonic saline and bradyki~n activate receptors easily from outside the portal vein, raising the question if changes in physical and chemical properties of the intra-peritoneal fluid may be involved in overall body fluid and electrolyte control. No studies have, to our knowledge, been performed to answer this question. The vasopressin released in response to bradykinin superfusion of the portal vein (Fig. 4) may possibly act on the mesenteric circulation’ or on intestinal absorption,* thus restricting blood flow and reducing the absorption of chemical irritants. In view of the potent vasodilatating properties of bradykinin, a regulatory vas~nst~cting action of vasopressin may be appropriate for local blood flow distribution within visceral organs. In this context it is interesting to note that bradykinin stimulations of skin and cornea, where capsaicin-sensitive substance P fibres have been found,” do not activate the hypothalamoneurohypophysial system (data not shown). Further experiments are clearly needed to determine the physiological importance of the hepatic portal vein area in chemosensation. Acknowledgements---Thisstudy was supported in part by the Swiss National Science Foundation, grants 3.195.82 and 3.800.80. We thank MS M. Friedli for excellent technical assistance.
REFERENCES
1. Altura B. M. (1978) Humoral, hormonal and myogenic mechanisms in microcirculatory regulation. In Microcirculation (eds Kaley G. and Altura B. M.) Vol. II, pp, 431-502. University Park Press. 2. Baertschi A. J. and D&fuss J. J. (1979) The antidromic compound potential of the hypothalamo-neurohypophysial tract, a tool for assessing posterior pituitary activity in t&o. Brain Res. 171, 437451. 3. Baertschi A. J. and Dreifuss J. J. (1981) Enkephalins, substance P, bradykinin and angiotensin II: Differential sites of action on the hypothalamo-neurohypophysial system. Brain Res. 2u), 107-l 19.
912
L. Stoppini or ul
4. Baertxhi A. J. and Vallet P. G. (1981) Osmosensitivity of the hepatic portal vein area and vasopressin release in rats. J. Physiol.. Lond. 315, 217-230. 5. Barja F. and Mathison R. D. (1982) Adrenergic and peptidergic (substance P and vasoactive intestinal polypeptide) innervation of the rat portal vein. Blood Vessels 19, 263-272. 6. Buck S.. Walsh J. H.. Yamamura H. and Buhks T. (1982) Neuropeptides in sensory neurons. f.ge Sci. 30. 1857. 1866. 7. Coons A. H. and Kaplan M. H. (1950) Localization of antigens in tissue cells. II. Improvement in a method for the detection of antigens by fluorescent antibodies. J. exp. Med. 91, I-13. 8. Dennhardt R., Lingdbach B. and Haberich F. J. (I 979) Intestinal absorption under the influence of vasopressin: Studies in unanaesthetized rats. Gut 20, 107--l 13. 9. Dobry P. J.. Piercey M. F. and Schroeder L. A. (1981) Pharmacological characterization of scratching behaviour induced by intracranial injection of substance P and somatostatin. Neurophormacology 20, 267-272. IO. Dogterom J. (1977) The release and presence of vasopressin in plasma and cerebrospinal fluid as measured by radioimmunoassay. Studies on vasopressin as a mediator of memory processes in the rat. Thesis, Utrecht. I I. Gamse R., Lackner D.. Gamse G. and Leeman S. E. (1981) Effect ofcapsaicin pretreatment on capsaicin-evoked release of immunoreactive somatostatin and substance P from primary sensory neurones. Vuunyn-Schmiedebergs Arch. Pharmuc.. 316, 38-41. 12. Gamse R., Leeman, S. E., Holzer P. and Lembek F. (1981) Differential effects of capsaicin on the content of somatostatin. substance P and neurotensin in the nervous system of the rat. Nuunyn-Schmiedebergs Arch Phurmuc. 317, 140-148. 13. Henry J. L. (1976) Effects of substance P on functionally identified units in cat spinal cord. Bruin Res. 114, 439-451. 14. Jancso N. (1966) Desensitization with capsaicin and related acylamides as a tool for studying the function of pain receptors. In Pharmacology oj’ pain, 3rd in!. Phurmuc. Meeting,. pp. 33-35. Pergamon Pr&, Oxford.
IS. Jesse1 Y. M.. lversen L. L. and Cuello A. C. (1978) Caosaicin-induced deDletion of substance P from mimarv . . s sensorv neurones. B&in Res. 152, 183-188. 16. Lundberg J. M. and Saria A. (1983) Capsaicin-induced desensitization of airway mucosa to cigarette smoke, mechanical and chemical irritants. Nature 302, 251-253. 17. Mathison R. D., Stofer P., Baja F. and lmmer H. (1983) Development of a new tachykinin antagonist. Experientiu. 18. Nagy J. I., Vincent S. R., Stain= W. M. A., Fibiger, H. C., Reisine T. D. and Yamamura H. 1. (1980) Neurotoxic action of capsaicin on spinal substance P neurones. Bruin Res. 186, 4354. 19. Regoli D. and Barabe J. (1980) Pharmacology of bradykinin and related kinins. Phormuc. Rev. 32, l-46. 20. Stoppini L., Baertschi A. J., Mathison R. D. and Baja F. (1983) Neural actions of several substance P antagonists in the rat spinal cord. Neurosci. Lprrs. 21. Vallet P. and Baertschi A. J. (1980) Sodium-chloride sensitive receptors located in hepatic portal vein of the rat. Neurosci. lerts 17, 283-288. 22. Vallet, P. and Baertschi A. J. (1982) Spinal afferents for peripheral osmoreceptors
in the rat. Bruin res. 239, 271-274. 23. Wright D. M. and Roberts M. H. T. (1980) Responses of spinal neurones to a substance P analogue. noxious pinch and bradykinin. Eur. J. of Phurmuc. 64, 165-167. 24. Yaksh T. L., Farb D. H., Leeman S. E. and Jesscll T. M. (1979) lntrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia. Science, N.Y. 206, 481483 (Accepted 5 October 1983)