Receptor-mediated PLGA nanoparticles for glioblastoma multiforme treatment.

Glioblastoma multiforme is the most lethal type of brain tumor and the established therapy only extends patients survival to approximately one year. Its first-line treatment is based on of chemotherapy with the alkylating agent temozolomide (TMZ). As many other chemotherapeutic drugs, TMZ presents several limitations as high toxicity and low bioavailability. The delivery of TMZ using poly(lactic-co-glycolic acid) nanoparticles is proposed in this work. Stable nanoparticles functionalized with a OX26 type monoclonal antibody for transferrin receptor were developed, targeting the glioblastoma tumor cells, since these cells are known for overexpressing this receptor. The release profile of TMZ from the nanoparticles was studied mimicking physiological conditions, and targeted cellular internalization was also investigated. Two glioblastoma cell lines - U215 and U87 - were used to evaluate the in vitro cytotoxicity of the drug, showing that the prepared nanocarriers enhance the anticancer activity of TMZ. The functionalization with the monoclonal antibody for transferrin receptor proved to be advantageous in enhancing the cellular internalization in glioblastoma cells.

Audiogenic and audiogenic-like seizures: locus of induction and seizure severity determine postictal prolactin patterns.

Audiogenic seizures (AS) are a model of generalized tonic-clonic seizures, evoked by high-intensity (110 dB) acoustic stimulation evaluated by means of behavioral severity indexes (SI). Postictal prolactin (PRL) is a marker of generalized seizures, both in animals and humans. Thus, in the present work we assayed postictal PRL in a) male Wistar AS susceptible (S, n = 5) and AS resistant (R, n = 13) rats made susceptible by specific midbrain lesions. b) In rats electrically stimulated in the central nucleus (CN) of the inferior colliculus (IC) (n = 20), or the cortical IC (CxIC, n = 18). In c) S rats pretreated with either bromocriptine (BRO; 4 mg/kg; SC), a PRL release inhibitor, or vehicle (V), 30 min before the electrical stimulation. Basal PRL was 2-10 ng/ml at time 0. In the S group, only animals with generalized seizures presented a postictal PRL elevation between 5 and 15 min (60-90 ng/ml; p < 0.05). R rats displayed a discrete PRL response lower than that of S animals. CxIC stimulation produced more severe seizures and greater postictal PRL enhancement than CNIC stimulation, always raising at 5-15 min (p < 0.01). BRO blocked the PRL increase even in the presence of higher seizure scores (p < 0.02). The positive correlation between seizure intensity (SI values), site of initiation (central or cortical IC nuclei), and postictal PRL patterns makes this a reliable model for studying the neurochemistry of the postictal phase and the interaction between hormones and epilepsy.

Participation of the median raphe nucleus and central serotoninergic pathways in the control of water electrolyte excretion.

1. The role of the median raphe nucleus (MRN) and of increased central serotonin (5HT) synthesis/release in the mediation of Na+ excretion (UNaV) and K+ excretion (UKV) and of urine output (UV) was evaluated for 120 min. 2. Male Wistar rats weighing 220-280 g were used in each group of 12-13 animals. The rats implanted with a cannula in the MRN were injected with saline (0.5 microliters) or with 5.0 and 15.0 ng/0.5 microliters kainic acid (KA), an excitatory amino acid (EAA). Another group of rats was injected ip with 200 mg/kg saline or tryptophan, the initial precursor of 5HT synthesis. 3. Injection of both kainic acid and tryptophan led to increased Na+ excretion, but the magnitude and time course were different for each treatment. 4. Both KA doses were effective in increasing UNaV (0.61 +/- 0.08, mean +/- SEM, and 0.95 +/- 0.19 microEq/min, respectively, vs 0.27 +/- 0.04 microEq/min for saline at 60 min). The effect on UKV was statistically significant with the 15.0 ng dose (0.44 +/- 0.05 microEq/min vs 0.25 +/- 0.03 microEq/min for saline) at 20 min. 5. Tryptophan administration caused an initial gradual increase in UNaV which became steady and significant after 60 min (1.02 +/- 0.15 microEq/min vs 0.36 +/- 0.06 microEq/min for saline), as well as an increase in UKV (0.58 +/- 0.06 microEq/min vs 0.26 +/- 0.04 microEq/min for saline) at 60 min and throughout the remainder of the observation period. 6. KA-induced MRN stimulation and systemic tryptophan overload significantly increased UV at 60, 80 and 100 min (30 to 97% above control values).(ABSTRACT TRUNCATED AT 250 WORDS)

Central serotonergic modulation of drinking behavior induced by water deprivation: effect of a serotonergic agonist (MK-212) administered intracerebroventricularly.

The present study was carried out to evaluate the participation of the serotonergic system (5-HT) in the modulation of the drinking response induced by water deprivation. Male Wistar rats implanted with a cannula in the 3rd ventricle were injected with the 5-HT1C/5-HT2 agonist 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212) at doses of 0.5, 5, 25, 50 and 125 nmol/2 microliters. MK-212 induced a significant reduction (P less than or equal to 0.05) in water intake over a period of 300 min. This result indicates that the central 5-HT system plays an important role, probably at the level of the periventricular hypothalamus, in the modulation of drinking behavior induced by water deprivation.

Central serotonergic modulation of drinking behavior induced by angiotensin II and carbachol in normally hydrated rats: effect of intracerebroventricular injection of MK-212.

The objective of the present study was to evaluate the role of the central serotonergic (5-HT) system in the modulation of drinking behavior induced by angiotensin II (Ang II) and carbachol. Male Wistar rats implanted with a delay cannula in the 3rd ventricle were injected with the 5-HT1C/5-HT2 agonist 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212) (50 nmol/2 microliters) before receiving an intracerebroventricular (icv) injection of Ang II or carbachol (100 ng/2 microliters). MK-212 induced a significant reduction in the drinking response evoked by Ang II or carbachol which was more marked in the case of the cholinergic agonist. The results obtained suggest that thirst and water intake produced by angiotensinergic or cholinergic activation are modulated by the action of 5-HT, possibly at the level of the periventricular hypothalamus.

Participation of the serotonergic system in the regulation of water and electrolyte balance.

To determine the possible participation of the serotonergic (5-HT) system in the regulation of water and electrolyte balance, rats were submitted to two sessions of water overloading and the 5-HT agonist MK 212 was administered intracerebroventricularly (icv) in 1.0 microliters 20 min after the second session. Urine volume and sodium and potassium excretion were then measured over a period of 120 min. Microinjection of MK-212 (1 micrograms/animal) caused a significant reduction (24 to 57%; mean, 43%) in natriuresis throughout the experimental period, and the administration of 10 micrograms/animal caused a 26-41% reduction (mean, 33%) in kaliuresis. At 20 micrograms/animal, MK-212 did not change any of the parameters investigated. No significant change in urine volume was detected after any of the treatments used.

Effect of central administration of serotoninergic agonists on electrolyte excretion control.

1. The participation of different central serotoninergic (5HT) receptors in the mediation of Na+ excretion (UNaV), K+ excretion (UKV) and urine output (UV) was evaluated. 2. Male Wistar rats weighing 220-280 g were used in each group of 9-18 animals. The rats were injected intracerebroventricularly (icv) with the 5HT agonists MK212 (1.5, 15.0 and 30.0 micrograms), 8-OH-DPAT (5.0 and 15.0 micrograms), 5HT (2.5, 12.5 and 25.0 micrograms) and DOI (10.0 and 25.0 micrograms). 3. At the lowest MK212 dose, UNaV was significantly reduced (0.18 +/- 0.04 microEq/min vs 0.35 +/- 0.04 microEq/min for saline) at 20 min. At the highest dose, MK212 provoked a significant increase in UNaV (0.60 +/- 0.06 microEq/min vs 0.34 +/- 0.03 microEq/min for saline) at 40 min. UKV values were significantly modified only at the 1.5-micrograms dose (0.18 +/- 0.04 microEq/min vs 0.35 +/- 0.04 microEq/min for saline) at 20 min. 4. Icv injection of 8-OH-DPAT provoked a significant reduction in UNaV (0.16 +/- 0.05 microEq/min vs 0.35 +/- 0.03 microEq/min for saline) and UKV (0.15 +/- 0.05 microEq/min vs 0.34 +/- 0.02 microEq/min for saline) at 40 min both doses. 5. Icv injection of 5HT at the highest dose provoked a significant increase in UNaV (0.92 +/- 0.10 microEq/min vs 0.33 +/- 0.04 microEq/min for saline) and in UKV (0.55 +/- 0.08 microEq/min vs 0.24 +/- 0.07 microEq/min for saline) at 40 min. 6. Icv administration of DOI caused a natriuretic response (0.69 +/- 0.12 microEq/min vs 0.31 +/- 0.04 microEq/min for saline) at 40 min, with no significant effect on UKV.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain serotoninergic stimulation reduces the water intake induced by systemic and central beta-adrenergic administration.

1. This study was undertaken to determine if the stimulation of central serotoninergic receptors affects the thirst-inducing action of systemically or intracerebroventricularly (icv) injected isoproterenol. 2. Male Wistar rats weighing 220-300 g were used in groups of 10-14 animals each. Normally hydrated rats implanted with a delay cannula into the third ventricle were injected icv with the 5HT1C/5HT2 agonist MK212 (50 nmol/2 microliters) prior to administration of isoproterenol sc (330 micrograms/kg body weight) or icv (10 and 25 and 50 micrograms/2 microliters). 3. Icv injections of MK212 reduced the water intake induced by isoproterenol injected systemically (56%) and by the two lowest doses of isoproterenol injected icv (76 and 86%, respectively). 4. The results suggest that the central serotoninergic system modulates the central beta-adrenergic system involved in water intake. 5. Taken together with previous results showing that the activation of 5HT1C/5HT2 receptors promotes a reduction of the dipsogenic response evoked by water deprivation or by icv injection of angiotensin II or carbachol, the present data suggest that the central serotoninergic system plays a ubiquitous role in the modulation of water intake behavior.

Drinking behavior in thyroidectomized rats: effects of central cholinergic stimulation and of water deprivation.

Normally hydrated thyroidectomized (TX) rats stimulated intracerebroventricularly with carbachol and dehydrated TX rats drank significantly smaller volumes of water than their respective controls. This suggests lower central sensitivity to thirst and to drinking behavior induced by both cholinergic activation and extracellular fluid depletion. Dehydrated TX rats excreted a significantly larger urinary volume than the controls, suggesting the existence of changes in the renal mechanisms of water retention. Such changes could be related to a reduction in vasopressin binding sites.

Thyroidectomy reduces stress-induced prolactin secretion in rats. Participation of brain serotonergic systems.

Plasma prolactin levels were measured in thyroidectomized and sham-operated rats under immobilization stress. Thyroidectomy significantly reduced prolactin secretion during stress. The pretreatment of thyroidectomized rats with 6-chloro-2-[1-piperazinyl]- pyrazine (MK 212), a serotonin agonist that easily penetrates the blood-brain barrier, reversed the reduction in stress-induced prolactin release in the thyroidectomized group.

Effects of central epinephrine synthesis inhibition on stress-induced prolactin secretion in male rats.

1. The present study was designed to examine the role of central epinephrine pathways in the control of stress-induced prolactin secretion in male adult Wistar rats. 2. Intracerebroventricular administration of two epinephrine synthesis inhibitors, SKF 64139 (5 and 10 micrograms/rat) and LY 134046 (10 and 20 micrograms/rat), 6 h before the onset of immobilization stress blocked prolactin secretion in a dose-dependent manner. Prolactin values before stress were about 4.0 ng/ml and increased to almost 50 ng/ml in the control group. SKF 64139 injection in the higher dose (10 micrograms/rat) induced a complete blockade of the stress-induced prolactin release, whereas partial blockade was observed after the higher dose (20 micrograms/rat) of LY 134046. 3. Salbutamol pretreatment (10 micrograms/rat) completely restored stress-induced prolactin secretion in animals receiving a central injection of both epinephrine synthesis inhibitors under the same conditions as described above. 4. It is suggested that epinephrine pathways in the brain play an important role in the control of prolactin release occurring during immobilization stress.

Desipramine blocks stress-induced prolactin release in rats: role of central beta-2 adrenoceptors.

1. The present study was designed to examine the relationship between beta-adrenoceptors and the enhanced, sustained prolactin secretion induced by immobilization stress in rats. 2. Chronic administration of desipramine (15 mg kg-1 day-1, intraperitoneally) for 7 days, a procedure that desensitizes central beta-adrenoceptors, partially inhibits stress-induced prolactin release. 3. Intracerebroventricular administration of the beta-2 adrenoceptor agonist salbutamol (1 microgram/rat) to rats pretreated with desipramine for 7 days, 15 min before immobilization, significantly relieved the inhibition by desipramine 5 and 10 min after the initiation of stress but the effect was not demonstrable after 20 and 40 min. 4. We conclude that beta-2 adrenoceptors play a role in the control of prolactin release in response to stress.

Effect of chronic oral administration of a low dose of captopril on sodium appetite of hypothyroid rats. Influence of aldosterone treatment.

Rats rendered hypothyroid by treatment with methimazole develop an exaggerated sodium appetite. We investigated here the capacity of hypothyroid rats (N = 12 for each group) to respond to a low dose of captopril added to the ration, a paradigm which induces an increase in angiotensin II synthesis in cerebral areas that regulate sodium appetite by increasing the availability of circulating angiotensin I. In addition, we determined the influence of aldosterone in hypothyroid rats during the expression of spontaneous sodium appetite and after captopril treatment. Captopril significantly increased (P<0.05) the daily intake of 1.8% NaCl (in ml/100 g body weight) in hypothyroid rats after 36 days of methimazole administration (day 36: 9.2 +/- 0.7 vs day 32: 2.8 +/- 0.6 ml, on the 4th day after captopril treatment). After the discontinuation of captopril treatment, daily 1.8% NaCl intake reached values ranging from 10.0 +/- 0.9 to 13.9 +/- 1.0 ml, 48 to 60 days after treatment with methimazole. Aldosterone treatment significantly reduced (P<0.05) saline intake before (7.3 +/- 1.6 vs day 0, 14.4 +/- 1.3 ml) and after captopril treatment. Our results demonstrate that, although hypothyroid rats develop a deficiency in the production of all components of the renin-angiotensin-aldosterone system, their capacity to synthesize angiotensin II at the cerebral level is preserved. The partial reversal of daily 1.8% NaCl intake during aldosterone treatment suggests that sodium retention reduces both spontaneous and captopril-induced salt appetite.

Thyroidectomy blocks stress-induced prolactin rise in rats: role of the central serotonergic system.

Thyroidectomized (TX) adult Wistar male rats and their sham-operated controls were submitted to immobilization stress during forty minutes. Thyroidectomy partially blocks stress-induced prolactin (PRL) secretion. Previous administration of MK 212, a serotonin agonist, reverts this picture. The effect of MK 212 is specifically due to its interaction with 5HT2 receptors, since the injection of LY 53857, a selective blocker of these receptors, 30 min before MK 212, prevents the effect of this serotonin agonist. LY 53857, injected alone, yields a partial blockade of PRL secretion during stress in sham-operated rats. TX rats receiving LY 53857 or saline have comparable low values of plasma PRL during stress. It is suggested that thyroidectomy disrupts the functional integrity of the central serotonergic pathways involved in the stress-induced PRL rise.

Participation of the ascending serotonergic system in the stimulation of atrial natriuretic peptide release.

Results obtained in our laboratories have provided evidence for the participation of the hypothalamic atrial natriuretic peptide (ANP) neuronal system in the regulation of water and electrolyte homeostasis. The anterior ventral third ventricular (AV3V) region, a site of the perikarya of the ANP neurons, receives important afferent input from ascending serotoninergic axons. We hypothesized that the ascending serotoninergic tract might be involved in control of the liberation of ANP. Therefore, electrolytic lesions were produced in the mesencephalic dorsal raphé nucleus (DRN), the site of perikarya of serotonin (5-HT) neurons whose axons project to the AV3V region. Rats with sham lesions constituted the control group. In a second group of animals, the serotoninergic system was depleted of 5-HT by lateral ventricular administration of p-chlorophenylalanine (PCPA), an amino acid that causes depletion of 5-HT from the serotoninergic neurons. Control animals were injected with an equal amount of isotonic saline. The DRN lesions induced an increase of water intake and urine output beginning on the first day that lasted for 1 week after lesions were produced. There was a concomitant sodium retention that lasted for the same period of time. When water-loaded, DRN-lesioned and PCPA-injected animals showed diminished excretion of sodium, accompanied by a decrease in basal plasma ANP concentrations, and blockade of the increase in plasma ANP, which followed blood volume expansion by intraatrial injection of hypertonic saline. The results are interpreted to mean that ascending stimulatory serotoninergic input into the ANP neuronal system in the AV3V region produces a tonic stimulation of ANP release, which augments sodium excretion and inhibits water intake. Therefore, in the absence of this serotoninergic input following destruction of the serotoninergic neurons by DRN lesions or intraventricular injection of PCPA, an antinatriuretic effect is obtained that is associated with increased drinking, either because of sodium retention per se or removal of ANP-induced inhibition of release of the dipsogenic peptide, angiotensin II. The serotoninergic afferents also play an essential, stimulatory role in volume expansion-induced release of ANP and the ensuing natriuresis.

Reduced prolactin release during immobilization stress in thyrotoxic rats: role of the central serotoninergic system.

Adult Wistar male rats in a thyrotoxic state T4 increases rats) induced by administration of T4 (350 micrograms/kg/day, i.p. for 7 days) as well as their euthyroid controls were submitted to immobilization stress during forty minutes. Prolactin (PRL) secretion during stress was significantly lower in T4 increases rats as compared to control animals. Treatment with MK 212, a serotoninergic agonist, entirely reverts this situation. The effect of MK 212 seems to be due to its interaction with 5-HT2 receptors since it is blocked by LY 53857, a selective 5-HT2 antagonist. Furthermore, the blockade of 5-HT2 receptors by LY 53857, a selective 5-HT2 antagonist, significantly diminishes prolactin (PRL) response to stress in euthyroid rats but has no effect in T4 increases animals. It is suggested that an increased concentration of thyroid hormone in plasma disrupts an endogenous serotoninergic brain input necessary to trigger stress-induced PRL rise.

Hypothyroidism attenuates stress-induced prolactin and corticosterone release in septic rats.

We investigated the effects of sepsis, through the lipopolysaccharide (LPS)-induced inflammatory response, on plasma corticosterone and prolactin (PRL) levels during acute immobilization stress in normal and thyroidectomized rats. Thyroidectomized (TX) or sham-operated (N) rats were subjected to 120 min of immobilization stress. Rats were treated with an intraperitoneal injection of either LPS (250 microg (100 g body wt)(-1)) or the same volume of vehicle (saline solution), 90 min before the induction of stress. Blood samples for hormone assays were collected before sepsis and stress induction for baseline measures (-90 min), and during sepsis and immobilization stress for the measurement of prolactin and corticosterone levels by radioimmunoassay. Our results show that the thyroid hormones are necessary for a proper response of PRL and corticosterone release during immobilization stress. Although sepsis enhanced PRL secretion, this was not true of corticosterone release in either group of rats. Low levels of thyroid hormones partially block the release of PRL, but do not block corticosterone secretion during sepsis.

Possible role of endothelin acting within the hypothalamus to induce the release of atrial natriuretic peptide and natriuresis.

Since endothelin has been localized in neurons in areas involved in water and electrolyte metabolism, areas which also contain atrial natriuretic peptide (ANP) neurons, we determined whether endothelin would release ANP and induce natriuresis. Endothelin-3 (ET-3) in doses ranging from 38 to 760 pmol was microinjected into the third ventricle (3V) of conscious, water-loaded male rats, and the effect on natriuresis and plasma ANP was determined. ET-3 evoked a dose-related natriuresis beginning within 20 min of injection. Even the lowest dose tested (38 pmol) was effective. At a dose of 95 pmol, it produced a rapid increase of plasma ANP within 5 min peaking at 20 min. A slight kaliuresis and antidiuresis was observed at the 2 highest doses of 380 and 760 pmol. The urinary changes following 3V injection of ET-3 were similar to those evoked by ANP, except for the antidiuresis with increased sodium concentration which followed injection of the 2 higher doses. These results suggest that these 2 higher doses also released vasopressin. Alternatively, activation of the sympathetic nervous system by these higher doses may have decreased glomerular filtration rate and been in part responsible for the antidiuresis. The results with 3V injection of ET-3 contrasted sharply with those obtained following intravenous injection of the 95-pmol dose injected intraventricularly. This intravenous dose of ANP induced a transient decrease in sodium and potassium excretion and urine volume, maximal at 20 min, and had no effect on plasma ANP concentrations at 5 or 20 min after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Lesions of the hypothalamus and pituitary inhibit volume-expansion-induced release of atrial natriuretic peptide.

Expansion of the blood volume causes a release of atrial natriuretic peptide (ANP) that is believed to be important in induction of the subsequent natriuresis and diuresis which, in turn, acts to reduce the increase in blood volume. Since stimulation of the anteroventral portion of the third cerebral ventricle (AV3V) induced a rapid elevation of plasma ANP, whereas lesions of the AV3V were followed by a marked decline in plasma concentration of the peptide, we hypothesized that release of ANP from the brain ANP neuronal system might be important to the control of plasma ANP. The perikarya of the ANP-containing neurons are densely distributed in the AV3V and their axons project to the median eminence and neural lobe. To test the hypothesis that these neurons are involved in volume-expansion-induced ANP release, by using electrolysis we destroyed the AV3V, the site of the perikarya, in male rats. Other lesions were made in the median eminence and posterior pituitary, sites of termination of the axons of these neurons, and also hypophysectomy was performed in other animals. In conscious freely moving animals, volume expansion and stimulation of postulated sodium receptors in the hypothalamus were induced by injection of hypertonic NaCl solution [0.5 or 0.3 M NaCl; 2 ml/100 g (body weight)]. Volume expansion alone was induced with the same volume of an isotonic solution (NaCl or glucose). In the sham-operated rats, volume expansion with hypertonic or isotonic solutions caused equivalent rapid increases in plasma ANP that peaked at 5 min and returned nearly to control values by 15 min. Lesions caused a decrease in the initial levels of plasma ANP on comparison with values from the sham-operated rats, and each type of lesion induced a highly significant suppression of the response to volume expansion on testing 1-5 days after lesions were made. Because a common denominator of the lesions was elimination of the brain ANP neuronal system, these results suggest that the brain ANP plays an important role in the mediation of the release of ANP that occurs after volume expansion. Since the content of ANP in this system is much less than that in the atria, there must be a remarkable increase in synthesis and release of brain ANP associated with this stimulus. It is also possible that blockade of volume-expansion-induced release of other neurohypophyseal hormones, such as endothelin, may block release of ANP from atrial myocytes. It is probable that volume expansion detected by stretch of atrial and carotid-aortic baroreceptors causes afferent input to the brain ANP system, thereby causing increased release of the peptide from the median eminence and neural lobe. Our results emphasize the importance of brain ANP to the control of ANP release to the blood.

Effect of losartan on sodium appetite of hypothyroid rats subjected to water and sodium depletion and water, sodium and food deprivation.

The involvement of angiotensin AT1 receptors in sodium appetite was studied in hypothyroid rats treated with the angiotensin II antagonist losartan. Losartan was administered chronically by the oral route or acutely by the subcutaneous route after water and sodium depletion or water, sodium and food deprivation. Three days after addition of losartan to the food at the dose of 1.0 mg x g(-1), the rats significantly reduced (P < 0.02) their spontaneous intake of 1.8% NaCl. Increasing the dose of losartan to 2.0 and 4.0 mg x g(-1) did not reduce NaCl intake; in contrast, the intensity of the sodium appetite gradually returned to previous levels. The simultaneous administration of captopril, an angiotensin converting enzyme inhibitor, and losartan significantly increased (P < 0.05) NaCl intake and after captopril removal NaCl intake returned to the levels observed with losartan treatment alone. The administration of losartan 4 days after the beginning of captopril treatment significantly reduced (P < 0.0001) NaCl intake. Following acute administration of losartan, water- and sodium-depleted rats significantly reduced their NaCl and water intake (P < 0.001). The administration of losartan also induced a significant reduction in NaCl and water intake in water, NaCl and food-deprived rats (P < 0.0001 and P < 0.001, respectively). The present results show that chronic treatment with oral losartan inhibited spontaneous sodium appetite in hypothyroid rats. Continuation of treatment rendered rats resistant to the blockade of AT1 receptors. Water and sodium depletion and water, NaCl and food deprivation induced sodium appetite, which in the short term depends on cerebral angiotensinergic activity mediated by the activation of AT1 receptors.