Reduction of a centrally induced pressor response by neurohypophyseal peptides: The involvement of lower brainstem mechanisms

European Journal of Pharmacology, 94 (1983) 133-140

133

Elsevier

R E D U C T I O N O F A CENTRALLY INDUCED P R E S S O R R E S P O N S E BY N E U R O H Y P O P H Y S E A L PEPTIDES: T H E INVOLVEMENT O F LOWER BRAINSTEM M E C H A N I S M S CEES A.M. VERSTEEG, K A R L I E N C R A N S B E R G , W Y B R E N DE J O N G and BI~LA BOHUS *

Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondelloan 6, Utrecht, The Netherlands Received 8 June 1983, accepted 15 August 1983

C.A.M. VERSTEEG, K. CRANSBERG, W. DE JONG and B. BOHUS, Reduction of a centrally induced pressor response by neurohypophyseal peptides: the involvement of lower brainstem mechanisms, European J. Pharmacol. 94 (1983) 133-140. Pressor and bradycardiac responses induced by electrical stimulation of the mesencephalic reticular formation in urethane-anesthetized rats were used as model of neurogenic hypertension. Oxytocin (OXT) and prolyl-leucylglycinamide (OXT-(7-9)) administered into the fourth cerebral ventricle markedly attenuated the magnitude of the pressor response. OXT-(7-9) was somewhat more potent than OXT and its effect was dose-dependent. Microinjection of OXT-(7-9) into the dorsal raphe nucleus reduced the pressor response as well. [Argg]vasopressin (AVP) did not affect the pressor response when administered via this route, while prolyl-arginyl-glycinamide (AVP-(7-9)) had an action that was similar to that of OXT-(7-9). None of these peptides affected the magnitude of the bradycardiac response. It is suggested that OXT and related fragments modulate neurogenic hypertensive responses through lower brainstem mechanisms. Neurogenic pressor response Dorsal raphe nucleus

Fourth cerebral ventricle [Arg 8]vasopressin

1. Introduction

It is well established that the neurohypophyseal hormones vasopressin and oxytocin besides having their classical endocrine activities serve as important modulators of brain functions related to learning, memory, tolerance and addiction (for reviews see De Wied, 1977; De Wied and Versteeg, 1979; Bohus, 1981). Behavioral (De Wied, 1977; Bohus et al., 1978; Kov/lcs et al., 1979), neurochemical (Kov/lcs et al., 1977; Tanaka et al., 1977; Dunn et al., 1982) and electrophysiological observations (Urban and De Wied, 1978) suggest that the peptide actions are of central nature. In addition, the discovery of peptidergic neuronal systems in the brain that contain vasopressin and * To whom all correspondence should be addressed at present address: Department of Animal Physiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands. 0014-2999/83/$03.00 © 1983 Elsevier Science Publishers B.V.

Oxytocin

OXT-(7-9)

AVP-(7-9)

oxytocin (Sofroniew and Weindl, 1981; Buijs, 1978; Buys et al., 1978) provided the functional morphological basis for central functions of the peptides or their fragments. An additional functional property of the central peptide action has recently been recognized. Neurohypophyseal hormones or their fragments may also function as modulators of neurally induced hypertensive responses. It was found that intracerebroventricularly administered [ArgS]vasopressin (AVP) profoundly attenuated the pressor responses induced by electrical stimulation of the mesencephalic reticular formation (Versteeg et al., 1982a). This action of AVP was shared with oxytocin (OXT) and the C-tcrmi.nal tfipeptide fragments of both AVP and OXT (Versteeg et al., 1982b). However, it appeared that AVP and the C-terminal tripeptides may act in a different way. Bilateral destruction of the dorsal hippocampus in rats prevented the modulatory action of AVP on

134

the neurogenic pressor response, while the effect of OXT-(7-9) was only slightly diminished by these lesions (Versteeg, 1983). The aim of the present experiments was to investigate whether lower brain structures around the fourth cerebral ventricle are involved in diminishing acute neurogenic hypertensive responses to neurohypophyseal peptides. Structures surrounding the fourth ventricle serve as primary regulators or modulators of the cardiovascular system. In addition, vast peptidergic (vasopressin- or oxytocinergic) innervation of both p r i m a r y cardiovascular regulatory or modulatory areas such as of the nucleus tractus solitarius (NTS), dorsal vagal nerve nucleus, lateral reticular nucleus, raphe nuclei, etc. has been demonstrated (Sterba 1974; Buijs et al., 1978; Sofroniew and Weindl, 1981). Accordingly, the influence of AVP, OXT and the C-terminal tripeptide fragments of these peptides on centrally evoked pressor responses were studied in urethane-anesthetized rats following fourth cerebral ventricular injection. In addition, we investigated the effect of OXT-(7-9) microinjected into the dorsal raphe nucleus. Some of the data described in this paper have been presented in preliminary from elsewhere (Versteeg et al., 1979; Bohus et al., 1982a).

2. Materials and methods 2.1. Animals and surgery Male Wistar rats of an inbred strain (CPB-TNO, Zeist, The Netherlands) weighing between 200 and 250 g were used. The rats were anesthetized with urethane (1.3 g / k g i.p.). The left iliac artery was catheterized for the measurement of systemic blood pressure. Blood pressure was recorded by means of a Statham P23 AC transducer coupled to a Grass polygraph 79C. Heart rate was monitored with a Narco-Biosystems biotachometer BT 1200 triggered by the pulse pressure. Rectal temperature was measured with a thermistor connected to a telethermometer (Yellow Springs 46 TUC) and was kept at 37°C by application of radiant heat. Stainless steel stimulating electrodes (diameter 150 /~m), insulated except for the flat end, were

stereotaxically placed in the mesencephalic reticular formation (MRF; AP + 1.4; RL 1.5; DV + 3.0 according to the coordinates of Albe-Fessard et al., 1966) with the aid of a David-Kopf stereotaxic instrument. A guide cannula with an outer diameter of 600 # m was inserted into the fourth cerebral ventricle ( A P - 2.0; RL 0.0; DV + 3.0) for intracerebroventricular (i.c.v.) administration of peptides or vehicle. In one series of experiments microinjections were given into the dorsal raphe nucleus ( D R N ) of the brainstem. For this purpose a guide cannula was inserted into the D R N and fixed to the skull with the aid of dental cement. In order to avoid damage to the transverse sinus the coordinates of Pellegrino (Pellegrino et al., 1979) were used. The coordinates were as follows: AP 5.6; RL 0.0; DV 5.8. The fixation of the guide cannula preceded the insertion of stimulation electrodes. 2.2. Electrical stimulation A Grass $88 stimulator was used to stimulate the MRF. The stimuli were rectangular biphasic pulses with a pulse duration of 1 ms and the interval between the electronegative and electropositive pulses was also 1 ms. Each stimulation period lasted for 5 s. The frequency of the stimuli (10, 30, 50, 70 and 90 Hz) was varied randomly. The current intensity that caused an increase of systolic blood pressure (pressor response) of 50 m m Hg at 50 Hz stimulation frequency was determined first for each animal and used subsequently for the other frequencies. This procedure served to obtain a comparable baseline pressor response. The current that was necessary to induce the pressor response varied between 40-100 ~tA with an average of 66 + 4 /~A. The first random series of stimuli was followed by intracerebroventricular treatment and the stimulation series were repeated 20, 40 and 60 min after treatment unless otherwise stated. 2.3. D ~ Peptides were freshly dissolved in 0.9% saline containing 0.02 vol of 0.01 N HCI. Acidified saline was used as vehicle. The injection volume for i.c.v.

135 treatment was 1/~1 and a dose of 25 ng of peptides was used, except in one experiment where a doseresponse curve was established for OXT-(7-9). For microinjection into the D R N the volume was 0.5 ~1 and the dose of the peptide OXT-(7-9) 1 ng. The peptide or vehicle were injected with a microsyringe. The injection needle protruded from the guide cannula by 100 /~m. A micromanipulator attached to the syringe secured slow injection of the peptide or vehicle (2 min). The peptides administered were: [ArgS]vasopressin (AVP; pressor activity 471 I U / m g ) ; oxytocin (OXT; avian depressor activity 525 I U / m g ) ; AVP-(7-9) (prolylarginyl-glycinamide) and O X T - ( 7 - 9 ) (prolylleucyl-glycinamide). 2.4. Histology After the completion of the experiment the brain was removed, fixed in 4% formalin solution, sectioned in 100/~m slices and stained with 0.1% thionin. The localization of the guide cannula in the ventricle or the D R N and that of the electrodes was verified microscopically. 2.5. Statistical analysis The difference between the prestimulation systolic blood pressure and the maximal increase during stimulation was determined and served as measure of the pressor response. The heart rate response was determined similarly. The data were expressed as differences in the magnitude of pressor (A pressor response) and heart rate responses (A heart rate response) of each rat before and different intervals after i.c.v, treatment. Mean arterial pressure and heart rate were measured before the first series of stimulation and subsequently 20, 40 and 60 min after treatment, again before the stimulation series. Statistical analysis of the data was performed by two-tailed t-tests and one- or two-way analysis of variance. The parametric test of Dunnett was applied for multiple comparisons (De Jonge, 1964).

3. Results 3.1. Basal blood pressure and heart rate Successive steps in the experimental procedure preceding the first random series of stimulation caused a slight increase in mean arterial blood pressure from 113.3 + 3.0 to 118.3 + 1.3 m m Hg (P < 0.05; paired t-test), but it subsequently declined to the starting level (110.7 + 2.5; 111.5 + 3.6 and 113.9 + 2.8 mm Hg respectively at 20, 40 and 60 min after i.c.v, treatment). AVP administered into the fourth ventricle slightly but significantly attenuated the decline in blood pressure that was found in vehicle-treated rats (P < 0.05; F = 4.85; df = 1 / 3 6 ; two-way analysis of variance). The values in rats that received AVP were as follows: 114.0 + 2.6 m m Hg; 117.2 + 2.2 mm Hg and 119.2 + 1.6 m m Hg. AVP-(7-9), OXT and OXT-(7-9) did not influence basal blood pressure. The heart rate also increased during the experimental procedure from 354.9 + 5.9 to 402.1 + 9.9 b e a t s / m i n (bpm; P < 0.05; paired t-test), returned to the starting level and again increased slightly (363.1 + 14.2 b p m at 20 min; 368.1 + 16.6 b p m at 40 min and 384.0 + 11.6 bpm at 60 rain after i.c.v. treatment). The elevation in heart rate 60 min after vehicle administration was significantly higher (P < 0.05; paired t-test) than before operative procedures. The heart rate of the AVP-treated rats was also significantly higher than that of the controls (P < 0.05; F = 4.55; df = 1 / 3 6 ; two-way analysis of variance). The values were as follows: 373.1 + 9.7 bpm; 406.0 + 12.8 b p m and 401.4 + 9.7 b p m respectively 20, 40 and 60 min after administration of AV,P. The other peptides had no effect on the baseline heart rate pattern observed in the vehicle-treated rats. 3.2. Effects of vehicle and peptides on pressor and heart rate responses Administration of the vehicle into the fourth ventricle resulted in a slight reduction in the pressor response 40 and 60 rain later as compared to the pretreatment values (table 1). Administration of AVP failed to induce significant changes in the magnitude of the pressor response in comparison

136 TABLE 1 Effect of argininea-vasopressin,oxytocin, AVPT_9, OXTT_9 and vehicle, administered into the fourth cerebral ventricle, on pressor responses evoked by electrical stimulation of the mesencephalicreticular formation. Time after treatment (min)

Treatment

20

Stimulation frequency (Hz) 30

50

70

Vehicle AVP AVP-(7-9) OXT OXTff7-9)

-1.4±1.5 a 1.9±2.1 -2.7±1.7 -3.3±1.6 -4.7±0.7

-2.3±1.7 -2.7±4.7 -16.3±3.1 h -16.0±4.5 b -23.6±4.5 ~

1.3±3.2 0.6±7.1 -17.6±6.5 b -21.1±6.1 b -24.0±3.6 ~

Vehicle AVP AVe(7-9) OXT OXT-(7-9)

-2.6±1.4 2.3±1.7 -5.3±2.3 -3.9±1.8 -8.4±1.5 b

-9.0±1.9 -4.9±4.3 -20.4±2.6 b -23.3±3.9 b -30.3±4.6 ¢

-3.1±3.5 -2.3±8.0 -24.1±6.2 b -30.6±7.5 c -34.9±4.8 c

Vehicle AVP AVP-(7-9) OXT OXT47-9)

-2.7±2.1 0.7±1.9 -6.6±2.1 -6.1±1.8 -8.6±1.6 b

-15.0±3.4 -9.9±3.2 -29.0±3.1 h -27.9±4.5 b -34.1±4.3 c

-8.7±4.8 -9.6±8.6 -33.4±4.3 b -38.0±7.6 c -43.3±5.2 ¢

• Mean+ S.E. of difference in the magnitude of the pressor response (mm Hg) as compared to the pretreatment values; n = 7 for each group. b p < 0.05, Dunnett's test. c p < 0.01, Dunnett's test.

to vehicle-treated rats. Marked a t t e n u a t i o n of the pressor response was observed following the adm i n i s t r a t i o n of OXT, A V P - ( 7 - 9 ) a n d O X T - ( 7 - 9 ) in a n a m o u n t of 25 ng. The m a g n i t u d e of the decreases appeared to be most p r o n o u n c e d at 70 Hz stimulation frequency. The effect of all these peptides was already significant 20 m i n after treatment, b u t the decreases were even larger after 40 a n d 60 min. The influence of peptides a n d vehicle o n the heart rate response was determined as well. The b r a d y c a r d i c response to stimulation was diminished in vehicle-treated rats in c o m p a r i s o n to p r e t r e a t m e n t values. However, n o n e of the peptides c h a n g e d this response p a t t e r n significantly (data are not shown).

m a g n i t u d e of the pressor response a n d heart rate response d e t e r m i n e d 20 min after the treatment (fig. 1). C o m p a r i s o n of the effect of various doses of O X T - ( 7 - 9 ) with vehicle showed that both 25 a n d 50 ng of O X T - ( 7 - 9 ) reduced the pressor response at all frequencies. There was practically no difference in the m a g n i t u d e of the effect of these two doses. The 12.5 ng dose a t t e n u a t e d the pressor response, b u t the difference with the vehicle group was significant only at the frequency of 70 Hz. O X T - ( 7 - 9 ) in a n y dose failed to affect the magnitude of the heart rate response as compared to that of vehicle-treated rats.

3.3. Dose-dependent actioity of OXT-(7-9)

As it shown in fig. 2 microinjection of OXT( 7 - 9 ) in a dose of 1 ng into the dorsal raphe nucleus resulted in a substantial decrease in the m a g n i t u d e of the pressor response induced by

I n one experiment, different doses of O X T - ( 7 - 9 ) were administered in the fourth ventricle a n d the

3.4. Local microinjection of OXT-(7-9) into the dorsal raphe nucleus

137

0

6.

12.5

25

0

50

30Hz

-10"

-10

6.

12.5

25.

~~

50.

..~ 30Hz

-20

-20

•

o

50Hz

50Hz 70Hz

7OHz

-/.0

-30

A heort rote response (beats/rain)

tx pressor response (rnm Hg )

Fig. 1. Changes in the magnitude of pressor and bradycardiac response as a function of the dose of prolyl-leucyl-glycinamide (OXT-(7-9)) and stimulation frequency. Vehicle or OXT-(7-9) were administered into the fourth cerebral ventricle immediately after the first random series of electrical stimulation of the MRF. The values represent the differences in response magnitude before and 20 min after treatment. Seven observations for each dose. Stimulation frequencies: 30 Hz (O); 50 Hz ( O ) and 70 Hz (A). The significance of the differences between vehicle- and peptide-treated rats was calculated by means of Dunnett's test. " P < 0.05; *" P < 0.01.

511MUlATION FREQUENCY 30

50

70 Hz

electrical stimulation of the MRF. The pressor response following vehicle injection into this nucleus was also diminished. The action of the peptide was however significant at stimulation frequencies of 50 and 70 Hz as compared to the effect in vehicle-treated rats.

-. • ...•. • 20

4. Discussion

-/.0

.6oL

°

A pres~or re~;pons_l~

(ram Hg) Fig. 2. Changes in the magnitude of the pressor response induced by electrical stimulation of the M R F following locally administered vehicle ( O ) or OXT-(7-9) (O) ito the dorsal raphe nucleus (DRN). Treatment-stimulation interval was 20 rain.

The main finding of these experiments was that, on injection in the fourth cerebral ventricle, OXT and the C-terminal tripeptides related to OXT and AVP markedly reduced the magnitude of pressor responses induced by electrical stimulation of the mesencephalic reticular formation while AVP failed to induce similar action following its administration via this route. The results sugsest that OXT and the triMean values :l=S.E. represent the differences between the magnitude of pressor response before and after treatment. Significance of differences: P < 0.05 (Student's t-test); seven observations per group.

138 peptides might have acted in the vicinity of the fourth cerebral ventricle and that AVP appears to be different in that it affected lower brainstem mechanisms involved in central cardiovascular regulation. The observed effect of AVP on the baseline blood pressure and heart rate however may have involved medullary structures like the NTS and the dorsal nucleus of the vagus. Recently, immunohistochemical observations have revealed a vasopressinergic innervation of these cell groups (Sofroniew and Weindl, 1981). The existence of neuronal connections between the hypothalamic neurosecretory nuclei and the NTS has been shown by electrophysiological studies (Zerihun and Harris, 1981; Kannan and Koizumi, 1981). A high density of oxytocin-containing fibers a n d / o r terminals in the mesencephalon, pons and medulla has been also demonstrated (Sofroniew and Weindl, 1981). Intracerebroventricular administration of AVP appears to accelerate noradrenaline turnover rate in the NTS and in the A 1 cell group in the medulla oblongata (Tanaka et al., 1977). This finding supports the possibility that AVP participates in lower brainstem cardiovascular regulation. Moreover, the AVP content of the brainstem is reduced in the stroke-prone substrain of the spontaneously hypertensive rats but not in control normotensive Wistar Kyoto rats (Lange et al., 1981). OXT and the C-terminal fragments of neurohypophyseai peptides appear to suppress the neurogenic pressor responses at the level of the lower brainstem. OXT, OXT-(7-9) and AVP-(7-9), administered in a lateral cerebral ventricle, attenuated the neurogenic pressor response to the same extent as was observed following fourth cerebral ventricular administration (Versteeg et al., 1982b). However, the peptide-induced suppression of pressor responses 20 min after treatment was less after lateral ventricular administration than after fourth ventricular administration. A site of action of these peptides may be the dorsal raphe nucleus (DRN). The medullary raphe areas have been implicated in cardiovascular regulation (Adair et al., 1977; Cabot et al., 1979) and the D R N is one of the lower brain regions from where modulation of behavioral functions by neurohypophyseal hormones can be evoked (Kov/lcs et al.,

1979; Bohus et al., 1982b). The injection of an amount of the peptide that was no longer effective intracerebroventricularly led to a small but significant suppression of the neurogenic pressor response. This finding and the fact that the D R N is rich in serotonergic cell bodies, the axons of which terminate both in forebrain and the spinal cord, may indicate that OXT, OXT-(7-9) and AVP-(7-9) affect the serotonergic system. That OXT-(7-9) acted through the neighbouring central grey areas cannot be fully excluded. Oxytocinergic terminals have been demonstrated in this area also (Sofroniew and Weindl, 1981). It is, however, puzzling why a marked diminition of the magnitude of the pressor response occurred following vehicle injection into the dorsal raphe nucleus. One cannot exclude the possibility of a volume action of the vehicle through the same cells or neurons where the peptide was effective. The maximal action of the various peptides occurred 60 min after their injection. In a former study it was found that upon lateral cerebral ventricular administration of the fragment AVP(7-9) the duration of action on the pressor response was 40 rain. The effect of peptides upon repeated stimulation for 60 min seemed to be a carry-over of the former activity rather than a long duration of action (Versteeg et al., 1982b). It is likely that the maximal action of OXT and AVP fragments upon fourth cerebral ventricular administration was also a carry-over of former activity. Whether this carry-over outlasts 60 min was not determined because of decreased responsiveness to stimulation after this time. This phenomenon may be related to urethane anaesthesia. The dose-response relationship of the tripeptides related to the C-terminal linear portion of O X T and AVP warrant discussion. OXT-(7-9) appeared the most potent peptide, at least on a weight basis, in diminishing the magnitude of pressor responses following fourth ventricular administration. The dose-response curve of this peptide similarly to the one obtained upon lateral ventricular administration (Versteeg et al., 1982b), was rather steep between 6 and 25 ng. Interestingly C-terminal di- or tripeptides of OXT and AVP displayed similar dose-response relations in the experiments of Flexner et al. (1977). These authors

139

studied the action of these neuropeptides, given peripherally, on puromycin-induced memory loss in mice. On the other hand, the action of AVP on the neurogenic pressor response following lateral ventricular administration displayed a 'classical' type, less steep dose-response relation. This difference between AVP and the tripeptides leads to a second aspect of the action of OXT-(7-9) and AVP-(7-9). Structure-activity studies following lateral ventricular peptide administration have suggested that OXT-(7-9) may be considered as the active site in the OXT molecule required to attenuate the centrally-induced pressor response. AVP-(7-9) has been considered as a second active site of the AVP molecule besides the active covalent ring structure AVP-(I-6) (Versteeg et ai., 1982b). The effectiveness of AVP-(7-9) but not of AVP following fourth ventricular administration, however, may be explained in a different way. It may be that AVP-(7-9) mimicked the influence of OXT-(7-9) because putative receptors for these peptides do not distinguish between the basic amino acids leucine and arginine. Alternatively, it may be that AVP-(7-9) is indeed the active fragment of AVP at the level of the lower brainstem, but that the full peptide sequence is not active because of the absence of proper enzymes to form this fragment. Recently available information may point to the tripeptides as pharmacological agents which act through a putative receptor system not identical with AVP or OXT sites. Burbach et al., (1980) and Burbach and Lebouille (1983) were unable to show the formation of the 7-9 fragments from AVP and OXT by peptidases in a synaptic membrane fraction of rat brain. In addition, larger fragments containing 7-9 sequence were formed by these peptidases. In behavioral tests these peptides were several orders of magnitude more potent than the intact neurohypophyseal hormones. One may speculate that the effects of AVP-(7-9) and OXT-(7-9) reported here reflect activities of these larger AVP and OXT fragments.

Acknowledgements The peptides were donated by Dr. H.M. Greven (Organon, Oss, The Netherlands). The secretarial help by Mrs. Joke Poelstra-Hiddinga is gratefully acknowledged.

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