Nitric oxide inhibition by L-NAME but not 7-NI induces a transient increase in cortical cerebral blood flow and affects the cerebrovasodilation induced by TRH

Peptides 24 (2003) 579–583

Nitric oxide inhibition by L-NAME but not 7-NI induces a transient increase in cortical cerebral blood flow and affects the cerebrovasodilation induced by TRH Lars-Owe D. Koskinen a,b,∗ , Mona L. Koch b a

Department of Pharmacology and Clinical Neuroscience, Division of Neurosurgery, Umeå University Hospital, SE-901 85 Umeå, Sweden b Institution of Threat Assessment, Division of NBC Defence, Swedish Defence Research Agency, Umeå, Sweden Received 10 December 2002; accepted 10 February 2003

Abstract The tripeptide thyrotropin releasing hormone (TRH) has multiple interesting and complex physiological effects. One of these is the cerebrovasodilating effect, which has been described under several different conditions. The final mechanism for this effect is unknown. In the present study, we found an initial atropine-resistant cerebral vasodilation (24%) elicited by the NOS inhibitor L-NAME in the rat. D-NAME and 7-NI did not produce this effect. TRH (300 ␮g kg−1 , i.v.) induced an increase in cerebral blood flow by 62%. L-NAME reduced this effect significantly. The cerebrovasodilating mechanism of TRH, at least in part, is endothelial NO dependent as the neuronal 7-NI NOS inhibitor does not affect the TRH response. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Nitric oxide; L-NAME; 7-NI; TRH; Cerebral blood flow; Peptides

1. Introduction Thyrotropin releasing hormone (TRH), a neuropeptide which can be found in many tissues, is involved in a variety of physiological and pathophysiological processes [10,11,34]. Clinically this neuropeptide and its analogues have been employed in the treatment of some cerebrovascular disorders [25,29] and motor disorders [2,5,33]. Animal studies have shown beneficial effects of TRH and some analogues in experimental stroke and brain injury [4,7,18,28]. Also, it was recently reported that TRH penetrates skin and exerts biological effects after transdermal delivery [26]. The cardiovascular effects of TRH and some of its analogues include a prominent cerebrovasodilating effect [12,15–17,22,23]. The mechanism of this cerebrovasodilation is not fully understood, though it appears not to be a direct effect on the vessel [19]. A brainstem-mediated activation of a cerebrovasodilating pathway is involved [19]. Nitric oxide (NO) is a mediator for wide range of physiological effects. NO is well known to induce an increase in cerebral blood flow [8,32]. Some peptides elicit their vascular effects by NO-mediated mechanisms. Whether the ∗

Corresponding author. Tel.: +46-90-7852826; fax: +46-90-122448. E-mail address: [email protected] (L.-O.D. Koskinen).

cerebrovasodilating effect of TRH in the rat is mediated by NO is unknown. The aim of the present study was to investigate the involvement of NO in the cerebrovascular effects of TRH. Secondly, the study evaluated whether endothelial or neuronal NO mechanisms are involved. Thirdly, the unexpected hyperemic response to the NOS inhibitor, L-NAME, was studied in some detail.

2. Methods Rats (male Sprague–Dawley; Møllegaard, Ejby, Denmark) weighing 337–414 g were used in the study. Animals were housed five rats per cage. The room was artificially lighted in a 12 h/12 h light/dark cycle and the room temperature was 22 ± 2 ◦ C with humidity 50 ± 5%. The animals had free access to the pelleted R34 diet (Lactamin, Vadstena, Sweden) and tap water. The experiments were performed between 08:00 and 14:00 h. Anesthesia was achieved with 120 mg kg−1 thiobutabarbital (Inactin® , RBI, Natick, MA) intraperitoneally. The tracheostomized animal was connected to a ventilator (model 683, Harvard Apparatus, South Natick, MA). Both femoral arteries were cannulated. One was used for continuous mean

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arterial blood pressure (MAP) monitoring with a Gould Statham P 23 ID pressure transducer (Gould Inc., Oxnard, CA) and an SE 120 recorder (ABB-Goerz-Metrawatt, Vienna, Austria). The MAP signal was also collected on-line using a personal computer. The other arterial access cannula was used for blood sampling. Arterial pO2 , pCO2 , and pH were determined at regular intervals with an ABL 500 blood gas analyzer (Radiometer, Copenhagen, Denmark). Continuous infusion of 0.5 ml h−1 100 g b. wt.−1 of Ringer’s solution (25 mM NaHCO3 , 120 mM NaCl, 2.5 mM KCl, 0.75 mM CaCl2 ) via a cannulated femoral vein was provided by a syringe pump (341A, SAGE Instruments, Cambridge, MA). Skeletal muscle relaxation was induced by pancuronium bromide (Pavulon® , Organon, Oss, Holland), 0.2 mg kg−1 intravenously. Additional doses of pancuronium were given when needed to facilitate controlled ventilation. The rectal temperature was measured and maintained at 37–38 ◦ C using a CMA 150 servo heating pad (Carnegie Medicine, Stockholm, Sweden). The outer ear meatuses and the “razzed” skin of the head were locally pre-treated with topical lidocain hydrochloride (Xylocain® salve 5%, Astra, Södertälje, Sweden). With the animal placed in a stereotaxic frame (I.H. Wells Jr., Mechanical Developments Co., South Gate, CA), the parietal bone was exposed, and a hole of 2–3 mm in diameter was made with a flat-bottomed drill. The center of the hole was 4 mm lateral to the sagital suture and 3 mm caudal to the coronal suture. Continuous cooling with physiological saline was performed while drilling. A very thin bone layer was left intact (0.1 mm) in order not to disturb the cortical blood flow. Dural and pial blood vessels were easily observed through the partial craniotomy. Cortical microcirculation was continuously measured using a laser-Doppler flowmeter (Periflux PF4001, Perimed, Järfälla, Sweden). A micromanipulator was used to position the measuring probe (PF 403, outer diameter 1.0 mm, fiber diameter 0.125 mm, fiber separation 0.25 mm) on the thin bone layer. Large dural and pial vessels were avoided. Appropriate position of the probe was checked by a brief period of apnea eliciting an increase in cerebral blood flow. The laser-Doppler method has been validated for measurement of the cerebral microcirculation [6,27]. At least 30 min elapsed from the positioning of the probe to the start of blood flow recordings. The laser-Doppler signal was recorded on a desktop computer and analyzed with the Perisoft software (Perimed, Järfälla, Sweden) and reported as perfusion units (PU). The vascular resistance was calculated as R = MAP (kPa)/Q (PU) where R is vascular resistance and reported as vascular resistance unit (VRU), MAP is mean arterial blood pressure, and Q is tissue blood flow. The CBF was measured continuously before and after the intravenous (i.v.) administration of 30 mg kg−1 L-NAME (n = 8) and D-NAME (n = 6) (dissolved in saline, 45 mg ml−1 ), and after the intraperitoneally administered 7-NI (n = 8). Thirty minutes later, 300 ␮g kg−1 TRH (dissolved in 0.9% NaCl, 200 ␮l) was administered i.v. and the

effect recorded. 7-NI was dissolved in peanut oil. In order to assure that peanut oil per se has no vascular effects, the vehicle was administered i.p. and blood flows measured (n = 4). The effect of TRH in animals pre-treated, as mentioned above, is compared with the effect of TRH in animals receiving TRH in the same dose without any pre-treatment (n = 8). In an attempt to elucidate whether the cerebral hyperemia discovered after L-NAME administration was due to a muscarinic mechanism, a group of animals was pre-treated i.v. with atropine sulfate (n = 8). The blood flows were recorded before (control) and 5 min after the i.v. administration of 0.5 mg−1 kg atropine followed 2 min later by the i.v. administration of 30 mg kg−1 L-NAME. TRH (Lot No. 33H5820), L-NAME (Lot No. 102H0930, 44H0102), D-NAME (Lot No. 54H0226), 7-NI (Lot No. B125127), atropine sulphate (Lot No. 55H0884), and peanut oil (Lot No. 50H0123) were acquired from Sigma Chemicals Co. (St. Louis, MO). The results of the various NO-related effects were evaluated for statistically significant differences by ANOVA. Student’s t-test with Bonferroni correction was used as a post hoc test. The paired two-tailed Student’s t-test was used to compare the effect of TRH with the baseline CBF or CBVR as the animal was its own control. In order to statistically evaluate whether the TRH effect differed between the control group and the pre-treated groups the Bonferroni corrected unpaired two-tailed Student’s t-test was employed. Results are reported as means ± S.E.M. The experiments were approved by the Regional Research Ethical Committee according to the national law.

3. Results Table 1 shows the blood gas values, MAP, and hemoglobin concentration. L-NAME caused a significant increase in MAP in the control group (F(7, 4) = 25.54, P < 0.0001), and atropine-treated group (F(7, 4) = 37.03, P < 0.0001). A concomitant decrease in HR was observed. D-NAME, 7-NI, and peanut oil had no pressor effect. There were no significant changes in blood gas values and pH during the experiments. Baseline cerebral blood flows are similar to previously reported results. The CBF in the L-NAME group was 151.0 ± 16.6 PU, D-NAME group 120.0 ± 16.1 PU, atropine group 177.9 ± 14.9 PU, 7-NI group 123.2 ± 8.3 PU, and peanut oil group 140.7 ± 30.1 PU. As depicted in Fig. 1, L-NAME induced a transient and significant increase in CBF (F(7, 3) = 46.89, P < 0.0001). The increase was 24 ± 6% (P < 0.02) as compared to control value. This increase appeared at 1.28 ± 0.12 min after L-NAME administration. Notably, the cerebral vascular resistance (CBVR) was unchanged during the hyperemia. Thus, the baseline CBVR was 0.11 ± 0.01 VRU and during hyperemia 0.10 ± 0.01 VRU. Atropine per se reduced the CBF slightly to 160.5 ± 12.3 PU (P < 0.05). In these atropine-pretreated animals, L-NAME elicited a CBF

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Table 1 Cardiovascular parameters MAP (kPa)

HR (min−1 )

pHa

pa CO2 (kPa)

pa O2 (kPa)

Hemoglobin (g l−1 )

L-NAME C Hyper 15 30 TRH

15.4 19.3 19.7 19.9 20.8

± ± ± ± ±

0.6 0.9∗∗ 0.5∗∗ 0.5∗∗ 0.4∗∗

389 349 328 319 328

± ± ± ± ±

11 12 5 5 7

7.45 7.46 7.43 7.43 7.44

± ± ± ± ±

0.02 0.01 0.02 0.02 0.01

4.74 4.79 4.65 4.41 4.64

± ± ± ± ±

0.10 0.10 0.23 0.29 0.16

12.66 13.36 13.35 13.01 13.02

± ± ± ± ±

0.42 0.31 0.38 0.56 0.30

146 151 148 146 148

± ± ± ± ±

5 3 3 4 4

D-NAME C Hyper 15 30 TRH

14.5 15.1 14.3 14.2 15.8

± ± ± ± ±

0.8 0.8 0.8 0.8 0.8

390 393 379 373 378

± ± ± ± ±

14 14 13 11 10

7.47 7.46 7.46 7.47 7.47

± ± ± ± ±

0.01 0.01 0.01 0.01 0.01

4.80 4.74 4.81 4.74 4.72

± ± ± ± ±

0.06 0.12 0.12 0.05 0.08

13.26 13.47 13.49 13.40 13.47

± ± ± ± ±

0.43 0.52 0.42 0.35 0.52

150 148 147 145 147

± ± ± ± ±

8 8 8 7 7

7-NI C Hyper 15 30 TRH

15.5 16.6 16.7 16.5 16.7

± ± ± ± ±

0.5 0.7 0.5 0.5 0.5

401 411 400 394 397

± ± ± ± ±

14 10 8 9 8

7.47 7.47 7.47 7.48 7.48

± ± ± ± ±

0.01 0.01 0.01 0.01 0.01

4.69 4.84 4.58 4.49 4.49

± ± ± ± ±

0.07 0.06 0.05 0.07 0.07

12.96 12.75 13.11 13.25 13.25

± ± ± ± ±

0.33 0.26 0.26 0.29 0.29

156 160 157 156 156

± ± ± ± ±

2 2 3 3 3

Peanut oil C Hyper 15 30

14.2 14.9 14.0 14.1

± ± ± ±

1.2 1.6 1.0 1.2

360 356 344 351

± ± ± ±

23 20 19 20

7.49 7.49 7.49 7.48

± ± ± ±

0.02 0.02 0.01 0.01

4.64 4.70 4.62 4.55

± ± ± ±

0.17 0.12 0.05 0.10

13.74 13.61 13.82 13.96

± ± ± ±

0.66 0.55 0.48 0.45

144 138 141 138

± ± ± ±

7 7 7 6

Atropine C Atropine Hyper 15 30

15.6 13.4 18.8 20.3 20.5

± ± ± ± ±

0.6 0.8 0.6∗∗ 0.4∗∗ 0.4∗∗

417 404 375 348 344

± ± ± ± ±

9 6 5 8 8

7.48 7.48 7.48 7.48 7.47

± ± ± ± ±

0.01 0.01 0.01 0.01 0.01

4.75 4.60 4.54 4.29 4.28

± ± ± ± ±

0.06 0.07 0.07 0.06 0.08

13.05 13.52 13.80 13.90 14.02

± ± ± ± ±

0.18 0.11 0.14 0.17 0.22

157 151 151 155 156

± ± ± ± ±

2 2 2 2 2

Cardiovascular parameters, blood gas values, and hemoglobin concentrations before (C) and during the hyperemic response or at an equivalent time point (hyper), 15 and 30 min after the administration of the drug (L-NAME, i.v., n = 8; D-NAME, i.v., n = 6; 7-NI, i.p., n = 8, 30 mg kg−1 ) followed by 300 ␮g kg−1 TRH, i.v. Atropine (0.5 mg−1 kg, n = 8) was followed by L-NAME. In the peanut oil group (n = 4), no other drug was administered. ∗∗ P < 0.01 as compared to control = C (Bonferroni test).

Fig. 1. Relative change in CBF after L-NAME (30 mg kg−1 , n = 8), D-NAME (30 mg kg−1 , n = 6), L-NAME (30 mg kg−1 ) pre-treated with atropine (0.5 mg kg−1 , n = 8) i.v., 7-NI (30 mg kg−1 , n = 8), and peanut oil (n = 4) i.p. In cases were no hyperemia accurred the value is 2 min after the administration of the drug. ∗∗ P < 0.02, ∗∗∗ P < 0.001 as compared to baseline (Bonferroni test).

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Fig. 2. Relative change in CBF after TRH (300 ␮g kg−1 , n = 8) i.v. in control situation and after pre-treatment with L-NAME (30 mg kg−1 , n = 8), D-NAME (30 mg kg−1 , n = 6) i.v., 7-NI (30 mg kg−1 , n = 8), and peanut oil (n = 4) i.p. ∗ P < 0.05, ∗∗ P < 0.01 as compared to baseline (paired Student’s t-test).

increase of 30 ± 6% (P < 0.02) which appeared at 1.70 ± 0.22 min after L-NAME administration (F(7, 3) = 43.48, P < 0.0001). The CBVR was 0.09 ± 0.01 VRU during control, and not significantly affected by atropine or during the hyperemia induced by L-NAME. As depicted in Fig. 1, the hyperemia gradually changed to a decrease in CBF. At 15 min after L-NAME administration, the CBF decreased by 19 ± 2% (P < 0.001) of the control value, and at 30 min the decrease was 23 ± 3% (P < 0.001). The CBVR values were 0.17 ± 0.02 VRU (P < 0.005) and 0.18 ± 0.02 VRU (P < 0.005) indicating a vasoconstriction (F(7, 3) = 33.12, P < 0.0001). In the atropine-pretreated animals, a similar decrease in CBF was observed, and the corresponding values were 14±5% (P < 0.05) and 14±4% (P < 0.05). The CBVR values were 0.16 ± 0.01 VRU (P < 0.002) and 0.16 ± 0.01 VRU (P < 0.002) (F(7, 3) = 29.25, P < 0.0001). D-NAME, 7-NI, and peanut oil induced no effect on the CBF or CBVR. As shown in Fig. 2, TRH caused a significant increase in CBF by 62 ± 16% (P < 0.01) above control value at 1.3±0.1 min after TRH in animals without pre-treatment. In these animals, the CBVR decreased from 0.093±0.012 VRU to 0.061 ± 0.008 VRU (P < 0.01), indicating a vasodilation. In the L-NAME group, the CBF increase was only 19 ± 5% (P < 0.01). This cerebrovasodilating effect of TRH was significantly reduced as compared to animals without pre-treatment (P < 0.01). CBVR changed from 0.17 ± 0.03 VRU to 0.15 ± 0.02 VRU. In D-NAME pre-treated animals, TRH elicited a significant vasodilation, a 75 ± 29% (P < 0.05) increase in blood flow compared before TRH administration. This is reflected as a decrease in CBVR from 0.13 ± 0.02 VRU to 0.09 ± 0.02 VRU (P < 0.02). 7-NI had no effect on the TRH response and the increase in

CBF was 90 ± 28% (P < 0.01) and CBVR declined from 0.14 ± 0.01 VRU to 0.08 ± 0.01 VRU (P < 0.01). Peanut oil had no effect on the cerebrovasodilating property of TRH.

4. Discussion Several studies have shown that NO is a potent cerebral vasodilator [8,32]. NO is also a mediator in a number of other physiological processes. Many effects in pathophysiological events have been described [3]. NO seems to have some beneficial effects in experimental brain injury but negative effects have also been reported [1,9,13,24]. A main finding in the present study is the temporary increase in CBF after L-NAME. The response was not caused by a muscarinic vasodilating mechanism. One can speculate that the autoregulation of the CBF is attenuated by NOS-inhibition during the hyperemic period as the blood pressure was significantly increased and the vascular resistance unchanged. The hyperemia seems not to be an unspecific effect because D-NAME and 7-NI are devoid of the hyperemic response. The increased CBF was normalized within minutes, and a definitive vasoconstriction elicited at 15 min after the L-NAME administration. This effect was long lasting. The reduction in cerebral blood flow after NOS-inhibition appears to be an endothelial effect since 7-NI did not mimic the effect of L-NAME. TRH has been reported to have pronounced cerebrovasodilating properties [14,17,22,23]. This is not a direct effect on the vasculature and TRH seems to activate a cerebrovasodilating pathway [19]. Although the TRH vasodilating mechanism has been studied, the exact mechanism is unknown. An atropine sulfate [14] but not methylatropine

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[17] sensitive mechanism has been proposed. Muscarinic blockade does not inhibit the increase in CBF in the rabbit [21]. Opiate antagonist has no major effect [20] while an ␣2 -mechanism is partially involved [31]. The present results clearly indicate that, at least in part, an NOS-sensitive mechanism is involved in the mediation of the TRH-induced cerebrovasodilation. The mechanism seems to be of endothelial origin since the inhibition of the neuronal NOS did not influence the response to TRH. A species difference can be prevailing as NOS-inhibition does not counteract the cerebrovasodilating response to TRH in the rabbit [30]. However, in that study, a 10-fold higher dose of L-NAME and a 3-fold higher TRH dose was used. In conclusion, L-NAME elicited a transient increase followed by a decrease in cerebral blood flow. The hyperemic response is not atropine sensitive. The cerebrovasodilating response of TRH is affected by endothelial NOS-inhibition.

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[19]

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