Functional evidence that the angiotensin antagonist losartan crosses the blood-brain barrier in the rat

Brain Research Bulkfin, Vol. 30, pp. 33-39, Printed in the USA. All rights reserved.

1993 Copyright

0361-9230/93 $6.00 + .OO 0 1992 Pergamon Press Ltd.

Functional Evidence That the Angiotensin Antagonist Losartan Crosses the Blood-Brain Barrier in the Rat ZHENHUI

LI, JAIDEEP S. BAINS AND ALASTAIR V. FERGUSON’

Department of Physiology, Queen’s University, Kingston, Ontario, Canada K7L 3N6 Received 29 June 1992; Accepted 12 July 1992 LI, Z., J. S. BAINS AND A. V. FERGUSON. Functional evidence that the angiotensin antagonist losartan crosses the bloodbrain barrier in the rat. BRAIN RES BULL 30( l/2) 33-39, 1993.-Losartan is a novel nonpeptidergic antagonist of angiotensin (ANG) II subtype I (AT,) receptors, which effectively lowers blood pressure in high-renin hypertensive rat and blocks the pressor response to systemic ANG II. It is well known that high densities of ANG II receptors exist in the hypothalamic paraventricular nucleus (PVN). In addition, activation of putative angiotensinergic afferents to the PVN originating in suhfomical organ (SFO) elevates blood pressure and facilitates the activity of PVN neurons. We report here that systemic administration of losartan (3 mg/kg) significantly attenuates the pressor response to electrical stimulation of SFO. The excitatory responses of PVN neurons to SF0 stimulation or local pressure microinjection of ANG II were also significantly inhibited in 58.8% and 88.9% of PVN cells, respectively, by intravenous administration of losartan. These pharmacological effects were rapid and reversible, and were accompanied by little change of basal arterial blood pressure or spontaneous neuronal activity. These observations suggest that systemic losartan crosses the blood-brain barrier (BBB) and acts at AT1 receptors within the PVN. Losartan (Dup-753) Electrophysiology

Angiotensin II

Hypothalamic paraventricular nucleus

ANGIOTENSIN (ANG) II interactions with CNS receptors located both inside and outside the blood-brain barrier (BBB) cause drinking, an increase in plasma vasopressin concentration, and an elevation in blood pressure (30). As in peripheral organs, brain ANG II receptors are divided into two subtypes according to their different binding with nonpeptide antagonists or reducing agents (4-6,38). Binding of ligand to ANG II subtype 1 (AT,) receptors is sensitive to relatively low concentrations of losartan (Dup-753) and is resistant to inhibition by low concentrations of PD 1233 19 or WL 19; while binding to ANG II subtype 2 (AT,) receptors is inhibited by PD 1233 19, but not by relatively low concentration of losartan (7,14,35,39,40). Several studies have shown that losartan effectively lowers mean arterial blood pressure in rats with high-renin hypertension, and blocks pressor and drinking responses to systemically administered ANG II (4 1,42). The specificity and the lack of partial agonist actions of losartan have suggested this antagonist to be potentially useful as a physiological probe and a therapeutic agent. However, there is at present conflicting evidence regarding the ability of this lipophilic substance to cross the BBB (12,43).

Subfornical organ

The hypothalamic paraventricular nucleus (PVN) is one of the critical nuclei involved in regulation of neuroendocrine and autonomic functions in the CNS. It contains large populations of vasopressin (AVP)-, oxytocin (OXT)-, and corticotropin releasing hormone (CRH)-secreting neurons and sends efferent fibers to both the brainstem and spinal cord (2,8,26,33,37). In addition, the existence of a high density of ANG II receptors and angiotensinergic innervation has also been demonstrated in this region of the rat brain (3,16,2 1,23). Local microinjection, or microiontophoresis of ANG II into the PVN excites neurosecretory cells, increases plasma AVP concentration, and elevates blood pressure in vivo (1,32). Previous studies have also demonstrated that activation of subfornical organ (SFO) efferent projections to the hypothalamus results in a rise in blood pressure (1 1,17) and facilitates the firing activity of PVN cells (9,10,34), effects which have been suggested to result from actions of ANG II as a neurotransmitter in this nucleus (24). In the present studies we have utilized this model system in which activation of angiotensinergic projections from SF0 to PVN results in well-

I To whom requests for reprints should be addressed.

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established physiological and electrophysiological evaluate the ability of losartan to cross the BBB.

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METHOD

Male Sprague-Dawley rats ( 150-300 g, n = 44) were anaesthetized with IP urethane (1.4 g/kg), and the femoral artery and vein catheterized (PE 50 lntramedic) to enable the continuous monitoring of arterial blood pressure and the administration of drugs, respectively. Throughout all experiments body temperature was maintained at 37 f 1°C by a feedback-controlled heating blanket.

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Electrical Stimulation of SF0 Animals were then placed in a stereotaxic frame and a small midline burr hole was made 0.8 mm posterior to bregma; a monopolar tungsten stimulating electrode (Rhodes Medical lnstruments-tip exposure 200 pm) was advanced into the region of SF0 in 200 pm increments according to the coordinates of Paxinos and Watson (29). Electrical stimuli (200 FA, IO Hz, 1 ms pulse duration for 10 s) were presented at each 200 pm step in the electrode track, and the optimal stimulation site was determined according to the ability of such stimulation to elicit characteristic changes in arterial blood pressure (11,17). Once this site was identified, and a control response to stimulation

F’IG. I. Photomicrographs showing representative examples of (A) SF0 lesion made by the stimulating electrode tip and (B) the recording electrode penetration through PVN. The arrow indicates the penetration track. HC: hippocampal commissure; PVN: hypothalamic pamventricular nucleus; SFO: subfornical organ; V: third ventricle. The horizontal scale bars represent 0.5 mm in (A) and 0.2 mm in (B).

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A concentric bipolar stimulating electrode (Rhodes Medical Instruments, SNEX- IOOX, tip exposure 0.25 mm) was directed toward the region of the SF0 as described above. The electrode was fixed to the skull by use of jeweller’s screws and dental cement and was connected to an isolated stimulation unit controlled by a digitimer. The animal was then placed supine in a stereotaxic frame, the trachea cannulated, and the medial basal hypothalamus exposed through a transpharyngeal approach. Extracellular single unit recordings were obtained from neurons in the PVN using NaCl (2.0 M) filled recording pipettes ( 15-20 MO; tip diameter < 1 pm) attached to a five-barrel glass microelectrode. Signals were amplified, displayed on a digital oscilloscope, and the pulse output of a window discriminator was led to an online computer programmed for spike train analysis. The other pipettes of this microelectrode were separately filled with ANG II (10 FM, L-glutamate (0.5 M), and 0.9% saline, and connected to a picospritzer (General Vale Corp.). Ejection of drugs was achieved by applying a continuous pressure (1-25 s, 2-40 psi) to the pipette tips (overall diameter, < 8 pm) which were set back from the recording electrode by 15-30 Wm. Glutamate was used for seeking cells, and saline for excluding the direct effect of pressure on cells. After at least 5 min baseline recording of spontaneous activity, two different protocols resulting in excitation of PVN neurons were undertaken. These involved either electrical stimulation of SF0 (200-400 PA; 100 ps pulse duration for 100-200 cycles) or local administration of ANG II by pressure injection. The effects of SF0 stimulation were evaluated by comparing the number of action potentials in a period of 300 ms observed in a peristimulus histogram prior to stimulation with the number in the same period after stimulation, while the effects of local ANG II were determined by comparing the number of action potentials recorded immediately before treatment with the number during and immediately following treatment. A 30% change was assigned as an arbitrary level for significant alteration in a neuron’s activity. In cells demonstrating an increased activity following either treatment, losartan was IV administrated (3 mg/ kg) to determine whether such systemically administered drug could reach and influence central AT 1 receptors. The excitation observed in response to SF0 stimulation or local administration of ANG II prior to pharmacological treatment was assigned as the control response. A reduction in these responses of greater than 50% following drug administration was designated as indicative of receptor blockade. The data were excluded from our analyses whenever spike amplitude changed abruptly after pressure application or systemic injection of agents.

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was obtained, losartan (3 mg/kg, DuPont) or saralasin (30 pg, Sigma) was administered systemically. A baseline was reestablished, and SF0 was again stimulated and the effects of such stimulation on blood pressure and heart rate were monitored. In order to observe recovery of the pressor response following pharmacological treatment, the SF0 was stimulated every 15 min following drug administration. ANG II receptor blockade by saralasin or losartan was confirmed by comparing the blood pressure response to 1V administration of ANG II (200 ng, Sigma) prior to and following administration of antagonists. Mean arterial blood pressure and heart rate were monitored throughout the experiment by a Buxco cardiovascular analyzer. Data were displayed and stored for later analysis using a computer based data acquisition system.

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FIG. 2. Bar graphs summarising the blood pressure response to electrical stimulation in SFO, and the effects of losartan or saralasin on this response. (A) Mean group data illustrating that pressor responses in 18 rats tested were significantly inhibited following losartan but not saralasin administration. (B) Mean group data illustrating the time course of recovery of SFO-induced pressor responses following losartan.

Histology At the conclusion of each experiment, a small anodal lesion was made at the tip of the SFO-stimulating electrode. The animal was then perfused through the left ventricle of the heart with saline followed by a 10% formalin solution. The brain was removed and stored in formalin for at least 24 h. Coronal sections ( 100 pm) were cut using a Vibratome, stained with cresyl violet, and the location of stimulating sites in the SF0 and recording electrode penetrations in the PVN were verified (Fig. 1). RESULTS

SF0 Stimulation In histologically verified SF0 sites, electrical stimulation resulted in a characteristic biphasic increase in blood pressure with

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FIG. 3. Peristimulus histograms illustrating that systemic losartan affects the excitatory response of three different PVN neurons to SF0 stimulation. The upper panel shows the control excitation induced by SF0 stimulation (400 PA, 100 ~LSpulse duration, 1.1 s/per cycle) in these PVN cells. Triangles indicate the time of SF0 stimulus presentation. The lower panel demonstrates blockade (A2, B2) or potentiation (C2) of excitatory neuronal response lo-12 min after IV administration of losartan. The horizontal axis is presented in ms and the vertical axis in spikes/bin. Peristimulus histograms in A consist of 200 sweeps with a resolution of 4 ms per bin; histograms in B and C 100 sweeps with a resolution of 2 ms. Dose of losartan: 3 mg/kg, IV.

a mean increase in arterial blood pressure of 16.6 + 2.3 mmHg (Fig. 2A). The maximum response was observed within IO s after the onset of stimulation and the overall response had a mean duration of 63.2 rt 7.5 s. Following intravenous injection of losattan, the pressor response was reduced to 9.3 t 1.6 mmHg, which was significantly different from the increase in blood pres-

sure prior to treatment (p < 0.001, n = 14). The basal blood pressure and heart rate, however, were unaffected treatment. These effects of losartan were observed within and pressor effects of SF0 stimulation did not return to values until approximately 75 min after administration 2B), indicating a long duration of drug action.

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The effects of systemic losartan were tested on 17 PVN neurons that showed an increase in excitability following electrical stimulation of SFO. This SFO-evoked excitation had a latency of 65.7 f 10.2 ms (range: 20- 170 ms). SFO-evoked effects were reduced by losartan in 10 neurons, unaffected in five and potentiated in two cells. Peristimulus histograms from 10 of 17 (58.8%) PVN cells revealed a reduction of 85.04 ? 11.48% in the SFO-evoked excitation after IV administration of losartan as illustrated in Fig. 3A and B. These effects of losartan began within 5 min of injection and gradually became more effective, such that 60 min after losartan administration no recovery of SFO-evoked excitation was observed.

Local Application oJANG II Ill

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The effect of systemic losartan on the direct action of ANG II on PVN cells was evaluated in nine PVN cells. Local microinjection of ANG II into the PVN increased neuronal excitability (2- 10 times) with an onset latency of 2-5 s. In most cases, the responses to ANG II lasted 3-10 s after pressure microinjection and were dose dependent. Losartan administration completely abolished the direct ANG II-induced excitations observed in eight of nine (88.9%) PVN neurons as shown in Fig. 4. If the local ANG II-induced increase in spontaneous firing rate prior to antagonist application was designated as lOO%, the increase after application was 0.64 f 14.29% (p < 0.001). These effects of losartan appeared wnhm 5 min and were most potent between IO and 30 min. Recoveries of ANG II-induced excitations were observed in three PVN neurons tested 50-90 min after application of losartan (Fig. 4).

30 min

30 SBC FIG. 4. Ratemeter records from a PVN neuron show changes in the ANG II-induced neuronal response following losartan administration. ANG II application (IO-“ M, 40 psi) is indicated by the horizontal lines. The excitatory response to local ANG 11is attenuated within 5 min of IV losartan injection (3 mg/kg) and completely abolished subsequently. A partial recovery is observed after 70 min.

The above data do not exclude the possibility that the attenuation of this pressor response is the result of blockade of peripheral rather than central AT1 receptors. We, therefore, examined the effect of systemic saralasin, a peptide antagonist of ANG II receptors which does not cross the BBB, in four animals. Results showed that IV administration of saralasin was without effect (control 14.2 * 2.2 mmHg, postsaralasin 14.4 f 2.9 mmHg, p > 0.5) on the SF0 initiated blood pressure response, supporting the view that losartan’s effects were due to blockade of central AT 1 receptors.

Electrophysiological Single-Unit Recordings Single-unit recordings were obtained from a total of 79 PVN neurons, the majority of which displayed spontaneous activity of less than five spikes/s, which was unaffected by losartan treatment (control 2.36 + 0.42 Hz versus 1.67 + 0.42 Hz at 5 min and 2.02 f 0.53 Hz at 10 min, p > 0.05, n = 26). SF0 stimulation increased the activity of 69.6% (32/46) of PVN neurons, while extracellular microinjection of ANG II excited 43. I % (22/5 1) of the cells tested. Effects of losartan were examined only on

DISCUSSION Losartan is a nonpeptidergic antagonist of ANG II which inhibits the specific binding of ANG II and blocks ANG IIinduced 45Ca2f efflux in smooth muscle (6). IV losartan antagonizes pressor responses to ANG II without affecting the responses to norepinepherine, vasopressin, and isoproterenol, or the activity of converting enzyme or renin (6,43). Furthermore, losartan given orally or intravenously lowers blood pressure in furosemide-treated and renal hypertensive animals with high renin activity but does not change blood pressure in conscious normotensive or salt-hypertensive rats with low plasma renin activity (43). These studies have suggested that unlike peptide antagonists (saralasin) or ACE inhibitors (captopril), losartan is a potent, orally active, selective, and competitive AT1 receptor antagonist with a long duration of action. With the development of this and other nonpeptide antagonists, the distribution and binding properties of brain ANG II receptors have been revealed more clearly ( 14,15,19,28,36,39). The pharmacological effects of losartan demonstrated in the peripheral renin-ANG system intimate that it may have similar actions centrally. Autoradiographic studies have revealed a high concentration of ANG II receptors, predominantly the AT, subtype within both parvocellular and magnocellular regions of the PVN (15,36). The PVN receives many fiber projections from the SF0 (25,27), and it has been shown that electrical stimulation of the SF0 (9,10,34) or local microinjection of ANG II into the PVN (1) evokes an increase in excitability in the majority of PVN neurosecretory neurons. In view of the presence of ANG II-immunoreactive neurons in the SF0 (22) the distribution of

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ANG II-immunoreactive fibers ( 13,2 1,24) and ANG II receptors within the PVN (3,15,16), as well as the demonstration of ANG release in the PVN induced by physiological and chemical stimuli (18) it has been proposed that this peptide may mediate excitatory signal transmission as a transmitter from the SF0 to the PVN. This provides us with a model system in which we can assess the potential effects of an ANG II antagonist such as losartan. In the present study we have observed the influences of systemically administered losartan on the blood pressure response elicited by electrical stimulation of SFO, as well as on neuronal excitability utilizing electrophysiological techniques. The results showed that losartan significantly attenuated the pressor response initiated by SF0 stimulation. This finding, coupled with the inability of saralasin, a peptide ANG II antagonist which does not cross the BBB. to affect the same response, suggests that losartan elicited its effects by blocking AT, receptors at sites located within the BBB. These data contrast those of Wong et al. (43) showing that losartan (IO mg/kg), administered orally, did not affect ICV ANG II-induced pressor responses. The discrepency may be due to differing routes of administration as well as differing doses. The dose (3 mg/kg) and route of administration (IV) utilised in the present study were comparable to those in a recent study showing that losartan inhibited ANG IIinduced water drinking in the rat ( 12). Our electrophysiological data also demonstrated that in the majority of PVN neurons tested, systemic losartan could effectively block cell excitation induced by SF0 stimulation and local microinjection of ANG II into the PVN. Such effects usually appeared within minutes

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of losartan application and lasted for an extended period of time. These data again indicate that losartan can pass the BBB, reach, and block brain ANG II receptors in the PVN. These conclusions are consistent with the basic chemical structure of losartan (low molecular weight, lipophilic isosteres), and are supported by a recent report demonstrating that peripherally administered losartan inhibited water drinking produced by ICV ANG II ( 12). It is not clear why losartan did not block the excitatory effects of SF0 stimulation on all of the PVN neurons examined and why it potentiated the excitation of SF0 stimulation in some cases. Such observations do, however, suggest that other neurotransmitters may also be utilized by this pathway. The SF0 has been reported to contain catecholamine, serotonin, histamine, and the enzymes necessary for synthesizing all of these putative neurotransmitters (20,3 1) which, thus, all represent possible cotransmitter candidates. In conclusion, our present studies indicate that systemically administered losartan crosses the BBB and acts at central AT, receptors to block excitatory effects of endogenous ANG II in the CNS of the rat. Such findings further emphasize the potential usefulness of this pharmacological agent to manipulate central actions of this peptide. ACKNOWLEDGEMENTS

We thank Pauline Smith for her excellent technical assistance and C. Loucks who participated in some experiments. This work was supported by the Heart and Stroke Foundation of Ontario, URIF, and Eli Lilly Co. Canada. Gratitude is also expressed to DuPont for the generous donation of losartan.

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