Reproduction and the renin-angiotensin system.

A unique aspect of the circulating renin-angiotensin system and the many independent tissue renin-angiotensin systems is their interactions at multiple levels with reproduction. These interactions, which have received relatively little attention, include effects of estrogens and possibly androgens on hepatic and renal angiotensinogen mRNA; effects of androgens on the Ren-2 gene and salivary renin in mice; the prorenin surge that occurs with but outlasts the LH surge during the menstrual cycle; the inhibitory effects of estrogens on thirst and water intake; the tissue renin-angiotensin systems in the brain, the anterior pituitary, and the ovaries and testes, that is, in all the components of the hypothalamo-pituitary-gonadal axis; the presence of some components of the renin-angiotensin system in the uterus and the fetoplacental unit; and the possible relation of renin and angiotensin to ovulation and fetal well-being. These interactions are described and their significance considered in this short review.

Effects of catecholamine-depleting agents and receptor blockers on basal and angiotensin II- or norepinephrine-stimulated luteinizing hormone release in female rats.

Depletion of hypothalamic norepinephrine (NE) and epinephrine by administration of diethyldithiocarbamate abolished the stimulatory effects of intraventricular (IVT) angiotensin II (AII) on LH release in ovariectomized rats pretreated with estradiol and progesterone. The increase in blood LH produced by IVT NE or iv LHRH was unaffected in these drug-treated animals. Selective depletion of hypothalamic epinephrine by the administration of LY134046 (8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride) potentiated the effect of AII on LH secretion. Blockade of alpha 1-, alpha 2-, or beta-adrenergic receptors resulted in a transient increase in basal LH levels. The stimulation of LH secretion induced by IVT AII or NE was unaffected by alpha 1-receptor blockade with prazosin, but was abolished by alpha 2-receptor blockade with yohimbine. beta-Receptor blockade with propranolol potentiated both NE- and AII-induced LH release. AII receptor blockade with IVT saralasin prevented the LH rise due to AII without modifying that due to NE. Taken together, these data suggest that IVT AII stimulates LH release in ovariectomized rats treated with estradiol and progesterone by releasing endogenous NE, which, in turn, acts on facilitatory alpha 2-receptors to affect LH secretion, presumably by increasing the secretion of LHRH. Exogenous NE also acts at this receptor. beta-Receptors provide inhibitory tone to this facilitatory system, and blockade of this receptor subtype results in potentiated LH responses to both AII and NE.

Neuroendocrine regulation of plasma angiotensinogen.

In previous studies we found that plasma angiotensinogen levels were reduced by lesions of the hypothalamic paraventricular nuclei. To determine if the decrease was caused by decreased secretion of hormones that normally stimulate angiotensinogen secretion by the liver, we correlated the changes in plasma angiotensinogen produced by paraventricular lesions with changes in plasma LH, ACTH, and thyroid hormones; compared the changes in plasma angiotensinogen and other hormones to those produced by hypophysectomy; and determined the effects of treatment with ACTH and T4 in animals with paraventricular lesions. In male Sprague-Dawley rats, bilateral lesions destroying more than 50% of the paraventricular nuclei decreased plasma angiotensinogen to 787 +/- 52 ng angiotensin-I/ml in 7 days compared to 1576 +/- 142 ng angiotensin-I/ml in sham-operated controls. Plasma T3 and T4 were also reduced, whereas there were no statistically significant changes in plasma ACTH or LH. Hypophysectomy produced a comparable decline in plasma angiotensinogen and thyroid hormone levels. Daily administration of a single dose of ACTH had no effect on plasma angiotensinogen in rats with paraventricular lesions, but T4 treatment restored plasma angiotensinogen to normal levels. The data indicate that the decline in circulating angiotensinogen produced by lesions of the paraventricular nuclei is caused by the decrease in the secretion of thyroid hormones produced by these lesions. They also demonstrate that in addition to regulating circulating renin via the sympathetic nervous system, the brain has an effect on circulating angiotensinogen via neuroendocrine control of thyroid function.

Evidence that the gonadotrophs are the likely site of production of angiotensin II in the anterior pituitary of the rat.

To determine whether the angiotensin II-like immunoreactivity (AII-IR) previously reported in rat gonadotrophs is generated locally, we stained for AII-IR in neonatal rat pituitary explants and dispersed adult pituitary cells maintained in serum-free medium. In both preparations, AII-IR was found in cells with the morphological characteristics of gonadotrophs. In the anterior pituitaries of adult male rats, AII-IR and LH beta immunoreactivity were found by electron microscopy to be located in the same secretory granules. Pituitary tissue was also extracted, and the AII content was measured by RIA. The gland contained 300 times more AII than could be accounted for by the extracellular fluid in the gland. On HPLC, the pituitary AII-IR comigrated with synthetic Ile5-AII. Thus, it appears that the AII-IR in rat pituitary gonadotrophs is AII and that it is likely to be produced within these cells.

Ultrastructural localization of somatostatin in pancreatic islets of the rat.

In order to determine the precise localization of somatostatin (SRIF) in the pancreas, electron microscopic immunocytochemistry was performed on thin sections of whole pancreas and isolated pancreatic islets of the rat using the peroxidase anti-peroxidase technique. SRIF was localized in secretory granules concentrated in cells sparsely distributed near the periphery of the islet. The granules were closely applied to their limiting membrane, exhibited moderate electron density, and were smaller than those in other islet cells. The location and relative number of SRIF-containing cells as well as the morphology of the granules suggest that SRIF is present in delta cells or a subgroup with small granules. These results provide evidence that SRIF is present in a discrete granule population in a specific type of secretory cell in the pancreatic islets.

Evidence that the effects of isoproterenol on water intake and vasopressin secretion are mediated by angiotensin.

The effect of isoproterenol (6 microgram/kg sc) on drinking, urine flow, and vasopressin secretion was examined in a group of trained dogs with chronically implanted third ventricular cannulae. Isoproterenol stimulated drinking in association with a reduction in urine flow and an increase in urine to plasma osmolality ratio. Plasma renin activity increased from 3.1 +/- 0.8 to 13.0 +/- 2.7 ng/ml/3 h and plasma vasopressin concentration increased from 11.3 +/- 1.3 to 40.3 +/- 12.5 pg/ml. The effect of isoproterenol was reexamined during an intracerebroventricular infusion of the angiotensin II antagonist, saralasin (0.02 microgram/kg/min). This treatment did not affect the isoproterenol-induced increase in plasma renin activity, but inhibited the drinking, antidiuresis, and increase in plasma vasopressin concentration. These data indicate that the effects of isoproterenol on drinking, urine flow, and vasopressin secretion are mediated via the renin-angiotensin system.

Pharmacological evidence that the sympathetic nervous system mediates the increase in secretion of renin produced by p-chloroamphetamine.

The increase in plasma renin activity produced by p-chloroamphetamine in unanesthetized rats is blocked by p-chlorophenylalanine and by lesions of the dorsal raphe nucleus and the mediobasal hypothalamus. To determine whether the pathway from these areas of the brain to the kidneys that mediates the renin response is sympathetic, the effect of beta-adrenergic blockade and ganglionic blockade on the renin response to p-chloroamphetamine was studied. The increase in plasma renin activity 60 min after the administration of p-chloroamphetamine (10 mg/kg, i.p.) was prevented by the beta-adrenergic antagonist (-)-propranolol (1 mg/kg, i.p.), but not by (+)-propranolol (1 mg/kg, i.p.), given 30 min before p-chloroamphetamine. Sotalol (30 mg/kg, i.p.) injected 30 min before p-chloroamphetamine also blocked the renin response. The ganglionic-blocking drug chlorisondamine lowered blood pressure and increased plasma renin activity by itself. However, p-chloroamphetamine administered 30 min after chlorisondamine produced no further increase in plasma renin activity. Chlorisondamine, by itself, did not produce maximal secretion of renin, since isoproterenol, 30 min after chlorisondamine, produced a large increase in plasma renin activity. The data indicate that the increase in plasma renin activity produced by p-chloroamphetamine is mediated via the sympathetic nervous system.

Pharmacological evidence that the sympathetic nervous system mediates the increase in renin secretion produced by immobilization and head-up tilt in rats.

To determine the mechanism by which immobilization and head-up tilt under inactin anesthesia increase plasma renin activity (PRA), the effect of these stimuli on plasma levels of vasoactive intestinal polypeptide (VIP) were measured and the effect of the beta-adrenergic blocking drug, propranolol on the response of plasma renin activity determined. Increases in circulating VIP are known to stimulate secretion of renin. After 10 min of immobilization, plasma renin activity was increased and VIP in plasma was unchanged. After 30 min of tilting, plasma renin activity was also increased and VIP in plasma was unchanged. The increases in plasma renin activity were blocked by propranolol. Inactin anesthesia by itself increased plasma renin activity and this response was unaffected by propranolol and associated with a small decrease, rather than an increase in VIP in plasma. The results indicate that the responses of plasma renin activity to immobilization and head-up tilt are due to increased secretion of renin mediated by the sympathetic nervous system. On the other hand, the increase in secretion of renin produced by inactin anesthesia does not appear to be mediated by the sympathetic nervous system. There was no evidence that VIP was responsible for any of the increases.

Differential distribution of immunoreactive angiotensin and angiotensin-converting enzyme in rat brain.

To help resolve the controversy about the brain renin-angiotensin system, the distribution of immunoreactive angiotensin in the brains of male rats was analyzed using twelve different antibodies to angiotensin II, two of which had previously been reported to stain nerve fibers in the central nervous system. The distribution of angiotensin-converting enzyme immunoreactivity was also examined using an antibody to rabbit lung converting enzyme, and the distribution of this immunoreactivity was compared to that of immunoreactive angiotensin. Weak angiotensin-like immunoreactivity was found in cell bodies of the hypothalamic magnocellular nuclei of colchicine-treated rats and in nerve terminals of the median eminence, neurohypophysis, central nucleus of the amygdala, bed nucleus of the stria terminalis and various other sites in the brain and spinal cord of untreated rats. Staining could be demonstrated with only three antisera. Antigenic specificity was carefully studied in these antisera. Each was similar in that staining could be blocked with angiotensins I, II or III and tetradecapeptide renin substrate, although angiotensins II and III were most potent. Because of the relatively few angiotensin II antisera which could stain brain and because they are blockable with angiotensin I and tetradecapeptide renin substrate, the precise nature of immunoreactive angiotensin remains an open question. Intense converting enzyme-like activity was localized in endothelial cells of capillaries throughout the brain, in the subfornical organ and in the 'brush border' of choroidal epithelial cells in contact with cerebrospinal fluid. No activity was detected in neural tissue other than the subfornical organ and occasional weak activity in some ependymal elements elsewhere. These findings indicate that angiotensin and converting enzyme immunoreactivities are not co-distributed and raises several questions regarding the nature of, and pathway for, formation of immunoreactive angiotensin in the brain.

Angiotensinogen production by rat astroglial cells in vitro and in vivo.

To investigate the production of angiotensinogen by the brain, primary cultures were prepared from the brains of one-day-old rats. Two to four weeks after plating, they were transferred to serum-free medium. The cultures, which contained approximately 15% neurons, 80% astroglia and 5% other types of cells, produced angiotensinogen at a steady rate for three to four days in serum-free medium. Cultures prepared from subcortical tissue produced more angiotensinogen than cultures prepared from cerebral cortical tissue. Angiotensinogen mRNA was also identified in those cultures. Forskolin treatment had no effect on angiotensinogen production. Astroglia-enriched cultures that contained no identifiable neurons also produced angiotensinogen and its mRNA. Astroglial cells from hypothalamus and thalamus produced more of both than astroglial cells from the cerebral cortex. In situ hybridization histochemistry on sections of the hypothalamus of adult male rats showed a diffuse distribution of cells containing angiotensinogen mRNA that was more consistent with a glial than a neuronal distribution. The data indicate that most if not all of the angiotensinogen in rat brain is produced by astrocytes.

[Sar,Ala]angiotensin II in cerebrospinal fluid blocks the binding of blood-borne [125I]angiotensin II to the circumventricular organs.

There is a specific, high affinity uptake of angiotensin II in the circumventricular organs when the peptide is injected systemically. The question of whether angiotensin II in cerebrospinal fluid can reach angiotensin receptors in the circumventricular organs was investigated in rats by determining the effect of intraventricular administration of the angiotensin II receptor blocking peptide [Sar,Ala]angiotensin II (saralasin) on the binding of blood-borne [125I]angiotensin II. Other rats received intraventricular saline, intraventricular ACTH as a peptide control, or intravenous saralasin. The brains of the rats were then sectioned and subjected to radioautography. ACTH had no effect on angiotensin II uptake. Intraventricular saralasin reduced the uptake of blood-borne angiotensin II in the median eminence and organum vasculosum of the lamina terminalis to the same degree as intravenous saralasin, and reduced uptake in the subfornical organ and area postrema to a lesser extent. Uptake was reduced 40% in the anterior lobe of the pituitary by intraventricular saralasin and 73% by intravenous saralasin, indicating that some saralasin entered the portal vessels. Uptake in the posterior lobe was unaffected by intraventricular saralasin, but reduced by intravenous saralasin. The data indicate that saralasin, and so presumably angiotensin II, in the cerebrospinal fluid can reach angiotensin II receptors in the circumventricular organs which bind blood-borne angiotensin II. Consequently, the effects of intraventricular angiotensin II that are also produced by intravenous angiotensin II can probably be explained by the peptide acting on the circumventricular organs.

Pharmacological evidence for inhibition of ACTH secretion by a central adrenergic system in the dog.

To obtain more information about the transmitter involved in catecholaminergic inhibition of ACTH secretion, the site of this inhibition, and the receptors involved, the secretion of ACTH was studied in pentobarbital-anesthetized dogs that were surgically stressed and treated with drugs which modify central catecholaminergic transmission. The index of ACTH secretion was adrenal venous output of corticoid hormones. Intravenous L-dopa inhibited ACTH secretion, and this inhibition was not modified by blockade of peripheral decarboxylation of L-dopa with carbidopa. Intravenous or centrally administered clonidine inhibited stress-induced ACTH secretion, whereas centrally administered apomorphine did not. When given into the third ventricle, phenoxybenzamine (but not phentolamine) blocked the inhibitory effect of L-dopa and clonidine. Pimozide had no effect. L-propranolol caused a small but significant decrease in stress-induced ACTH secretion. Intraventricular procaine blocked the stress response. The data support the conclusion that the site of catecholaminergic inhibition of ACTH secretion is central, 'inside the blood-brain barrier', instead of the pituitary or the median eminence. They indicate that dopamine is not the mediator involved, and suggest that it is probably norepinephrine, although epinephrine is not ruled out. The receptor on which the released catecholamines act, presumably on the surface of the cells that secrete the hypothalamic hormone that regulates ACTH secretion, appears to be a type of alpha-adrenergic receptor.

Pharmacological evidence for stimulation of growth hormone secretion by a central noradrenergic system in dogs.

The role of brain catecholamines in the regulation of growth hormone secretion was investigated in pentobarbital-anesthetized dogs by using drugs which modify the function of adrenergic neurons and receptors. Intravenous administration of L-dopa produced a prompt, statistically significant increase in plasma growth hormone concentration. This response was not significantly reduced by blockade of peripheral dopa decarboxylase activity with carbidopa. Clonidine, an alpha-agonist which penetrates the brain, increased plasma growth hormone secretion. Norepinephrine, epinephrine, dopamine and isoproterenol, catecholamines which do not penetrate the blood-brain barrier, failed to affect plasma growth hormone concentration when administered intravenously. Apomorphine did not produce a statistically significant increase in plasma growth hormone concentration when administered directly into the the third ventricle, and pimozide failed to abolish the increase in plasma growth hormone produced by L-dopa. The increase in plasma growth hormone concentration produced by intravenous L-dopa and clonidine was prevented by administration of phentolamine or phenoxybenzamine directly into the third ventricle. The response to L-dopa was also abolished by intraventricular procaine. In dogs in which central beta-adrenergic blockade was produced by intraventricular L-propranolol, the growth hormone response to L-dopa was greater than it was in control dogs treated with intraventricular D-propranolol. The data indicate that in pentobarbital anesthetized dogs, the increase in growth hormone secretion produced by L-dopa is mediated by norepinephrine, rather than dopamine, that the site of action of the norepinephrine is central, above the median eminence and inside the 'blood-brain barrier', and that the norepinephrine acts via alpha-adrenergic receptors.

Relation of central alpha-adrenoceptor and other receptors to the control of renin secretion.

The location and nature of the receptors in the brain on which clonidine acts to decrease renin secretion have been investigated in dogs. Clonidine was injected into the vertebral and carotid arteries, and its effects were compared with those of norepinephrine and epinephrine when injected into the third ventricle. It was also injected intravenously (IV) after transection of the brain stem and following treatment with intraventricular 6-hydroxydopamine. The results suggest that the renin-regulating receptors are located in the brain stem in a region different from the receptors mediating the depressor response, that they are alpha 2-adrenoceptors, and that they are postsynaptic in location. Central alpha 1-adrenoceptors appear to mediate increased renin secretion. Central serotonergic receptors also mediate increased renin secretion, but it is not known how the alpha 1- and alpha 2-adrenoceptors interact with the serotonergic systems.

Renin-angiotensin system in the anterior pituitary of the rat.

In rats, angiotensin II appears to be synthesized in the anterior pituitary gland and stored in gonadotropes in the same granules as the beta-subunit of luteinizing hormone (LH). The gonadotropes also contain renin-like and angiotensin-converting enzyme-like immunoreactivity, but angiotensinogen-like immunoreactivity is found in a separate population of cells and does not colocalize with any of the known anterior pituitary hormones. This suggests that angiotensinogen shuttles to the gonadotropes in a paracrine fashion. There are angiotensin II receptors on lactotropes and corticotropes, but no definite function has been established for pituitary angiotensin II in the regulation of prolactin and adrenocorticotropic hormone.

Vasoactive intestinal peptide and renin secretion.

VIP has now been shown to produce an increase in renin release in a number of species, including humans. Our work suggests that VIP is capable of producing this effect by a direct action on the renin-secreting juxtaglomerular cells of the kidney. We have found no evidence to support the possibility that VIP produces this effect as a neurotransmitter in the kidney. In this regard, it should be noted that VIP has been identified as a cotransmitter primarily in cholinergic neurons. The kidney is thought to lack cholinergic innervation, and acetylcholine has no effect on renin secretion. We have explored two conditions where renin secretion is known to increase and found that circulating levels of VIP did not increase along with the increase in PRA. Thus, at least in hemorrhage and dietary sodium restriction, VIP does not appear to affect renin secretion through a humoral mechanism. There could be other untested situations where a humoral effect of VIP might come into play since we have shown that the whole animal is capable of increasing plasma VIP to levels that affect renin release. Studies employing recently developed VIP antagonists have the potential to determine in which physiological or pathological situations VIP contributes to the control of renin secretion. For example, in endotoxic shock, plasma levels of both VIP and PRA are significantly elevated. Could the increase in PRA be partly dependent on an action of circulating VIP?

The role of angiotensin II in the regulation of ACTH secretion.

1. In rats and in at least some other species, IV and IVT AII stimulate ACTH secretion. 2. Although AII increases ACTH secretion by a direct action on pituitary cells in vitro, it appears to act instead by stimulating CRH secretion in vivo. 3. The CRH stimulating effect of circulating AII is mediated by an action of the AII on one or more of the circumventricular organs of the brain. 4. AII administered into the cerebral ventricles and, presumably, centrally generated AII, increase CRH secretion by acting on AII receptors inside the blood-brain barrier as well as in the circumventricular organs. 5. A possible role for the renin-angiotensin system in mediating the increase in ACTH secretion produced by stress has been suggested, and the degree of involvement may vary from one stress to another. However, as yet we have been unable to obtain any evidence that either circulating AII or centrally generated AII plays a role in the increase in ACTH secretion produced by ether stress in rats and surgical stress in dogs.

Interference of eluates from octadecyl cartridges with an angiotensin II radioimmunoassay.

Variable values for angiotensin II were obtained when a radioimmunoassay was performed after extraction of biological samples on octadecyl cartridges. Dried residues of eluates from the cartridges were found to decrease the binding of the tracer to the antibody in assay tubes, causing high blank values. When the tubes used for the standard curve contained dried residues of eluates extracted in the same fashion as the unknowns, the interference and the high blanks were eliminated.

Ultrastructural localization of luteinizing hormone-releasing hormone in the median eminence of the rat.

The nature of cells in the hypothalamus that produce hypothalamic hypophysiotropic hormones remains unsettled. To investigate this problem electron microscopic immunocytochemistry was performed on thin sections of the median eminence of proestrous rats using antibodies to synthetic luteinizing hormone-releasing hormone (LHRH) and the peroxidase-antiperoxidase (PAP) technique. PAP complexes indicating the presence of LHRH were found over small, homogeneously dense neurosecretory granules 75-90 nm in diameter that occurred in clusters within neurons. Positive staining was more prevalent in the palisade zone in the anterior median eminence than at more posterior levels. LHRH positive granules were not obsereved in neuronal processes in the neurohemal contact zone, but appeared closer to the pericapillary space in the posterior median eminence than at more anterior levels. Occasional groups of LHRH positive granules were also found in the internal layer and hypendymal zone. No staining was observed in tanycytes or glial elements. These results support the hypothesis that hypothalamic hypophysiotropic hormones are produced by neurons and are stored in granules of homogeneous size and density in nerve processes located in the median eminence in the proximity of the hypophysial portal plexus.

Colocalization of angiotensinogen and glial fibrillary acidic protein in astrocytes in rat brain.

Angiotensinogen is produced in the brain, but its precise localization and the cells in the central nervous system producing it are unknown. We have performed a double staining test for angiotensinogen and glial fibrillary acidic protein in rat brain and report here that these proteins colocalize in astrocytes.