LFS-14349; No of Pages 8 Life Sciences xxx (2015) xxx–xxx

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Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats Marek Toczek a, Eberhard Schlicker b, Emilia Grzęda a, Barbara Malinowska a,⁎ a b

Department of Experimental Physiology and Pathophysiology, Medical University of Białystok, Mickiewicz Str. 2A, 15-222 Białystok, Poland Department of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany

a r t i c l e

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a b s t r a c t

Aims: This study was performed to examine whether hypertension affects the sympathetic transmission to resistance vessels of pithed rats via inhibitory presynaptic cannabinoid CB1 receptors and whether endocannabinoids are involved in this response. Materials and methods: We compared uninephrectomised rats rendered hypertensive by high salt diet and deoxycorticosterone acetate (DOCA) injections with normotensive animals (uninephrectomy only). Experiments were performed on vagotomised and pithed animals. Increases in diastolic blood pressure (DBP) were Chemical compounds studied in this article:: induced four times (S1–S4) by electrical stimulation or phenylephrine injection. 11-Deoxycorticosterone acetate (PubChem CID: 3001) Key findings: Electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 impulses) of the preganglionic sympathetic nerve fibres CP55940 (PubChem CID: 104895) innervating the blood vessels more strongly increased DBP in normotensive than in DOCA–salt rats. PhenylephAM251 (PubChem CID: 2125) rine (0.01 μmol/kg) induced similar increases in DBP in both groups. The cannabinoid receptor agonist CP55940 URB597 (PubChem CID: 1383884) Phenylephrine hydrochloride (PubChem CID: 5284443) (0.01–1 μmol/kg) did not modify the rises in DBP induced by phenylephrine. However, it inhibited the electrically Prostaglandin F2α (PubChem CID: 5282415) stimulated increases in DBP, more strongly in DOCA–salt than in normotensive animals (maximally by 50 and 30%, respectively). The effect of CP55940 was attenuated by the CB1 antagonist AM251 (3 μmol/kg). AM251 enKeywords: hanced the neurogenic vasopressor response during S4 by itself in hypertensive rats only. URB597 (3 μmol/kg), Cannabinoid receptors which inhibits degradation of the endocannabinoid anandamide, did not modify the electrically stimulated CP55940 increases in DBP. DOCA–salt hypertension Significance: The function of inhibitory presynaptic CB1 receptors on sympathetic nerves is enhanced in DOCA– Pithed rat salt hypertensive rats. Thus, the CB1 receptor-mediated inhibition of noradrenaline release from the sympathetic Presynaptic receptors nerve fibres innervating the resistance vessels might play a protective role in hypertension. © 2015 Elsevier Inc. All rights reserved. Article history: Received 12 January 2015 Received in revised form 20 March 2015 Accepted 26 March 2015 Available online xxxx

1. Introduction Sympathetic overactivity plays a significant pathophysiological role in hypertension (for a review, see [27,28]). Presynaptic receptors located on sympathetic nerve endings participate in the development and maintenance of the enhanced sympathetic activity, as shown in, e.g., the model of deoxycorticosterone acetate (DOCA)–salt hypertension in rats. Thus, an impaired function of inhibitory presynaptic α2adrenoceptors [23,31,34] and adenosine A1 heteroreceptors [37] and an increased function of facilitatory presynaptic β2-adrenoceptors [30] have been shown. Another example of presynaptic receptors in the sympathetic nervous system are the cannabinoid CB1 receptors on the sympathetic nerve fibres innervating the resistance vessels [25,32] and heart [26, 29,32,43]. Their activation reduces the electrically stimulated increases ⁎ Corresponding author at: Department of Experimental Physiology and Pathophysiology, Medical University of Białystok, Mickiewicz Str. 2A, 15-089 Białystok, Poland. Tel./fax: +48 85 7485699. E-mail address: [email protected] (B. Malinowska).

in blood pressure (BP) and heart rate (HR) and/or plasma noradrenaline concentration in pithed rats [25,26,32] and rabbits [43] and in human atrial appendages [29]. Presynaptic CB1 receptors might play a role in hypertension, which is associated with an up-regulation of the endocannabinoid system (for a review see e.g. [33,49]). Thus, the plasma level of the endocannabinoid anandamide (AEA) is increased in hypertensive patients [8,21] and animals [21,45]. Moreover, the defective gene variant FAAH 129T of fatty acid amide hydrolase (FAAH; an enzyme responsible for AEA degradation) is associated with lower BP in young males [38]. In addition, the expression of cannabinoid CB1 receptors in the myocardium and aortic endothelium [2] and the efficiency of brain CB1 receptor coupling [11] are higher in spontaneously hypertensive rats (SHR) when compared with normotensive rats. Moreover, the decreases in BP (and intraocular pressure) after Δ9-tetrahydrocannabinol inhalation were greater in hypertensive than those in normotensive open-angle glaucoma patients [5]. Similarly, the CB1 receptor-mediated hypotension elicited by AEA and its stable analogue methanandamide was stronger in anaesthetised SHR compared with normotensive controls [2,19]. Although AEA and methanandamide have a CB1 receptor-

http://dx.doi.org/10.1016/j.lfs.2015.03.022 0024-3205/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022

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M. Toczek et al. / Life Sciences xxx (2015) xxx–xxx

dependent hypotensive effect in conscious hypertensive rats [14,19], they increase BP in conscious normotensive rats. The CB1 receptor antagonists rimonabant and AM251 [2] further increase and the FAAH inhibitors URB597 [2] and AM3506 [11] decrease the elevated BP and cardiac contractility in hypertensive anaesthetised animals without affecting any hemodynamic parameters in normotensive controls. On the other hand, rimonabant reduces BP in conscious angiotensin IIdependent hypertensive (mRen2)27 rats but not in their normotensive controls [39]; although the results of the latter study cannot be easily reconciled with the data of the other studies, they nonetheless point to a role of the endocannabinoid system in hypertension. We have previously shown in pithed and vagotomised rats that the activation of presynaptic CB1 receptors by endogenously formed cannabinoids plays a role during the early phase of septic shock [12] and myocardial ischemia [36]. Using the same experimental model, we studied whether presynaptic inhibitory CB1 receptors are also involved in hypertension. First, we examined whether vascular CB1 receptors are active in a model of DOCA–salt hypertension. Second, we were interested whether these receptors may be activated by endocannabinoids. 2. Materials and methods 2.1. Animals All surgical procedures and experimental protocols were in accordance with the European and Polish legislation and were approved by the local Animal Ethic Committee in Białystok (Poland). DOCA–salt hypertension was induced in normotensive male Wistar rats. They had free access to food pellets and water and were maintained under a 12-h/12-h light/dark cycle. 2.2. DOCA–salt hypertension Animals initially weighing 130–200 g (aged 4–6 weeks) were anaesthetised by intraperitoneal (i.p.) injection of pentobarbitone sodium (300 μmol or ~70 mg/kg). The right kidney was removed in all rats via a right lateral abdominal incision. After a one week recovery period, hypertension was induced over a time period of 4 weeks by subcutaneous (s.c.) injections of DOCA (67 μmol or ~25 mg/kg in 0.4 ml/kg of dimethylformamide; DMF) twice weekly and replacement of drinking water with 1% NaCl solution. Normotensive control rats were also uninephrectomised (UNX) but received the vehicle for DOCA (DMF, 0.4 ml/kg, s.c) twice weekly and drank tap water. Systolic blood pressure (SBP) was measured in conscious rats by the non-invasive tail-cuff method using a Rat Tail Blood Pressure Monitor (Hugo Sachs Elektronik — Harvard Apparatus, March-Hugstetten, Germany). Rats treated with DOCA for 4 weeks which had a SBP equal to or higher than 150 mm Hg were considered as hypertensive.

leaving the spinal canal to study the electrically evoked increases in DBP (neurogenic vasopressor response). For stimulation, an electrical field was generated between the pithing rod in the vertebral column and an indifferent electrode placed on the thorax ventrally under the skin by means of a stimulator (Stimulator V type STV100, Bio-SysTech, Białystok, Poland). After 15–25 min of equilibration, during which the cardiovascular parameters were allowed to stabilize, the experiments were performed. 2.4. Experimental protocol The experimental protocol is shown in Fig. 1. At the beginning each rat was treated with the CB1 receptor antagonist AM251 (3 μmol or ~1.7 mg/kg i.v.) or its vehicle. Five minutes later the animals received an injection of pancuronium (0.8 μmol or ~ 0.5 mg/kg i.v.) to avoid twitches associated with electrical stimulation. Pancuronium was also administered to those rats in which an increase in DBP was induced by phenylephrine to ensure identical experimental conditions. Five minutes later the first stimulus (S1) was applied. Increases in DBP were induced four times (S1–S4) by electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses) of the preganglionic sympathetic nerve fibres innervating the resistance blood vessels or by administration of phenylephrine (0.01 μmol or ~2 μg/kg i.v.), which caused an increase in DBP of comparable magnitude to that induced by electrical stimulation. Stimulations S2–S4 were administered at intervals of 7 min. Increasing doses of the cannabinoid receptor agonist CP55940 (0.01, 0.1 and 1 μmol/kg corresponding to ~ 4, 40 and 400 μg/kg i.v.) or its vehicle were given 5 min before S2, S3 and S4, respectively. Because basal DBP was lower in pithed DOCA–salt than that in normotensive rats (for details, see the Results section), in some DOCA–salt hypertensive animals basal DBP was increased to the corresponding values of normotensive rats by i.v. infusion of PGF2α (0.06 to 1.7 μmol/kg/h corresponding to ~0.03 to 0.8 mg/kg/h) starting from 15 min before S1. The fatty acid amide hydrolase (FAAH) inhibitor URB597 (3 μmol or ~1 mg/kg) or its solvent were given i.p. 35 min before S1. 2.5. Data analysis The results are given as the mean ± SEM (n = number of animals). In order to quantify the effects of the agonist on the electrically or phenylephrine induced increase in DBP, the ratios S2/S1, S3/S1 and S4/S1 were determined. These ratios were expressed as percentages of the corresponding ratios obtained from the vehicle-treated animals (% of control). Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA). Student's t-test for paired and unpaired data was used to compare the mean values. Differences were considered significant when P b 0.05. When two or more treatment groups were compared to the same control, a oneway analysis of variance (ANOVA) followed by the Dunnett test was used.

2.3. Pithed rat 2.6. Materials The rats were anaesthetised with pentobarbitone sodium (300 μmol/kg; i.p.) and then injected with atropine (2 μmol or ~1 mg/kg; i.p.). The procedures and equipment for pithing, vagotomy and artificial ventilation of rats, the measurement of diastolic blood pressure (DBP) and heart rate (HR), the intravenous (i.v.; 0.5 ml/kg) and i.p. (2.0 ml/kg) drug administration, the prostaglandin F2α (PGF2α) infusion and the maintenance of constant body temperature were the same as those employed in our previous studies (e.g. [36]). Briefly, following anaesthesia, the animals were tracheotomised, vagotomised and pithed by inserting a stainless-steel rod through the orbit and foramen magnum into the vertebral canal. The pithing rod was enamelled except for an uncovered segment situated at vertebrae T2–L6. In most experiments, the pithing rod was used as an electrode for electrical stimulation of the preganglionic sympathetic nerve axons

Drugs and chemicals were obtained from the following sources: atropine sulphate, Cremophor EL, 11-deoxycorticosterone acetate (DOCA), dimethyl sulphoxide (DMSO), N,N-dimethylformamide (DMF), 5-(1,1-dimethylheptyl)-2-[5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-phenol (CP55940), 2-hydroxypropyl-β-cyclodextrin (cyclodextrin), pancuronium bromide, (R)-(−)-phenylephrine hydrochloride and Tween-80 from Sigma-Aldrich (Steinheim, Germany); N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl1H-pyrazole-3-carboxamide (AM251) and prostaglandin F2α (PGF2α) from Tocris Cookson Inc. (Bristol, UK); (3′-(aminocarbonyl)[1,1′biphenyl]-3-yl)-cyclohexylcarbamate (URB597) from Cayman Chemical Company (Ann Arbor, MI, USA); ethanol from POCH (Gliwice, Poland); pentobarbitone sodium from Biowet (Puławy,

Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022

M. Toczek et al. / Life Sciences xxx (2015) xxx–xxx

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Fig. 1. Experimental protocol used to examine the influence of the cannabinoid receptor agonist CP55940 on the increase in diastolic blood pressure (DBP) induced by electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses) of the preganglionic sympathetic nerve fibres or by bolus injection of phenylephrine (0.01 μmol/kg) in uninephrectomised pithed and vagotomised normotensive and deoxycorticosterone (DOCA)–salt hypertensive rats. DBP was increased four times (S1–S4) at intervals of 7 min. Each animal received an injection of pancuronium 5 min before S1. Increasing doses of CP55940 or its vehicle were injected 5 min before S2, S3 and S4. The cannabinoid CB1 receptor antagonist AM251 or its solvent was injected 10 min before S1. The fatty acid amide hydrolase inhibitor URB597 (3 μmol/kg) or its solvent was given 35 min before S1 (not shown). In some DOCA–salt rats basal DBP was increased by infusion of PGF2α (0.06 to 1.7 μmol/kg/h) started 15 min before S1 (not shown). Drugs were administered i.v., except for URB597 which was given i.p. The numbers in the scheme refer to the time intervals (in minutes) elapsing between two subsequent procedures (marked by arrows).

Poland) and sodium chloride (NaCl) from Chempur (Piekary Śląskie, Poland). Drugs were dissolved in saline with the following exceptions: AM251 in a mixture of DMSO, ethanol and Cremophor EL (2:1:1) and then diluted in saline (1:9) immediately before the experiment; CP55940 in a 19% w/v solution of cyclodextrin in saline; DOCA in DMF; URB597 in a mixture of DMSO and Tween 80 (1:2) and then diluted in saline (3:7) immediately before the experiment. The vehicle for URB597 increased basal HR (but not DBP) in DOCA–salt hypertensive but not in UNX normotensive rats (Table 1). Other vehicles did not affect basal cardiovascular parameters in pithed rats. 3. Results 3.1. General Conscious DOCA–salt hypertensive rats had a much higher SBP than normotensive animals (205 ± 4 mm Hg; n = 64 and 125 ± 2 mm Hg; n = 47, respectively; P b 0.001) and comparable HR (349 ± 5 beats/min and 351 ± 5 beats/min, respectively). By contrast, basal DBP and HR

immediately before S1 in anaesthetised, vagotomised, nephrectomised and pithed rats treated with pancuronium (0.8 μmol/kg, i.v.) and the solvent for AM251 were lower in DOCA–salt (37 ± 1 mm Hg and 235 ± 5 beats/min, respectively; n = 23) than those in normotensive rats (49 ± 1 mm Hg and 293 ± 5 beats/min, respectively; n = 23; P b 0.001 in the case of both parameters). The antagonist of CB1 receptors AM251 (3 μmol/kg, i.v.) did not affect basal cardiovascular parameters (Table 1). The FAAH inhibitor URB597 (3 μmol/kg, i.p.) decreased basal HR (but not DBP) by 8% in DOCA–salt rats but did not modify basal DBP and HR in normotensive rats (Table 1). All experiments described below were performed on pithed, vagotomised and nephrectomised rats. Electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses) of the preganglionic sympathetic nerve fibres innervating the resistance blood vessels increased DBP in normotensive rats by 34 ± 1 mm Hg (S1, n = 15). The neurogenic vasopressor response in DOCA–salt hypertensive animals was 60% lower than that in normotensive rats (for details, see Table 1 and Fig. 2). In order to ensure comparable experimental conditions and to exclude the possibility that the lower response to electrical stimulation in DOCA–salt

Table 1 Basal diastolic blood pressure (DBP), heart rate (HR) and electrically or phenylephrine induced increases in DBP by S1 in uninephrectomised (UNX) pithed and vagotomised normotensive and deoxycorticosterone (DOCA)–salt hypertensive rats.

Group

UNX

UNX-DOCA-salta

DBP before S1 (mmHg)

S1 Increase in DBP (mmHg)

HR before S1 (beats/min)

8

49 ± 2

29 ± 4

293 ± 8

15

49 ± 1

34 ± 1

292 ± 6

13

45 ± 2

30 ± 2

291 ± 7

veh1,2

6

46 ± 4

23 ± 2

295 ± 10

URB597, veh1

5

41 ± 2

24 ± 2

303 ± 11

Type of stimulation

Antagonist or inhibitor or their vehicle

Phenylephrine

veh1

Electrical

veh1 AM251

n

Phenylephrine

veh1

10

36 ± 2***

23 ± 3ΔΔ

240 ± 8***

Electrical

veh1

13

38 ±2***

14 ± 2***

231 ± 6***

AM251

18

34 ± 1***

12 ± 2***

238 ± 6***

veh1,2

4

38 ± 2*

15 ± 2*

292 ± 2ΔΔΔ

URB597, veh1

6

33 ± 2*

18 ± 5

269 ± 8*, ΔΔ

46 ± 2Δ

17 ± 3***

226 ± 6***

48 ± 1ΔΔΔ

24 ± 2**, Δ

290 ± 6ΔΔΔ

UNX-DOCA-saltb

Electrical

veh1

6

UNX-DOCA-salt + PGF2α

Electrical

veh1

13

Basal DBP and HR were measured immediately before the first stimulation (S1). Experiments were performed in the presence of the CB1 receptor antagonist AM251 (3 μmol/kg) or its vehicle (veh1) and the inhibitor of fatty acid amide hydrolase URB597 (3 μmol/kg) or its vehicle (veh2). Increases in DBP were induced by electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses) of the preganglionic sympathetic nerve fibres or by intravenous injection of phenylephrine (0.01 μmol/kg). Three groups of DOCA–salt rats were considered. DOCA–salta — all DOCA–salt rats without further treatment; DOCA–saltb — DOCA–salt rats without further treatment and with the highest basal DBP; DOCA–salt + PGF2α — DOCA–salt rats, in which basal DBP was increased by PGF2α (0.06 to 1.7 μmol/kg/h) infusion. Data are given as the means ± SEM of n rats; *P b 0.05 and ***P b 0.001 vs. the respective values in UNX rats; ΔP b 0.05, ΔΔP b 0.01 and ΔΔΔP b 0.001 vs. the respective values of electrically stimulated DOCA–salt rats treated with vehicle for AM251. For the sake of clarity, the S1 values for the electrically induced rise in DBP in normotensive and the 3 groups of hypertensive rats are highlighted in grey colour.

Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022

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M. Toczek et al. / Life Sciences xxx (2015) xxx–xxx

Fig. 2. Traces from experiments representing the influence of CP55940 and its interaction with AM251 on the increase in diastolic blood pressure (DBP) induced by electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses; A, B) of the preganglionic sympathetic nerve fibres or by bolus injection of phenylephrine (0.01 μmol/kg; C) in uninephrectomised (UNX) pithed and vagotomised normotensive (upper trace) and deoxycorticosterone (DOCA)–salt hypertensive rats (lower trace). DBP was increased four times (S1–S4) at intervals of 7 min. Increasing doses of CP55940 (0.01, 0.1 and 1 μmol/kg; A.II, B.II and C.II) or its vehicle (veh, A.I, B.I and C.I) were given 5 min before S2, S3 and S4. AM251 (3 μmol/kg; B) or its solvent (A, C) was injected 10 min before S1.

rats is related to lower basal DBP in these animals (see above) we studied two additional DOCA–salt groups with a basal DBP similar to that in normotensive rats. The first group was composed of all DOCA–salt rats with particularly high basal DBP. In the second group, PGF2α infusion increased not only basal DBP but also basal HR to a level similar to that in normotensive rats (Table 1). However, in spite of a comparable level of basal DBP in normotensive and in the two additional DOCA–salt rat groups, the S1 values in the two latter groups were still lower by 50 and 30% than those in the normotensive animals, respectively (Table 1). Intravenous injection of phenylephrine (0.01 μmol/kg) increased DBP by about 30 mm Hg in normotensive animals and to a slightly lower extent in DOCA–salt rats (the difference did not reach statistical significance; Table 1). The degree of vasopressor responses both in normotensive and hypertensive rats not treated with any cannabinoid receptor ligands did not markedly change upon repeated electrically or phenylephrine-induced stimulations (S2–S4); in other words, the

ratios S2/S1, S3/S1 and S4/S1 were close to or slightly higher than unity (Figs. 2A.I, B.I, C.I, 3, 5). 3.2. Influence of the cannabinoid receptor agonist CP55940 on the electrically or phenylephrine-induced increases in DBP in normotensive and DOCA–salt rats The cannabinoid receptor agonist CP55940 (0.01, 0.1 and 1 μmol/kg) caused a dose-dependent inhibition of the electrically evoked rise in DBP (Figs. 2A.II, 3A) both in normotensive (by about 15, 30 and 25% of S1, respectively) and hypertensive (by about 30, 45 and 50% of S1, respectively) rats. Thus, the inhibitory effect of each dose of the agonist was by about 15–20% lower in normotensive than in DOCA–salt rats. This was also true for the two additional groups of DOCA–salt hypertensive rats (Figs. 3A and 4A). A comparison of the standard DOCA–salt group and of the DOCA–salt group with PGF2α infusion is particularly

Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022

M. Toczek et al. / Life Sciences xxx (2015) xxx–xxx

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Fig. 3. Influence of the cannabinoid receptor agonist CP55940 on the electrically (A) or phenylephrine (B) induced increase in diastolic blood pressure (DBP) in uninephrectomised (UNX) pithed and vagotomised normotensive and deoxycorticosterone (DOCA)–salt hypertensive rats. DBP was increased four times (S1–S4) at intervals of 7 min by electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses) of the preganglionic sympathetic nerve fibres or by intravenous injection of phenylephrine (0.01 μmol/kg). Increasing doses of CP55940 or its vehicle were injected 5 min before S2, S3 and S4. Results are expressed as ratios S2/S1, S3/S1 and S4/S1 (in % of S1) and given as the means ± SEM of 4–9 rats. **P b 0.01, ***P b 0.001 vs. the respective values in the control groups (animals treated with vehicle for CP55940); ΔP b 0.05, ΔΔP b 0.01 vs. the respective values in normotensive rats (UNX only) treated with CP55940.

interesting. Thus, the dose–response curves for CP55940 were coincident whereas the level of the S1 value was lower by 60 and 30%, respectively, than the S1 value of normotensive rats (Fig. 4A, Table 1). By contrast, CP55940 did not inhibit the increases in DBP induced by phenylephrine both in the normotensive and hypertensive animals (Figs. 2C.II and 3B). The CB1 receptor antagonist AM251 (3 μmol/kg) attenuated the inhibitory effect of CP55940 on the electrically induced rise in DBP both in normotensive and in DOCA–salt rats (Fig. 4B).

3.3. Influence of the CB1 receptor antagonist AM251 and the anandamide degradation inhibitor URB597 on the electrically induced increases in DBP in normotensive and DOCA–salt rats The CB1 receptor antagonist AM251 (3 μmol/kg) did not affect the increase in DBP induced by the first electrical stimulation (S1) in normotensive and DOCA–salt rats (Table 1). The same holds true for the three subsequent stimulations (S2–S4) in normotensive rats. By

Fig. 4. Influence of the basal diastolic blood pressure (DBP, A) and the CB1 receptor antagonist AM251 (B) on the inhibitory effect of CP55940 on the electrically induced increase in DBP in uninephrectomised (UNX) pithed and vagotomised normotensive and deoxycorticosterone (DOCA)–salt hypertensive rats. DBP was increased four times (S1–S4) at intervals of 7 min by electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses) of the preganglionic sympathetic nerve fibres. Increasing doses of CP55940 or its vehicle were injected i.v. 5 min before S2, S3 and S4. The cannabinoid CB1 receptor antagonist AM251 (3 μmol/kg) or its solvent was injected i.v. 10 min before S1. Three groups of DOCA–salt rats were considered. DOCA–salta — all DOCA–salt rats without further treatment; DOCA–saltb — DOCA–salt rats without further treatment and with the highest basal DBP; DOCA–salt + PGF2α — DOCA–salt rats, in which basal DBP was increased by PGF2α (0.06 to 1.7 μmol/kg/h) infusion. The ratios S2/S1, S3/S1 and S4/S1 were determined. These ratios were expressed as percentages of the corresponding ratios obtained from the vehicle-treated animals (% of control). Data are given as the means ± SEM of 3–9 rats; **P b 0.01, ***P b 0.001 vs. the respective values in groups not treated with AM251; Δ P b 0.05, ΔΔP b 0.01 vs. the respective values in UNX rats.

Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022

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M. Toczek et al. / Life Sciences xxx (2015) xxx–xxx

Fig. 5. Influence of the cannabinoid CB1 receptor antagonist AM251 (A) and the fatty acid amide hydrolase inhibitor URB597 (B) on the increase in diastolic blood pressure (DBP) induced by electrical stimulation (0.75 Hz, 1 ms, 50 V, 5 pulses) of the preganglionic sympathetic nerve fibres in uninephrectomised (UNX) pithed and vagotomised normotensive and deoxycorticosterone (DOCA)–salt hypertensive rats. DBP was increased four times (S1–S4) at intervals of 7 min. AM251 (3 μmol/kg) or its vehicle was injected i.v. 10 min before S1. URB597 (3 μmol/kg) or its vehicle was given i.p. 35 min before S1. Each animal received the vehicle for CP55940 5 min before S2–S4. Note that S1 was neither affected by AM251 (3rd vs. 2nd and 8th vs. 7th line of Table 1 for normotensive and DOCA–salt rats, respectively) nor by URB597 (5th vs. 4th and 10th vs. 9th line of Table 1 for normotensive and DOCA–salt rats, respectively). Data are expressed in % of S1. Means ± SEM of 4–9 rats. **P b 0.01 vs. the corresponding AM251-free control.

contrast, as shown in Fig. 5A, AM251 tended to increase (S2 and S3) or significantly increased (S4) the rise in DBP induced by the next three stimulations in DOCA–salt hypertensive animals, maximally by about 20% (S2 and S3) and 40% (S4) in comparison to its own S1 (also in the presence of AM251). The fatty acid amide hydrolase inhibitor URB597 (3 μmol/kg) did not influence the magnitude of the electrically induced increase in DBP during S1 in normotensive and in DOCA–salt rats (Table 1). The same was true for the three subsequent stimulations (the ratios of S2/S1, S3/S1 and S4/S1 were close to 100%; Fig. 5B). 4. Discussion The aim of the present study was to examine (1) whether hypertension affects the function of inhibitory presynaptic cannabinoid CB1 receptors on the sympathetic neurons supplying the resistance vessels and (2) whether these receptors are tonically activated by endocannabinoids in hypertension. The rats were anaesthetised with pentobarbitone, which unlike urethane does not attenuate the function of presynaptic receptors [18]. Like in our previous studies [12,36], we used pithed and vagotomised rats because we wanted to study peripheral mechanisms only. Cannabinoids induce complex cardiovascular changes via various peripheral and/or central receptors/mechanisms; the central effects can override the peripheral ones [24]. In our model, the electrical stimulation of the preganglionic sympathetic nerve fibres increases BP, which is predominantly associated with the release of catecholamines from neuronal endings of the resistance vessels because 10 μmol/kg prazosin diminished these responses by 95% (data not shown). Moreover, the neurogenic vasopressor response is not related to an increase in cardiac output and catecholamines released from the adrenal medulla [26]. 4.1. Characterization of cardiovascular parameters in DOCA–salt hypertension We applied the model of the volume overload DOCA–salt hypertension, because an increase in sympathetic nerve activity plays a crucial role in its development [42,47]. The higher sympathetic tone is at least partially due to an impaired function of inhibitory presynaptic

α2-adrenoceptors [22,31,34,44] and adenosine A1 heteroreceptors [37] and an increased function of facilitatory presynaptic β2adrenoceptors [30]. We can exclude the possibility that a reduction of renal mass affects our results, since unilateral nephrectomy was performed both in the hypertensive and normotensive animals. Four weeks after the beginning of the hypertension induction conscious DOCA–salt rats had a higher SBP than normotensive animals, whereas HR was similar in both groups. Spinal cord destruction more strongly reduced both cardiovascular parameters in hypertensive than in normotensive rats. We have concentrated on changes in DBP, since they reflect changes in the peripheral resistance. A lower level of basal DBP and HR in pithed and vagotomised DOCA–salt than that in normotensive rats (by 24 and 20%, respectively) proves the crucial role of the sympathetic nervous system in the pathogenesis of this model of hypertension [47]. Our results are in line with other observations. BP and HR were also lower in female pithed DOCA–salt hypertensive than their normotensive controls fifteen weeks after the beginning of the hypertension induction [7]. A selective depletion of central catecholamines by intraventricular administration of 6-hydroxydopamine prevented the development of DOCA–salt hypertension [20]. Acute ganglion blockade with hexamethonium decreased BP and HR more in DOCA–salt hypertensive than normotensive rats [10]. Indeed, according to the most recent hypothesis, higher salt intake enhances the excitability of central sympathetic networks and consequently BP (e.g. [41]). The neurogenic vasopressor response was stronger by 60% in normotensive than in DOCA–salt hypertensive rats. In order to exclude the possibility that the lower level of basal DBP is the reason for the discrepancy, we considered two additional groups of DOCA–salt rats with a basal DBP comparable to that of normotensive animals: (1) the DOCA– salt rats with the highest basal DBP were analysed separately. (2) In an extra group of DOCA–salt rats the level of basal DBP was adjusted to that of normotensive animals by PGF2α infusion. We have chosen PGF2α, because it does not modify the neurogenic vasoconstrictor response [15,36] and is not involved in the pathomechanism of DOCA–salt hypertension [47]. Moreover, it increases not only BP but also HR in pithed rats [9]. However, although the basal DBP in the two special groups of DOCA–salt rats did no longer differ from that of the normotensive rats the pressor responses induced by S1 were still lower by 50 and 30%,

Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022

M. Toczek et al. / Life Sciences xxx (2015) xxx–xxx

respectively. By contrast, the lower basal DBP only slightly and insignificantly diminished the pressor response to the α1-adrenoceptor agonist phenylephrine. Thus, the lower response to electrical stimulation in DOCA–salt rats is related mainly to presynaptic mechanisms such as a decrease in noradrenaline content (found in mesenteric and renal arteries and cardiac tissue; [4,16,23,46]) and a defect in the storage and retention of noradrenaline by granules in the sympathetic nerves [17]. The lower neurogenic vasopressor response connected with a diminished sensitivity to the noradrenaline uptake inhibitor cocaine (but without any difference in the response to noradrenaline) in pithed DOCA–salt hypertensive when compared to normotensive rats [7] also fits into this picture. This also holds true for the strongly reduced responses of femoral or renal arteries to sympathetic nerve stimulation (but unaltered or only slightly decreased responses to noradrenaline) in anaesthetised DOCA–salt dogs [13] and rats [16] in comparison to control animals. The evidence is, however, not unequivocal, since the maximum neurogenic response was greater in arteries from DOCA– salt hypertensive than from normotensive rats (e.g., [23,34,44]). 4.2. Function of presynaptic cannabinoid CB1 receptors in DOCA–salt hypertension To determine whether hypertension affects the presynaptic inhibitory cannabinoid CB1 receptor on the sympathetic nerve endings we applied the same cannabinoid receptor agonist (CP55940) and antagonist (AM251) and the same protocol as in our previous studies [25,48]. Like in our previous studies, CP55940 dose-dependently inhibited the neurogenic vasopressor response in normotensive rats, maximally by 30%. However, the inhibitory effect of CP55940 was enhanced by up to 50% in DOCA–salt hypertensive animals; this was also true for the two additional DOCA–salt groups. Thus, we can exclude the possibility that the lower basal DBP in pithed DOCA–salt rats is the reason for the higher inhibitory effect of CP55940. Moreover, the extent of effect of CP55940 appears to be independent from the level of the electrically induced rise in BP. Thus, the dose–response curves for CP55940 in the standard group of DOCA–salt rats and the group of DOCA–salt rats with PGF2α infusion coincided although the level of the electrically stimulated pressor response was significantly higher in the second (24 ± 2 mm Hg) than in the first group (14 ± 2 mm Hg). Both in normotensive and hypertensive rats presynaptic CB1 receptors located on pre- and/or postganglionic nerve fibres are responsible for the inhibitory effect of CP55940 because in both cases (1) the action of CP55940 was reduced by AM251 and (2) CP55940 failed to modify the pressor response to phenylephrine. The enhanced function of presynaptic inhibitory CB1 receptors suggests that these receptors may play a protective role in hypertension. By contrast, presynaptic α2- [22,31,34,44] and β2-adrenoceptors [30] and adenosine A1 heteroreceptors [37] rather increase hypertension. The differential behaviour of α2 (or A1) as opposed to CB1 receptors is indeed surprising since each of the three receptors is Gi/o proteincoupled [1]. On the other hand, the possibility that the discrepancy is related to the well-known inhibitory interaction between α2 and CB1 receptors on noradrenergic nerve endings may be considered. Thus, the CB1 receptor-mediated inhibitory effect on noradrenaline release is increased when the presynaptic α2-adrenoceptors are blocked [40]. In other words, the enhancement of the CB1 receptor response in DOCA–salt hypertension may result (at least partially) from the impaired function of the α2-adrenoceptor in these rats [22,31,34,44]. The endocannabinoid system is up-regulated in hypertension (for literature, see the Introduction section). Thus, our next question was whether endocannabinoids play a role in the enhanced inhibitory effect of presynaptic CB1 receptors in hypertension. For this purpose, we examined the CB1 receptor antagonist AM251 and the FAAH inhibitor URB597. URB597 was used at a dose three times higher than that used in our previous paper, in which it tended to increase the CB1

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receptor mediated inhibitory effect of acute myocardial infarction on neurogenic vasopressor responses in pithed rats [36]. AM251 and URB597 did not markedly affect basal DBP and HR both in pithed DOCA–salt and normotensive rats. The only exception was the small reduction of basal HR by URB597 in DOCA–salt rats. A pressor response to CB1 blockade and/or normalization of the increased BP by FAAH inhibitors was observed in conscious [14] and anaesthetized SHR as well as in rats in which hypertension was induced by angiotensin II infusion and high salt diet [1,10,18]. The lower values of basal cardiovascular parameters are probably the reason for the lack of direct effects of CB1 antagonism and FAAH inhibition in pithed rats. AM251 and URB597 did not have any influence on the magnitude of the neurogenic vasopressor responses (S1–S4) in normotensive rats, suggesting that the endocannabinoid system is not activated under physiological conditions. In DOCA–salt rats, URB597 had no effect; by contrast, AM251 did not affect S1 but gradually, maximally by 40%, raised the electrically stimulated increases in DBP induced by the three subsequent stimulations (S2–S4). The effect of AM251 may point to the involvement of endogenously formed cannabinoids acting via CB1 receptors. Indeed, the plasma level of anandamide is higher in hypertensive patients and animals including rats on high sodium intake (for literature, see the Introduction section). The reason for the gradual increase of the neurogenic vasopressor response associated with AM251 is unclear especially in the light of the fact that the antagonism of CP55940 against AM251 is already evident at S2 and S3. We suppose that AEA, which is formed in a depolarization-dependent manner [6], may increasingly accumulate upon repeated electrical stimulation. However, an alternative possibility has to be also considered, i.e., that the facilitatory effect of AM251 can be ascribed to its inverse agonism at CB1 receptors [35]. If an agonist at a given receptor inhibits noradrenaline release (e.g., CP55940), an inverse agonist at the same receptor (e.g., AM251) will have a facilitatory effect instead [35]. The fact that the inhibition of AEA degradation by URB597 did not influence the neurogenic vasopressor response would be in harmony with the latter notion. A final decision whether an increased formation of endogenous cannabinoids, a marked constitutive activity of presynaptic CB1 receptors or both mechanisms play a role cannot be reached on the basis of the present results.

5. Conclusions Our results show that the function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves is enhanced in DOCA–salt hypertensive rats. Thus, the CB 1 receptor-mediated inhibition of the excessive noradrenaline release from the sympathetic nerve fibres innervating the resistance vessels might play a protective role in hypertension. On the other hand, one should keep in mind, that peripherally restricted CB1 receptor antagonists, which may become new therapies for controlling obesity and related disorders (e.g., [3]), may increase sympathetic nerve activity and thereby precipitate or worsen an essential hypertension and a metabolic syndrome.

Conflict of interest There are no conflicts of interests.

Acknowledgements The work has been supported by the National Science Centre (Poland), research grant NCN 2012/05/B/NZ7/03102. We wish to thank Mrs. I. Malinowska and Mrs. T. Makar for their excellent technical assistance.

Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022

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Please cite this article as: M. Toczek, et al., Enhanced function of inhibitory presynaptic cannabinoid CB1 receptors on sympathetic nerves of DOCA–salt hypertensive rats, Life Sci (2015), http://dx.doi.org/10.1016/j.lfs.2015.03.022