Adrenergic vasoconstrictor activity in the cerebral circulation after inhibition of nitric oxide synthesis in conscious goats

Autonomic Neuroscience: Basic and Clinical 89 Ž2001. 16–23 www.elsevier.comrlocaterautneu

Adrenergic vasoconstrictor activity in the cerebral circulation after inhibition of nitric oxide synthesis in conscious goats Nuria Fernandez, Marıa Luis Monge, Angel Luis Garcıa-Villalon, ´ ´ Angeles Martınez, ´ ´ ´ ) Godofredo Dieguez ´ Departamento de Fisiologıa, de Madrid, Arzobispo Morcillo, 2, 28029 Madrid, Spain ´ Facultad de Medicina, UniÕersidad Autonoma ´ Received 24 November 2000; received in revised form 6 February 2001; accepted 2 March 2001

Abstract The interaction between nitric oxide ŽNO. and adrenergic activity in the cerebral circulation was studied using conscious goats, where blood flow to one brain hemisphere Žcerebral blood flow. was electromagnetically measured, and the effects of phentolamine and hexamethonium on cerebrovascular resistance were evaluated before Žcontrol. and after inhibition of NO synthesis with N W-nitro-Larginine methyl ester ŽL-NAME.. L-NAME Ž12 goats, 40 mg kgy1 administered i.v.. reduced cerebral blood flow from 62 " 3 to 44 " 2 ml miny1, increased mean systemic arterial pressure from 100 " 3 to 126 " 4 mm Hg, decreased heart rate from 79 " 5 to 50 " 4 beats miny1, and increased cerebrovascular resistance from 1.63 " 0.08 to 2.91 " 0.016 mm Hg mly1 miny1 Žall P - 0.01.. These hemodynamic variables normalized 48–72 h after L-NAME administration. Phentolamine Žsix goats, 1 mg., injected into the cerebral circulation, increased cerebral blood flow without changing systemic arterial pressure, but its cerebrovascular effects were augmented for about 24 h after L-NAME. The decrements in cerebrovascular resistance induced by phentolamine, in mm Hg mly1 miny1, were: under control, 0.42 " 0.05; immediately after L-NAME, 1.38 " 0.09 Ž P - 0.01 compared with control.; by about 24 h after L-NAME, 0.71 " 0.09 Ž P - 0.05 compared with control.; and by about 48 h after L-NAME, 0.40 " 0.07 Ž P ) 0.05 compared with control.. Hexamethonium Žsix goats, 0.5–1 mg kgy1 miny1 i.v.. decreased mean systemic arterial pressure to about 75 mm Hg and caused tachycardia similarly before and after L-NAME, but the decrements in cerebrovascular resistance were augmented for about 24 h after y1 L-NAME. The decrements in cerebrovascular resistance induced by hexamethonium, in mm Hg ml miny1, were: under control, Ž . 0.61 " 0.09; immediately after L-NAME, 1.33 " 0.16 P - 0.01 compared with control ; by about 24 h after L-NAME, 1.18 " 0.10 Ž P - 0.01 compared with control.; and by about 48 h after L-NAME, 0.99 " 0.10 Ž P ) 0.05 compared with control.. Therefore, these results suggest that adrenergic vasoconstrictor tone in cerebral vasculature may be augmented after inhibition of NO synthesis, and that this increment may contribute to the reduction of cerebral blood flow after inhibition of NO formation. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Cerebral blood flow; Cerebrovascular resistance; Cerebral vasoconstriction

1. Introduction Experimental data suggest that nitric oxide ŽNO. and sympathetic adrenergic activity may interact in the vascular wall, and this interaction may be involved in the regulation of vascular function. It has been suggested that NO, in addition to producing direct vasodilatation ŽMoncada et al., 1991., may modulate both sympathetic adrenergic activity and vasoconstriction ŽHansen et al., 1994; Zanzinger et al., 1994.. On the other hand, adrenergic activation, in addition to inducing vasoconstriction, )

Corresponding author. Fax: q34-91-397-5324. .. E-mail address: [email protected] ŽG. Dieguez ´

may stimulate the release of NO in blood vessels ŽVanhoutte et al., 1986; Lacolley et al., 1991.. With regard to the cerebral circulation, experimental evidence suggests that NO may be released in the cerebral vessel wall from the endothelium and perivascular nerves, and that it may be involved in the regulation of cerebral blood flow by producing a basal vasodilator tone and modulating cerebrovascular reactivity ŽKovach ´ et al., 1992; Faraci and Brian, 1994; Kelly et al., 1994.. On the other hand, cerebral blood vessels appear to receive a dense adrenergic innervation and, although the functional significance of this innervation remains a controversial topic, some in vitro and in vivo experiments suggest that the cerebral vasculature exhibits a basal adrenergic constrictor

1566-0702r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 Ž 0 1 . 0 0 2 4 4 - 2

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tone, and constricts to noradrenaline by direct alpha-adrenergic activation, and to sympathetic nerve stimulation or tyramine by releasing noradrenaline from perivascular nerve terminals ŽEdvinsson et al., 1993b; Goadsby and Edvinsson, 1997; Jordan et al., 2000.. Therefore, NO and sympathetic adrenergic activity may be two counterregulatory forces that may play a role in the regulation of the cerebral circulation. Studies exploring the possible interaction between NO and adrenergic activity in cerebral blood vessels are sparse, and the results reported are inconclusive ŽBauknight et al., 1992; Kelly et al., 1994; Wagerle et al., 1995; Aldasoro et al., 1996.. In relation to this, there are data suggesting that NO may inhibit the cerebral vasoconstriction in vivo to exogenous noradrenaline ŽBauknight et al., 1992., and that it may also inhibit the cerebral vasoconstriction in vitro to nerve stimulation ŽAldasoro et al., 1996.. Also, it has been reported that the relation between cerebral blood flow and arterial pressure during hypertension after inhibition of NO synthesis is similar in intact brain areas and sympathectomized brain cortical areas ŽKelly et al., 1994.. In our laboratory, we have observed that the reactivity of the cerebral vasculature of awake goats to exogenous and endogenous noradrenaline may be increased after inhibition of NO synthesis ŽDieguez et al., 1998.. Also, we have ´ recently reported that the role of NO in the cerebrovascular response to hypotension may differ depending on how hypotension is induced, for example, during hypotension after ganglionic blockade the cerebral vasodilator tone mediated by NO is diminished ŽDieguez et al., 1999.. ´ The present study was performed to examine further the interaction between NO and adrenergic activity in the cerebral circulation by evaluating the changes in resting adrenergic constrictor activity in cerebral vasculature after inhibition of NO synthesis. This study provides new data that extend and complement previous experiments from our laboratory ŽDieguez et al., 1998, 1999.. The experi´ ments were performed using an experimental model in the goat where blood flow to one cerebral hemisphere can be continuously measured on a beat-to-beat basis ŽReimann et al., 1972., and where it has been previously reported that adrenergic mechanisms ŽLluch et al., 1973, 1975. and NO ŽDieguez et al., 1993; Fernandez et al., 1993. may be ´ ´ involved in the regulation of cerebral blood flow under normal conditions.

2. Materials and methods 2.1. Experimental preparation Blood flow to one cerebral hemisphere was measured in 12 female goats Ž34–53 kg body weight. with an electromagnetic flow transducer previously placed on the ipsilateral internal maxillary artery. In the goat, each internal maxillary artery provides the total blood flow to each

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cerebral hemisphere, and the vertebral arteries do not contribute to brain blood flow ŽDaniel et al., 1953.. The circle of Willis in the goat is similar to that of humans except that the blood flows in a caudal direction in the basilar artery ŽDaniel et al., 1953; Reimann et al., 1972.. Analysis of the distribution of radioactively labeled microspheres in the cerebral circulation of the goat after the surgical procedure described by Reimann et al. Ž1972. indicates that nearly all of the blood flow carried by the internal maxillary artery passes directly to cerebral tissue ŽMiletich et al., 1975.. Extracerebral blood flow is minimal, accounting for less than 5% of total flow. The operative procedure to measure cerebral blood flow has been described in detail elsewhere ŽReimann et al., 1972.. Briefly, the extracerebral branches from one of the left internal maxillary arteries were ligated and thrombosed with 1000 units of thrombin ŽThrombin, ICN Biomedicals. dissolved in 1 ml of 0.9% NaCl solution. This manoeuver produces an almost immediate obliteration of the ethmoidal, ophthalmic, and buccinator arteries and thus eliminates blood flow to the eye and other facial structures. This is confirmed on recovery from surgery by the presence of ipsilateral blindness. However, obliteration of the extracerebral vessels from the internal maxillary artery does not cut off the vascular supply to half of the face. The areas supplied by the ethmoidal, buccinator, dental, and temporal arteries are nourished by anastomotic channels that are normally in a state of dynamic balance but in which the direction of blood flow can be quickly changed, depending on the pressure differential from one side of the union to the other ŽDaniel et al., 1953; Reimann et al., 1972.. There is no necrosis, and the functions related to these areas such as eating, drinking, and ruminating are intact. Obliteration and thrombosis of the ophthalmic artery permanently cuts off vascular supply to the ipsilateral eye. This procedure becomes necessary for the successful isolation of the cerebral circulation ŽReimann et al., 1972; Miletich et al., 1975.. An electromagnetic flow tranducer ŽBiotronex, Silver Spring, MD. was placed on the left internal maxillary artery to measure blood flow to the ipsilateral hemisphere. A polyethylene catheter ŽPE-90. inserted in the temporal artery permitted the injection of drugs directly into the internal maxillary artery in the awake goat; the same catheter was used to measure arterial blood pressure with a Statham P23 ID transducer, and to obtain blood samples for gasesrpH measurements. A snare-type occluder was placed on the external carotid, close to the temporal artery, to obtain zero-flow base lines. The external connecting leads from the flow transducer and occluder, and the temporal artery catheter were led out subcutaneously and secured to the horn of the goat. Heart rate was measured from arterial pressure pulse with a ratemeter. Flow measurements were made with a Biotronex electromagnetic flowmeter Žmodel BL-610.. Cerebral blood flow, systemic arterial blood pressure, and

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N. Fernandez et al.r Autonomic Neuroscience: Basic and Clinical 89 (2001) 16–23 ´

heart rate were recorded on a Dynograph recorder Žmodel R611, Sensor Medics, Bilthoven, The Netherlands.. Electromagnetic flow transducers provide blood flow measurements in absolute values Žml miny1 ., which are obtained from the recordings and applying the calibration of the flow tranducer used ŽReimann et al., 1972.. The experiments on the awake goats were started 2–3 days after the operative procedure, at which time the goats had fully recovered and were in good condition. The various measurements were made with the goat unrestrained in a large cage, except for a Lucite stock fitting loosely around the neck that limited forward and backward motion. Once placed in the cage, the animal stood quietly during the experiments and showed no signs of disturbance. However, the experiments were stopped whenever the animal showed signs of excitation or uneasiness evidenced by alterations in the recordings of blood pressure and heart rate. In this event, the goat was brought back to the animal quarters, and a period of more than 24 h was allowed before attempting a new experiment.

The experimental procedure used in the present study was approved by the local animal research committee of our institution. 2.3. Data analysis All hemodynamic measurements before and after LNAME treatment were compared using the same animal as its own control. An ANOVA for repeated measures, followed by a Dunnett test was applied to the hemodynamic data Žin absolute and in percentage. obtained before and after L-NAME to evaluate the hemodynamic effects of inhibition of NO formation. The same procedure was applied to evaluate the hemodynamic data with phentolamine and hexametonium, both under control conditions and after L-NAME; then the effects of these two interventions on cerebrovascular resistance, in absolute values, before and after L-NAME were compared to evaluate the effects of inhibition of NO production on the action of phentolamine and hexametonium. P - 0.05 was considered statistically significant.

2.2. Experimental protocol 2.4. Chemicals In this work, the following experiments were performed in conscious goats. After resting control measurements were recorded, the effects of phentolamine Žsix goats. and of hexamethonium Žsix goats. on cerebral blood flow and systemic arterial pressure were recorded under control conditions and after inhibition of NO formation with N Wnitro-L-arginine methyl ester ŽL-NAME.. Phentolamine Ž1 mg., dissolved in physiological saline at a concentration of 1 mg mly1 , was directly injected into the cerebral circulation through the catheter placed in the left temporal artery. The ganglion blocker hexamethonium, dissolved in physiological saline at a concentration of 30 mg mly1 , was infused by i.v. route at a rate of 0.5–1 mg kgy1 miny1 until mean arterial pressure decreased to about 75 mm Hg. L-NAME, dissolved in physiological saline at a concentration of 10 mg mly1 , was administrated by i.v. route during 15–20 min, and the animals received a dose of 40 mg kgy1 . The experiments with phentolamine were performed in one group of six animals and those with hexamethonium were performed in another group of six animals. The experiments with these two substances before Žcontrol. and after L-NAME in each animal were separated by 3–4 days. The effects of phentolamine and hexamethonium after L-NAME were tested immediately after L-NAME administration when hemodynamic parameters reached a steady state, and then at about 24, 48 and 72 h after treatment with L-NAME. The cerebrovascular effects of phentolamine and hexamethonium under control conditions and after L-NAME treatment were evaluated as changes in cerebrovascular resistance. This resistance was calculated as mean systemic arterial pressure in mm Hg divided by blood flow to one cerebral hemisphere in ml miny1 .

N W -nitro-L-arginine methyl ester ŽL-NAME., phentolamine hydrochloride, and hexametonium chloride, were all obtained from Sigma.

3. Results 3.1. Effects of L-NAME In 12 awake goats, i.v. injection of L-NAME Ž40 mg kgy1 . caused hypertension, bradycardia and decreases in cerebral blood flow. The maximum effects of L-NAME were reached 4–6 min after ending the injection, and cerebral blood flow decreased by 29 " 3%, mean systemic arterial pressure increased by 25 " 3%, heart rate decreased by 37 " 4%, and calculated cerebrovascular resistance increased by 78 " 6% Žall P - 0.01.. These hemodynamic variables returned to control after this treatment: arterial pressure normalized by about 72 h, cerebral blood flow by about 48 h, heart rate at about 48 h, and calculated cerebrovascular resistance by about 72 h. Table 1 summarizes the hemodynamic values obtained in conscious goats under control conditions and at distinct moments after L-NAME treatment. L-NAME treatment did not affect significantly the levels of blood gases and pH as compared with control conditions ŽTable 1.. 3.2. Effects of phentolamine In six goats under control conditions, phentolamine Ž1 mg., injected into the cerebral circulation, increased cerebral blood flow by 19 " 2 ml miny1 Ž P - 0.01. and did

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Table 1 Hemodynamic values Žmeans" SEM. obtained in 12 awake goats under control conditions and after L-NAME treatment Control

After L-NAME treatment Immediate

CBF Žml miny1 . MAP Žmm Hg. CVR Žmm Hg mly1 miny1 . HR Žbeats miny1 . pO 2 Žmm Hg. pCO 2 Žmm Hg. pH

62 " 3 100 " 3 1.63 " 0.08 79 " 5 84 " 3 32 " 2 7.43 " 0.002

))

44 " 2 126 " 4 ) ) 2.91 " 0.16 ) ) 50 " 3 ) ) 82 " 5 33 " 3 7.42 " 0.003

; 24 h ))

49 " 3 116 " 4 ) 2.36 " 0.17 ) ) 62 " 6 ) 84 " 2 34 " 3 7.41 " 0.002

; 48 h

; 72 h

55 " 4 111 " 3 ) 2.02 " 0.15 ) 74 " 6 81 " 4 35 " 4 7.40 " 0.003

60 " 4 101 " 5 1.67 " 0.16 73 " 5 83 " 3 34 " 3 7.44 " 0.002

CBF s cerebral blood flow; MAP s mean systemic arterial pressure; CVR s cerebrovascular resistance; HR s heart rate. ) P - 0.05 compared with its corresponding control. )) P - 0.01 compared with its corresponding control.

not affect systemic arterial pressure and heart rate. Cerebrovascular resistance after phentolamine decreased by 0.42 " 0.05 mm Hg mly1 miny1 Ž P - 0.01.. In the same six goats after L-NAME treatment, phentolamine Ž1 mg. also increased cerebral blood flow without affecting systemic arterial pressure and heart rate, but its cerebrovascular effects were higher than under control conditions. Immediately after L-NAME, phentolamine increased cerebral blood flow by 39 " 3 ml miny1 Ž P - 0.001., which was significantly greater than under control Ž P 0.05., and decreased cerebrovascular resistance by 1.38 " 0.09 mm Hg mly1 miny1 Ž P - 0.001., which was significantly greater than under control Ž P - 0.01.. By about 24 h after L-NAME, phentolamine increased cerebral blood flow by 28 " 4 ml miny1 Ž P - 0.005., which was not significantly different from under control Ž P - 0.05., and decreased cerebrovascular resistance by 0.71 " 0.09 mm Hg mly1 miny1 Ž P s 0.005., which was significantly greater than under control Ž P - 0.05.. By about 48 h after L-NAME, phentolamine increased cerebral blood flow by 22 " 5 ml miny1 Ž P - 0.05., which was not significantly distinct from under control Ž P ) 0.05., and decreased cerebrovascular resistance by 0.40 " 0.07 mm Hg mly1 miny1 Ž P - 0.01., which was not significantly distinct from under control Ž P ) 0.05..

mly1 miny1 Ž P - 0.001., this decrement being significantly greater than under control Ž P - 0.01.. By about 24 h after L-NAME, hexamethonium decreased mean arterial pressure to 72 " 3 mm Hg Ž P - 0.01., increased non-significantly cerebral blood flow Ž P ) 0.05., and decreased cerebrovascular resistance by 1.18 " 0.10 mm Hg mly1 miny1 Ž P - 0.001., this decrement being significantly greater than under control Ž P - 0.01.. By about 48 h after L-NAME, hexamethonium decreased mean arterial pressure to 73 " 4 mm Hg Ž P - 0.01., increased non-significantly cerebral blood flow Ž P ) 0.05., and decreased cerebrovascular resistance by 0.99 " 0.10 mm Hg mly1 miny1 Ž P - 0.01., this decrement being not significantly different from under control Ž P ) 0.05.. Hexamethonium, before and after L-NAME, did not affect significantly the levels of arterial blood gases and pH as compared to control conditions Žthese data are not shown..

3.3. Effects of hexametonium In six goats under control conditions, hexamethonium infused i.v. decreased mean arterial pressure to 72 " 3 mm Hg Ž P - 0.01., increased heart rate to 96 " 6 beats miny1 Ž P - 0.05., and did not affect significantly cerebral blood flow Ž P ) 0.05.. Cerebrovascular resistance decreased by 0.61 " 0.09 mm Hg mly1 miny1 Ž P - 0.01.. In the same six goats after L-NAME treatment, hexamethonium also caused hypotension and tachycardia. Immediately after L-NAME, hexamethonium decreased mean arterial pressure to 79 " 5 mm Hg Ž P - 0.01., increased non-significantly cerebral blood flow Ž P ) 0.05., and decreased cerebrovascular resistance by 1.33 " 0.16 mm Hg

Fig. 1. Decreases in cerebrovascular resistance induced by phentolamine ŽZ, six animals. and hexametonium ŽI, six animals. obtained in conscious goats under control conditions and at different moments after )) L-NAME treatment. P - 0.01 and ) P - 0.05 compared with its corresponding effects under control conditions.

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Fig. 1 summarizes the effects of phentolamine and hexametonium on cerebrovascular resistance before Žcontrol. and after L-NAME treatment.

4. Discussion The present study in conscious goats shows that LNAME reduces resting cerebral blood flow and produces hypertension and bradycardia, confirming previous observations from our laboratory ŽDieguez et al., 1993, 1999; ´ Fernandez et al., 1993.. These effects of L-NAME on ´ cerebral blood flow are probably related, at least in part, to the inhibition of basal NO production, thus suggesting that NO produces a basal vasodilator tone in cerebral vasculature under normal conditions. This agrees with that suggested by others ŽFaraci and Brian, 1994; Faraci and Heistad, 1998.. As it has been previously reported ŽFernandez et al., 1993; Dieguez et al., 1998., the present ´ ´ experiments also show that the hemodynamic effects of L-NAME persisted for a relatively long period and that NO production recovers after its inhibition with this L-arginine analogue. We have used a single dose of L-NAME because it has shown to be effective for producing evident cerebral and peripheral vascular effects for about 24–48 h in our experimental preparation ŽFernandez et al., 1993; Dieguez ´ ´ et al., 1998., which permits to examine the role of NO in the regulation of the cerebral circulation during this period without the necessity of repeated administrations of this drug. Studies performed in awake Merino ewes indicate that the vasopressor effect of a single i.v. dose of L-NAME Žthis dose was lower than that used in the present study. remained for at least 24 h ŽMeyer et al., 1994., and those performed in rats indicate that the cerebrovascular effects of a single intraperitoneal dose of this drug remained for 15 h ŽKelly et al., 1995.. W L-NAME, a methyl ester of N -nitro-L -arginine, has been used frequently as an inhibitor of NO synthesis to evaluate the role of NO in the regulation of blood vessels tone, in general ŽMoncada et al., 1991., and of the cerebral blood vessels in particular ŽFaraci and Brian, 1994; Faraci and Heistad, 1998.. This L-arginine analogue inhibits in a competitive manner the constitutive NO synthase Žthis inhibitory capacity may be similar for the endothelial, NOS III, and neuronal, NOS I, isoforms., and in a minor degree for the inducible NO synthase, NOS II ŽMoncada et al., 1997.. This capacity of L-NAME has been tested in cortical tissue of dogs, cats and pigs, which was in a doseand time-dependent manner, and the duration of this action was at least 6 h ŽTraystman et al., 1995.. As L-NAME may inhibit the two isoforms of constitutive NO synthase, we cannot distinguish the role played by the endothelial and neuronal isoforms in the observed cerebrovascular effects of L-NAME. The dose of L-NAME used in the present study is higher than that used in other experiments ŽMoncada et al., 1991; Faraci and Brian, 1994; Conner et

al., 2000., and we have previously reported that the effects of a single dose of L-NAME on the cerebral circulation and arterial pressure are reversed by L-arginine, the substrate for NO formation, in awake ŽFernandez et al., 1993. ´ and anesthetized ŽDieguez et al., 1993. goats. Therefore, ´ the observed cerebrovascular effects of L-NAME in the present study are probably related to inhibition of endothelial and neuronal NO synthases and the subsequent inhibition of NO formation, but we cannot distinguish the role played by each of these two enzyme isoforms in these effects. Whereas it is generally accepted that cerebral vasculature has a dense adrenergic innervation, discrepancy exists about the role of this innervation in the regulation of the cerebral circulation ŽEdvinsson et al., 1993b; Goadsby and Edvinsson, 1997; Jordan et al., 2000.. One of the main reasons for this discrepancy, especially in relation to the presence of basal adrenergic vasoconstrictor tone, may be the use of experimental animals with and without anesthesia. As anesthesia depresses activity of the nervous system, including that of sympathetic nervous system, the use of unanesthetized, awake animals may facilitate the evaluation of sympathetic activity in blood vessels. In the present study, we have used awake goats and data with phentolamine and hexamethonium obtained under control conditions confirm previous results from our laboratory ŽLluch et al., 1975; Dieguez et al., 1999., and they are in favour ´ of the idea that cerebral vasculature may exhibit an adrenergic vasoconstrictor tone under resting conditions ŽAlborch et al., 1977.. We measured blood flow through one internal maxillary artery, which in our experimental preparation provides nearly all of the blood supply to the ipsilateral cerebral hemisphere, and as the blood supply to the goat brain occurs via an intracranial carotid rete, it raises the question of whether or not the observed effects of phentolamine and hexamethonium on cerebral blood flow in the awake goat could reflect the overall response of retial and cerebral vasculature to these drugs. It has been reported earlier that goat retial arteries, in contrast to goat pial arteries, have a very poor adrenergic innervation ŽConde et al., 1978; Santamarıa ´ et al., 1987., and that adrenergic innervation may regulate cerebral blood flow by affecting cerebral vasculature rather than retial vessels ŽUrquilla et al., 1974; Lluch et al., 1975, 1985; Conde et al., 1978; Dieguez et al., 1983, 1988.. ´ The magnitude of basal adrenergic cerebral vasoconstrictor activity may be evaluated by the decrement in cerebrovascular resistance produced by antagonists of aadrenoceptors Že.g., phentolamine. or by blockers of sympathetic ganglia Že.g., hexamethonium.. We found that phentolamine increased resting cerebral blood flow without changing systemic arterial pressure, suggesting that the decreased cerebrovascular resistance after this drug is probably related with cerebral vasodilatation after inhibition of alpha-adrenergic vasoconstrictor tone present in cerebral blood vessels under normal conditions. Hexam-

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ethonium did not affect resting cerebral blood flow in spite of hypotension suggesting that the observed decrement in cerebrovascular resistance is related to cerebral vasodilatation after inhibition of sympathetic vasoconstrictor activity in this vasculature. The results with hexamethonium agree with those previously found in conscious goats after treatment with trimetaphan, another ganglioplegic blocking agent ŽLluch et al., 1978.. Studies to explore the relationship between cerebral blood flow and arterial pressure suggest the presence of an autoregulatory response in the face of arterial pressure changes, and that in this response myogenic, metabolic, neurogenic and endothelial factors may be involved ŽEdvinsson et al., 1993a.. Our results with hexamethonium suggest that neurogenic factors may be involved in the observed relationship between cerebral blood flow and arterial pressure. We cannot, however, exclude the involvement of other factors such as NO ŽDieguez et al., 1999. or the possible direct effects of ´ hexamethonium on cerebral vasculature on that relaxation. Previous findings from our laboratory indicate that inhibition of NO synthesis increases cerebrovascular reactivity to exogenous and endogenous noradrenaline probably due, at least in part, to changes at the post-junctional level in the adrenergic innervation of the cerebral vessel wall, and that this increased adrenergic reactivity might contribute to the decrease in cerebral blood flow induced by inhibition of NO synthesis ŽDieguez et al., 1998.. Also, data obtained ´ in awake goats suggest that the NO-mediated vasodilator tone in the cerebral circulation is decreased during hypotension after ganglion blockade with hexamethonium probably due, at least in part, to attenuation of the shear stress-mediated and sympathetic-mediated NO release in cerebral blood vessels ŽDieguez et al., 1999.. These two ´ studies ŽDieguez et al., 1998, 1999. suggest that NO and ´ sympathetic activity may interact, thus contributing to regulate cerebral blood flow. This idea is supported by the present data with phentolamine and hexamethonium, as the decreases in cerebrovascular resistance induced by these two drugs were higher after L-NAME treatment than under control conditions. The observed changes in the cerebral circulation were analyzed using changes in cerebrovascular resistance because they probably reflect better the in vivo vascular effects, especially when arterial pressure is affected ŽLautt, 1989.. Consequently, our data suggest that adrenergic vasoconstrictor tone in the cerebral circulation increases after inhibition of NO synthesis with L-NAME, and this increment was present immediately after L-NAME treatment and persisted for about 24 h. This feature parallels the evolution of the effects of inhibition of NO formation on cerebral blood flow ŽDieguez et al., 1993, 1997; ´ Fernandez et al., 1993. and of the increased adrenergic ´ reactivity of cerebral vasculature after inhibition of NO formation ŽDieguez et al., 1998.. The increased cerebral ´ vasodilatation induced by phentolamine and hexamethonium after L-NAME may be related to the inhibition of NO synthesis rather than to an unspecific vasoconstrictor effect

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of L-NAME as we have previously found that this drug modifies selectively the response of the cerebral vasculature to different vasodilator stimuli ŽFernandez et al., 1993; ´ Dieguez et al., 1993, 1997.. Also, we have observed ´ previously in awake goats that phentolamine permitted increase in cerebral blood flow during hypertension induced with adrenaline or noradrenaline, but not during hypertension induced with angiotensin II ŽDieguez et al., ´ 1979.. Therefore, we suggest that the observed increased cerebrovascular effects of phentolamine, and probably those of hexamethonium are related, at least in part, to the presence of an augmented adrenergic vasoconstrictor tone in the cerebral vasculature after inhibition of NO synthesis with L-NAME. In the study of Dieguez et al. Ž1998., the reactivity of ´ the cerebral vasculature to exogenous and endogenous noradrenaline was examined, and in the present work, we have evaluated the magnitude of the adrenergic vasoconstrictor activity produced by endogenous adrenergic mechanisms under normal, resting conditions. Both of these studies permit analysis of different aspects of adrenergic mechanisms that are intimately related, and provide different, complementary information about the interaction between NO and adrenergic activity in the cerebral circulation. Based on the study by Dieguez et al. Ž1998., we can ´ suggest that the observed increased cerebral adrenergic vasoconstrictor activity after inhibition of NO production in the present study might be due, at least in part, to the increased adrenergic reactivity of this vasculature to endogenous adrenergic mechanisms that may be present under basal conditions. Studies related to the functional interaction between NO and adrenergic activity in the cerebral circulation are sparse, and the results reported are inconclusive ŽBauknight et al., 1992; Kelly et al., 1994; Wagerle et al., 1995; Aldasoro et al., 1996.. Data from anesthetized rabbits show that noradrenaline does not change the diameter of pial arteries under basal conditions, but it reduces their diameter during inhibition of NO synthesis, suggesting that under normal conditions noradrenaline releases NO from to the endothelium, which then inhibits the vasoconstriction to adrenergic stimulation ŽBauknight et al., 1992.. In line with this study, Wagerle et al. Ž1995. observed that inhibition of NO synthesis with L-NAME markedly augmented the contractile response to noradrenaline in the isolated middle cerebral artery from near-term ovine fetus. These experiments ŽBauknight et al., 1992; Wagerle et al., 1995. suggest that NO acts at the post-junctional level in the vessel wall thus inhibiting the response to noradrenaline. This conclusion agrees with that obtained from experiments where the effects of noradrenaline on systemic arterial pressure in anesthetized cats ŽZanzinger et al., 1994. and of electrical stimulation on the rat caudal artery ŽVo et al., 1992. have been studied. In summary, the present study suggests that inhibition of NO synthesis increases resting adrenergic vasocon-

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strictor tone in cerebral blood vessels, which may contribute to the decrease of cerebral blood flow after this inhibition. Therefore, our data support the idea that NO and adrenergic mechanisms may interact, and that this interaction may be involved in the regulation of the cerebral circulation.

Acknowledgements The authors are grateful to Mrs. E. Martınez and H. ´ Fernandez-Lomana for technical assistance. This work was ´ supported, in part, by CICYT ŽNo. 95r0032., FIS ŽNo. 96r0474. and CAM ŽNo. AE 00 236r96..

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