Differential loss in function of angiotensin II receptor subtypes during tissue storage.

In vitro receptor autoradiography was performed on rat brain and kidney sections stored frozen at -20 degrees C for extended time periods (17, 40, 64, 121, 183, 251, and 333 days). The results indicate that prolonged tissue storage has a differential effect upon 125I sar1ile8 angiotensin II binding to AT1 and AT2 receptor sites. Binding at AT1 receptor rich tissues studied (renal medulla, renal cortex, anterior pituitary, ventral hippocampus, spinal trigeminal nucleus, and nucleus of the solitary tract) shows a first order exponential decay pattern. The logarithmic linear regression slope (log(e) specific binding versus time), is significantly different from zero (p<0.05) in all AT1 rich tissues except for nucleus of the solitary tract (p=0.086). There is no detected loss of 125I sar1ile8 angiotensin II binding at the AT2 prominent regions in the superior colliculus, medial geniculate nucleus, and the inferior olivary nucleus. The half lives of AT1 receptors are highly variable, ranging from 36 days in the anterior pituitary to 442 days in the nucleus of the solitary tract, and this might be related to variable stability of AT1A and AT1B receptors. These observations should be taken into account when assessing and comparing AT1 and AT2 receptor subtype densities.

Angiotensin III depressor action in the conscious rabbit is blocked by losartan but not PD 123319.

Vasodilator and vasodepressor properties of angiotensins have been reported, and mediation by prostaglandins or nitric oxide has been proposed. Other studies indicate that angiotensin AT(2) receptors might mediate a depressor action, and the present study was designed to delineate and explore this possibility in a conscious rabbit model. Large intravenous boluses of angiotensin III (15 nmol/kg) produced a predictable pressor peak (82+/-4 mm Hg) followed by a depressor phase (20+/-3 mm Hg), whereas equipressor doses of angiotensin II were less effective at producing depressor responses. Angiotensin-(1-7) did not exert a depressor action, and the reduced potency of angiotensin IV (relative to angiotensin III) was similar for both the pressor and depressor phases ( approximately 100-fold). It is clear that specific angiotensin IV or angiotensin-(1-7) receptors do not mediate depressor effects in this model. The AT(1) antagonist losartan (1 mg/kg) blocked both the pressor and depressor components of the angiotensin III response, whereas the AT(2) antagonist PD 123319 (35 mg/kg) had no effect on either element of the response. The data obtained with the angiotensin receptor subtype-selective compounds, losartan and PD 123319, suggest that the depressor action is an AT(1)-mediated effect and give no indication that AT(2) receptors could be involved. Paradoxically, the greater potency of angiotensin III as a vasodepressor belies the conclusion that the response is AT(1)-mediated, because AT(1) receptors have a greater affinity for angiotensin II versus angiotensin III.

Angiotensin-(1-7) binding at angiotensin II receptors in the rat brain.

Angiotensin-(1-7) (Ang-(1-7)) is reported to be equipotent with angiotensin II (AII) in producing some central biological effects but the receptors responsible for these actions have not been defined. Three classes of receptor have been proposed: AT1, AT2, and a putative Ang-(1-7) selective receptor. This study specifically evaluates Ang-(1-7) competition at AII binding sites (AT1 and AT2) in the rat brain. 125I Sar1 Ile8 AII (269-312 pM) was used to conduct receptor autoradiographic binding assays in brain sections. Competition with Ile5 AII and Val5 AII was similar at nuclei in which either AT1 or AT2 receptor subtypes predominate (Ki = 11-18 nM). Ang-(1-7) competed 150-fold less effectively than native AII at AT1 predominant brain nuclei (Ki = 2.4 microM). At brain regions where AT2 receptors predominate, Ang-(1-7) showed a very low affinity (Ki = 104 microM) for the majority of the 125I Sar1 Ile8 AII binding sites (AT2). A small proportion of 125I Sar1 Ile8 AII binding sites showed an affinity of 2.0 microM, presumably AT1 receptors present in those brain regions. For biological responses where Ang-(1-7) is reported to be equipotent with AII, it is unlikely that these actions are mediated by the widely distributed AT1 or AT2 receptor subtypes which recognize 125I Sar1 Ile8 AII.

Effects of peptidase inhibitors on binding at angiotensin receptor subtypes in the rat brain.

Sulfhydryl reducing agents affect angiotensin II (AII) receptor binding differentially at AT1 and AT2 sites. Consequently, sulfhydryl reducing agents are now used infrequently in AII receptor binding assays. In this regard, the present autoradiographic study evaluates the effects of additional peptidase inhibitors on AII receptor binding and radioligand integrity. EDTA at 5 mM enhanced binding similarly, by about 70%, at both AT1 and AT2 binding sites, whereas bacitracin (10(-4) M) did not affect binding at either site. In contrast, addition of phenanthroline and bovine serum albumin (BSA) increased binding at AT1 sites 2.3-fold, whereas binding at AT2 sites was affected minimally. Degradation of 125I-[Sar1,Ile8]-AII (125I-SIAII) was determined by HPLC analysis of samples before and after incubation with tissue in each buffer. Omission of bacitracin from buffers reduced the recovery of intact radioligand to 83-87%, while recovery exceeded 94% in the presence or absence of all other buffer constituents. These results suggest that degradation of 125I-SIAII is minimal in large volume in vitro receptor autoradiography studies of rat brain AII receptors. Further, the beneficial effects on radioligand binding caused by buffer constituents such as EDTA, phenanthroline, and BSA were not due to their ability to protect the radioligand from enzymatic degradation. Because these constituents (and possibly others) had differential effects on binding with respect to receptor subtypes, caution should be used when interpreting or comparing binding data obtained from various laboratories utilizing different buffer components.

Angiotensin II binding sites in the hamster brain: localization and subtype distribution.

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [125I][sar1,ile8]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AT1 subtype) and PD123177 (AT2 subtype). Binding was quantified by densitometric analysis of autoradiograms and localized by comparison with adjacent thionein stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [125I][sar1,ile8]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT1 binding sites in the hamster but AT2 in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.

Analysis of angiotensin II receptor subtypes in individual rat brain nuclei.

Previous studies have used new angiotensin II (AII) receptor subtype selective compounds to localize AII receptor subtypes within discrete rat brain nuclei. The purpose of this autoradiographic study was to extend these preliminary findings and provide a comprehensive analysis of AII binding sites in 22 rat brain nuclei and the anterior pituitary, to include estimates of the binding affinity for 125I sar1 ile8 AII (125I SIAII) at each nucleus, and determine the fractional distribution of each subtype at each nucleus. Estimates of KD in separate experiments revealed that AT1 nuclei had a consistently higher affinity for 125I SIAII than AT2 nuclei (0.66 vs. 2.55 nM). Displacement of subsaturating concentrations of 125I SIAII by 10(-8)-10(-4) M DuP753 (selective for the AT1 subtype) or PD123177 (selective for the AT2 subtype) indicated that approximately half of the brain regions surveyed contained predominantly AT1 sites and half contained predominantly AT2 sites. Binding was partially displaced by both compounds in several regions and two site analyses were performed to estimate the distribution of subtypes within each nucleus. The data were then corrected for differential occupancy by 125I SIAII. Brain nuclei associated with cardiovascular or dipsogenic actions of AII, e.g., subfornical organ, organum vasculosum of the lamina terminalis, median preoptic nucleus, nucleus of the solitary tract and area postrema, contained pure, or almost pure, populations of AT1 receptors. The functions of AII in brain regions containing predominantly AT2 binding sites, e.g., thalamus, colliculi, inferior olive and locus ceruleus, remain undefined. Thus, AII binding sites in the rat brain have been differentiated into two subtypes with similar characteristics to those reported in peripheral tissues. However, the unexpected finding that they can be differentiated on the basis of their affinity for 125I SIAII raises questions concerning their coidentity with peripheral receptor subtypes.

Sulfhydryl reducing agents distinguish two subtypes of angiotensin II receptors in the rat brain.

Two angiotensin II receptor subtypes were distinguished in the rat brain using in vitro receptor autoradiography based on the differential effects of sulfhydryl reducing agents on 125I-sarcosine1,isoleucine8 angiotensin II binding in various brain nuclei. At several nuclei, e.g. the hypothalamus, circumventricular organs and the dorsal medulla, 125I-sarcosine1,isoleucine8 angiotensin II binding was strongly inhibited by 30 mM beta-mercaptoethanol or 5 mM dithiothreitol, whereas at other nuclei, e.g. the lateral septum, colliculi, locus coeruleus and medial amygdala, sulfhydryl reducing agents had either little effect on radioligand binding or enhanced the binding. The distribution of the sulfhydryl reducing agent inactivated subtype corresponds exactly with the distribution of DuP 753 sensitive (designated as AII alpha) 125I-sarcosine1,isoleucine8 angiotensin II binding sites25. The subtype not inhibited by sulfhydryl reducing agents corresponds with the DuP 753 insensitive (designated as AII beta) sites in the brain25. The sulfhydryl reducing agent effect on brain angiotensin II receptor subtypes is similar to that seen in angiotensin II receptor subtypes in peripheral tissues. These observations indicate that many previous studies of brain angiotensin II receptor binding that included 5 mM dithiothreitol in the assay medium overlooked the sulfhydryl reducing agent inactivated (AII alpha) receptor subtype.

Discrimination of angiotensin II receptor subtype distribution in the rat brain using non-peptidic receptor antagonists.

The non-peptidic angiotensin II receptor subtype selective antagonists, DuP 753 and PD123177, were used to characterize angiotensin II receptor binding sites in the rat brain. Competitive receptor autoradiography with 125I-Sar1-Ile8 angiotensin II defined a regional distribution of binding sites that were sensitive to either DuP 753 (designated AII alpha subtype) or PD123177 (designated AII beta subtype). Whereas most brain nuclei could be assigned to a category containing a predominant subtype, a multiple receptor subtype analysis indicated that some regions are homogeneous, while others contain a mixture of both AII alpha and AII beta subtypes.

Angiotensin II and the locus coeruleus.

The locus coeruleus (LC) is a putative site of action for angiotensin II in the brain. Immunocytochemical studies have identified angiotensin II-like immunoreactive material in nerve terminals innervating the LC, and the LC contains one of the highest densities of angiotensin II receptor binding sites in the rat brain. Recent studies using selective neurotoxins suggest that the binding sites for angiotensin II in the LC are present on noradrenergic perikarya. Angiotensin II receptors are now known to exist as two subtypes that are distinguishable both pharmacologically and biochemically. Radioligand binding studies using agonists and antagonists selective for these angiotensin II receptor subtypes indicate that the rat LC contains a mixture of the two known angiotensin II receptor subtypes, but that the PD123177-sensitive AII beta receptor subtype is predominant. Comparisons of spontaneously hypertensive rats with normotensive rats indicates that angiotensin II and its receptors in the LC are elevated in the hypertensive rat strain. Studies of the biochemical and physiological actions of angiotensin II in the LC have not yet established an agreed-upon function for angiotensin II in this nucleus.

Novel angiotensin II binding sites in the mesopontine area of the rat brain.

Angiotensin II (AII) immunoreactivity in the mesopontine area of the rat brain is distributed through several areas where co-localization of AII receptors has not been established. The current in vitro receptor autoradiography study re-examined the distribution of AII binding using 125I-Sar1,Ile8-AII ([125I]SIAII). When incubations were conducted without sulfhydryl reducing agents, [125I]SIAII binding was observed in the locus coeruleus, inferior colliculus, superior colliculus and the central gray in agreement with previous reports. Novel [125I]SIAII binding sites were detected in the parabrachial nucleus, pedunculopontine tegmental nucleus and the caudal linear raphe nucleus, corresponding with previously reported localization of AII immunoreactivity in these nuclei. [125I]SIAII binding was also found in the paragenual nucleus where the peptide has not been detected. Thus, the observation of novel AII receptors which are sensitive to sulfhydryl reducing agents, resolves several AII-AII receptor mismatches.

Angiotensin II receptor subtypes in the rat brain.

The non-peptide angiotensin II (AII) receptor subtype selective antagonist, DuP 753, was used to characterize AII receptor binding sites in the rat brain. DuP 753 competed for specific 125I-[Sar1,Ile8]AII (125I-SIAII) binding in many brain nuclei (IC50 = 20-30 nM), but was a weak competitor at remaining sites (IC50 greater than 10(-4) M). DuP 753 sensitive binding sites (designated AII alpha subtype) correspond with areas where binding is inhibited by sulfhydryl reducing agents, whereas DuP 753 insensitive sites (AII beta) correspond with areas where binding is not inhibited by sulfhydryl reducing agents.

Autoradiographic localization of angiotensin II receptor binding sites on noradrenergic neurons of the locus coeruleus of the rat.

The locus coeruleus (LC) of the rat was lesioned by microinjection of selective neurotoxins into the brainstem. 6-Hydroxydopamine (6-OHDA), 3 micrograms/microliter, given unilaterally at two sites 0.6 mm apart on the rostro-caudal axis of the LC, was used to lesion catecholamine-containing neuronal elements. Ibotenic acid, 2.5 micrograms/0.5 microliters, administered similarly was used to lesion nerve cell bodies. Two weeks after administration of the neurotoxin, lesion efficacy was determined based on the norepinephrine content of the cerebral cortex ipsi- and contralateral to the lesion. 6-OHDA lesions of the LC caused a 46% reduction in ipsilateral cortical norepinephrine and a 60% reduction in specific 125I-[Sar1, Ile8]-angiotensin II (125I-SIAII) binding in the LC. Ibotenic acid lesions of the LC caused a 73% reduction in ipsilateral cortical norepinephrine and a 81% reduction in specific 125I-SIAII binding in the LC. These results indicate that AII receptor binding sites in the LC are localized on noradrenergic nerve cell bodies or their dendritic and axonal ramifications within the LC.

The influence of indomethacin on blood pressure during the infusion of vasopressors.

The effect of indomethacin and its vehicle on blood pressure was studied in conscious rabbits during the infusion of three vasopressors. The cyclooxygenase inhibitor raised mean arterial pressure 12 (vehicle: 3) mm Hg during norepinephrine infusion, 5 (vehicle: 0) mm Hg during angiotensin II infusion, and 5 (vehicle: -8) mm Hg during arginine vasopressin infusion. When saline was given in place of vasopressors, indomethacin failed to alter blood pressure. Since indomethacin elevated pressure in the presence, but not the absence, of all three vasopressors, the possibility that elevation of blood pressure per se stimulates synthesis of vasodilator prostaglandins was considered. A pressor action of indomethacin was observed in ganglion-blocked animals, in which absolute blood pressure remained below normotensive levels during angiotensin II infusion. Thus, indomethacin raised arterial pressure during the infusion of norepinephrine, angiotensin II, and vasopressin, and this action was not influenced by manipulation of blood pressure. These results suggest that each vasopressor promotes prostaglandin synthesis independently to a degree sufficient to restrain its pressor action.

The effect of prostaglandin and kinin synthesis inhibition on blood pressure during infusion of angiotensin II in the conscious rabbit.

The contribution of vasodilator prostaglandins and kinins to blood pressure regulation was studied during the infusion of different doses of angiotensin II in conscious rabbits. Angiotensin II was infused for 60 min. in each experiment. Indomethacin, a prostaglandin synthesis inhibitor, or Trasylol, a kallikrein inhibitor, was given at the 30 min. interval. Indomethacin caused a sustained increase in blood pressure during the infusion of pressor doses of angiotensin II. The range of the mean increase after prostaglandin synthesis inhibition was 3.4 to 6.0 and 3.0 to 9.4 mm Hg at angiotensin II infusion rates of 10 and 50 ng/kg/min respectively. In contrast, indomethacin did not alter blood pressure when the peptide was administered at subpressor levels. Trasylol did not alter blood pressure during infusion of angiotensin II. These results suggest that when blood pressure is maintained at supranormal levels by angiotensin II, the pressor action is attenuated by one or more prostaglandins; an event which is not mediated or assisted by changes in kinin metabolism.

Biphasic blood pressure response to angiotensin II in the conscious rabbit: relation to prostaglandins.

The injection of a large bolus of angiotensin II causes a biphasic blood pressure response in the conscious rabbit. To investigate contribution of prostaglandins (PGs) to the depressor phase of the blood pressure response, we studied the blood pressure effect of i.v. bolus injections of angiotensin II before and after the administration of an inhibitor of cyclooxygenase, indomethacin or sodium meclofenamate (10 mg kg-1), and in relation to associated changes in the plasma concentration of immunoreactive PGs. In conscious rabbits, angiotensin II (0.05-5.00 microgram kg-1) produced a dose-related pressor response which at both 1.5 and 5.0 micrograms kg-1 was followed by lowering of blood pressure to below the preinjection level. Neither indomethacin nor meclofenamate affected the maximal rise in pressure produced by angiotensin II, but both cyclooxygenase inhibitors augmented the duration of the pressor phase and abolished the depressor phase of the hemodynamic response to angiotensin II at 1.5 to 5.0 micrograms kg-1. After administration of angiotensin II, 5 micrograms kg-1, the plasma concentration of 6-keto-PGF1 alpha increased (P less than .01) from 218 +/- 21 pg/ml by 221 and 235% during the pressor and the depressor phases of the blood pressure response, respectively. Increments in plasma 6-keto-PGF1 alpha correlated inversely with the duration of the pressor phase and directly with the maximal lowering of blood pressure during the depressor phase. Plasma levels of PGE2 and PGF2 alpha also were increased by the peptide but the increments were not correlated with any aspect of the blood pressure response. These data suggest that a mechanism involving PGs both curtails the pressor phase and mediates the depressor phase of the hemodynamic response to pharmacological doses of angiotensin II in the conscious rabbit.

The effect of angiotensin II infusion on plasma catecholamines in the conscious rabbit.

Plasma levels of norepinephrine, epinephrine, and dopamine were measured in conscious, male rabbits. Twenty-minute infusions of 5, 50, and 500 ng/kg/min angiotensin II elevated mean arterial blood pressure by 8 (P less than 0.05), 23 (P less than 0.01), and 33 mm Hg (P less than 0.01), respectively. Plasma catecholamines were unchanged at all levels of angiotensin II infusion except epinephrine which rose from 15 +/- 3 to 52 +/- 17 pg/ml at 500 ng/kg/min. These data suggest an action of the peptide at the adrenal medulla but offer no evidence of a generalized stimulation discharge by angiotensin II.

Effects of indomethacin, renal denervation, and propranolol on plasma renin activity in conscious dogs with chronic thoracic caval constriction.

The role of renal prostaglandins and the adrenergic nervous system in the control of renin release was studied in conscious dogs with thoracic caval constriction. Indomethacin reduced plasma renin activity (PRA) in intact animals with thoracic caval constriction by 43% but failed to change PRA after surgical renal denervation and during chronic propranolol administration; adrenergic blockade reduced the initial control level of PRA before indomethacin from 15 to 4 ng angiotensin I/ml per hr. Renal hemodynamic function was markedly reduced by indomethacin both before and after adrenergic blockade. These observations indicate that prostaglandins are involved in the control of renin release, but they appear to have a more important role in the control of renal arterial resistance. The adrenergic nervous system also plays a role in the hyperreninemia of caval constriction and, possibly, a greater role than the renal prostaglandins. In the first experimental design, surgical renal denervation and daily oral propranolol administration in dogs with caval constriction reduced PRA to normal in two of seven dogs and a natriuresis occurred. In four of the five remaining animals, PRA fell, but not to normal, and renal sodium excretion failed to increase. In a second experimental design, the kidneys were denervated and propranolol was given before the dogs were subjected to caval constriction and propranolol was continued for 5 days; PRA increased markedly, sodium retention occurred, and ascites formed. Under these circumstances, compensatory mechanisms secondary to caval constriction led to increased PRA in spite of adrenergic blockade.

Sodium and angiotensin in the pathogenesis of experimental renovascular hypertension.

The effects of simultaneous angiotensin blockade and sodium depletion on the development of one-kidney renovascular hypertension were studied in rats. In sodium-replete rats, systolic blood pressure (SBP) increased from 102 +/- 2 to 153 +/- 11 mmHg by the 12th day after unilateral nephrectomy and subsequent partial occlusion of the renal artery with a 0.22-mm silver clip. When changes in body fluid volume were minimized by sodium restriction in a second group of rats, the increase in SBP from 98 +/- 4 to 149 +/- 7 mmHg after clipping was not different from that in sodium-replete animals. Inhibition of the angiotensin-converting enzyme with SQ 14,225 during sodium restriction prevented the SBP from increasing above 101 +/- 3 mmHg by the 12th day after nephrectomy and clipping. Once SQ 14,225 administration was discontinued, SBP rose significantly to 148 +/- 5 mmHg within 5 days. Because previous studies have shown that neither sodium depletion nor angiotensin blockade alone prevented the development of one-kidney renovascular hypertension, it is concluded that the increase in blood pressure resulting from renal artery constriction and contralateral nephrectomy was prevented only by suppression of both the renin-angiotensin system and body fluid volume.

Effects of indomethacin and meclofenamate on renin release and renal hemodynamic function during chronic sodium depletion in conscious dogs.

We studied the control of renin release and renal hemodynamic function by administering prostaglandin synthetase inhibitors to conscious sodium-depleted dogs with blockade of the adrenergic nervous system induced by bilateral renal denervation and propranolol administration. Indomethacin (10 mg/kg) reduced plasma renin activity (PRA) by 59% from a high sodium-depleted value, but PRA was still 3 times the normal sodium-repleted level. Arterial pressure, CCr, CPAH, urine flow, and potassium excretion fell strikingly. Similar results were obtained with meclofenamate. When SQ 14,225 was given to another group of conscious, sodium-depleted dogs with adrenergic nervous system blockade, PRA increased from the high sodium-depleted level of 5.7 to 29.3 ng of Angiotensin I (AI)/ml per hour; indomethacin (10 mg/kg) appeared to reduce PRA (0.05 less than P less than 0.1) but to only 12.1 ng of AI/ ml per hour, which is 17 times the normal level. This high level of PRA after blockade of the adrenergic nervous system and injection of indomethacin suggests that important mechanisms other than norepinephrine and renal prostaglandins control renin release; it is proposed that both the renal vascular receptor and the macula densa are involved. The marked decreases in CCr and CPAH in response to indomethacin emphasize the important role of renal prostaglandins in the control of renal hemodynamic function during sodium depletion.