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J M Saavedra

Publications and source records attributed to J M Saavedra.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterization of AT2 receptor expression in NIH 3T3 fibroblasts.

1. A high expression of angiotensin II receptors and of angiotensin-converting enzyme (ACE) activity was detected in confluent NIH 3T3 fibroblasts. 2. Characterization with selective ligands, dithiothreitol, and GTP gamma S, indicated that only the AT2 subtype was expressed. 3. AT2 receptors and ACE expression were strictly dependent on the cell density and growth phase of the cells, with AT2 receptors being expressed earlier than ACE. In contrast, high expression of AT2 receptors irrespective of their growth state was observed in NIH 3T3 cells lacking contact inhibition upon neoplastic transformation with ras. 4. Our results imply a possible relation of AT2 receptors to cell growth and cell-cell contact.

3T3 Cells↗
PubMed1999

Local renin-angiotensin system is involved in K+-induced aldosterone secretion from human adrenocortical NCI-H295 cells.

NCI-H295, a human adrenocarcinoma cell line, has been proposed as a model system to define the role of the renin-angiotensin system in the regulation of aldosterone production in humans. Because the precise cellular localization of the components of the renin-angiotensin system in human adrenal cortical cells remains unclear, we investigated their localization in this defined cell system. NCI-H295 cells expressed both angiotensinogen and renin as shown by reverse transcriptase polymerase chain reaction and immunohistochemistry. Human angiotensin-converting enzyme (ACE) was not detectable by immunocytochemistry, ACE binding, or reverse transcriptase polymerase chain reaction. However, 3.5 mmol/L K+ stimulated the formation of both angiotensin I and angiotensin II 1. 9- and 2.5-fold, respectively, and increased aldosterone release 3. 0-fold. The K+-induced stimulation of aldosterone release was decreased by captopril and enalaprilat (24% and 26%, respectively) and by the angiotensin type 1 (AT1)-receptor antagonist losartan (28%). Angiotensin II-induced stimulation of aldosterone release was abolished by losartan treatment. Specific [125I]Sar1-angiotensin II binding was detected by receptor autoradiography. The binding of [125I]Sar1-angiotensin II was completely displaced by the AT1 antagonist losartan but not by the AT2 receptor ligand PD 123319, confirming the expression of angiotensin II AT1 receptors in NCI-H295 cells. Our results demonstrate that NCI-H295 cells express most of the components of the renin-angiotensin system. Our failure to detect ACE, however, suggests that the production of angiotensin II in NCI-H295 cells may be ACE independent. NCI-H295 cells are able to produce angiotensin II, and K+ increases aldosterone secretion in part through an angiotensin-mediated pathway. The production of angiotensin II in NCI-H295 cells demonstrates that this human cell line can be useful to characterize the role of locally produced angiotensin II in the regulation of aldosterone release.

Adrenal Cortex↗
PubMed1998

Molecular cloning and pharmacological characterization of an atypical gerbil angiotensin II type-1 receptor and its mRNA expression in brain and peripheral tissues.

In the gerbil brain, most of the [125I]Sarcosine1-Angiotensin II binding sites are atypical, not sensitive to displacement with selective Angiotensin II AT1 and AT2 receptor ligands. A similar atypical binding profile exists in the gerbil kidney, where binding is highly expressed. We isolated a 2197 base pair clone from a gerbil kidney cDNA library which encodes a 359 amino acid protein with higher than 90% homology to other mammalian angiotensin II AT1 receptors. When expressed in COS-7 cells, stimulation by Angiotensin II of both the cloned gerbil receptor or the human AT1 receptor enhanced IP3 production to a similar degree. In COS-7 cells, the gerbil receptor also had a ligand affinity profile similar to that of the human AT1 receptor, but showed greatly reduced affinity for losartan (IC50=3480+/-174 nM). In the gerbil brain, in situ hybridization revealed receptor mRNA in circumventricular organs, selective hypothalamic, midbrain and brain stem areas, and in the hippocampus, where high mRNA expression was detected in the stratum pyramidale of the CA1 and CA2 subfields, and in the stratum granulosum of the dentate gyrus. The expression pattern of receptor mRNA corresponded well with that of atypical [125I]Sar1-Ang II binding. In situ hybridization and Southern blot experiments using riboprobes against the open reading frame and the 3'-untranslated region of the cloned gerbil Ang II receptor cDNA suggest that gerbils have, like other rodents, two AT1 receptor subtypes. The receptor mRNA distribution of the cloned gerbil Ang II receptor corresponds to the distribution of AT1A receptors described in other rodent species.

Adrenal Cortex↗
PubMed1998

Chemical lesion of the inferior olive reduces [125I]sarcosine1-angiotensin II binding to AT2 receptors in the cerebellar cortex of young rats.

In young rats, AT2 receptors and AT2 receptor mRNA are discretely localized in neurons of the inferior olive, with highest expression in the medial nucleus. We previously detected AT2 receptor binding, but not AT2 receptor mRNA, in the molecular layer of the cerebellar cortex. To determine whether AT2 receptors are expressed in climbing fiber terminals which arise to the molecular layer from the inferior olive and innervate Purkinje cells, we chemically destroyed olivary neurons of 2-week-old rats by intraperitoneal (i.p.) injection of the neurotoxin 3-acetylpyridine. Lesions of the inferior olive reduced [125I]Sar1-Ang II binding to AT2 receptors and AT2 receptor mRNA levels in this area by 50%, and produced a similar decrease in AT2 receptor binding in the molecular layer of the cerebellar cortex. The extent of binding reduction was similar 3 days and 7 days after the lesion. 3-Acetylpyridine lesions did not change [125I]Sar1-Ang II binding to AT1 receptors in the molecular layer of the cerebellar cortex or AT1 receptor mRNA levels in Purkinje cells. AT2 receptor binding and AT2 receptor mRNA levels in the deep cerebellar nuclei were also not affected by 3-acetylpyridine. Our results support the hypothesis that AT2 receptors are produced by inferior olivary neurons and transported through climbing fibers to the molecular layer of the cerebellar cortex. The high expression of AT2 receptors in the inferior olivary-cerebellar pathway during a crucial time in postnatal development of climbing fiber-Purkinje cell connectivity suggest a role of AT2 receptors in the development of this pathway.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1998

Characterization and distribution of angiotensin II receptor subtypes in the mouse brain.

We localized and characterized angiotensin II receptor subtypes (AT1 and AT2) in the mouse brain, with the use of autoradiography after incubation with [l25I][Sar1]-angiotensin II or [125I]CGP 42112 and displacement with selective angiotensin AT1 (losartan and candesartan) or angiotensin AT2 (CGP 42112(1) and PD 123319(2)) receptor ligands. In the mouse, the receptor subtype affinity for the different ligands was similar to that of the rat. The receptor subtype distribution was also similar to that in the rat, with some notable exceptions, such as the presence of angiotensin AT1 but not AT2 receptors in the locus coeruleus, and the expression of angiotensin AT1 receptors in the caudate putamen. These results confirm that careful consideration of the specific distribution of receptor subtypes in different species, even those closely related such as the mouse and the rat, should be conducted before meaningful comparisons could be proposed. Our data also form the basis for future studies of mouse models such as those with angiotensin receptor gene deficiencies.

Angiotensin II↗
PubMed1998

Decreased expression of natriuretic peptide A receptors and decreased cGMP production in the choroid plexus of spontaneously hypertensive rats.

Atrial natriuretic peptide receptor (ANP) subtypes and their signal transduction response were characterized in choroid plexus of spontaneously hypertensive (SHR) and normotensive (WKY) rats. We found two ANP receptor subtypes, guanylate cyclase coupled and uncoupled, in both rat strains. Binding of ANP was lower in SHR choroid plexus when compared to WKY. The lower ANP binding in SHR was the result of a decrease of binding to the guanylate cyclase-coupled receptor subtype A, a decrease that correlated well with the decreased ANP-induced cGMP formation in SHR. Forskolin stimulated cGMP production to the same extent in both strains. In WKY rats, ANP increased basal and forskolin-stimulated cAMP production; conversely, in SHR, ANP did not affect the basal level of cAMP and inhibited the forskolin-stimulated cAMP production. These results demonstrate differences in ANP receptor subtype expression, and ANP signal transduction in choroid plexus of hypertensive and normotensive rats, which is of possible significance to the central mechanisms of blood pressure control.

Animals↗
PubMed1998

The angiotensin AT1 receptor antagonist CV-11974 regulates cerebral blood flow and brain angiotensin AT1 receptor expression.

We studied cerebral blood flow autoregulation by laser Doppler flowmetry, and expression of brain angiotensin II AT1 receptors by quantitative autoradiography, after administration of an angiotensin AT1 receptor antagonist, CV-11974 (Candesartan, 0.5 or 1.0 mg/kg.day) for two weeks via subcutaneously implanted osmotic pumps in adult normotensive Wistar Kyoto and spontaneously hypertensive male rats (SHR). In SHR, the autoregulation curve was shifted towards higher blood pressures, when compared with that of normotensive Wistar Kyoto rats. Administration of CV-11974 shifted the autoregulation curve toward lower blood pressures in both Wistar Kyoto and SHR, partially normalizing the autoregulation curve in SHR. CV-11974 treatment markedly decreased the expression of AT1 receptors in Wistar Kyoto rats, both in areas outside the blood brain barrier (subfornical organ, 95% decrease) and inside the blood brain barrier (nucleus of the tractus solitarius, 87% decrease, and paraventricular nucleus, 96% decrease). Our results demonstrate that blockade of AT1 receptors tends to normalize the shift to higher pressures in the autoregulation curve of genetically hypertensive rats, and has a profound modulatory role in brain angiotensin II AT1 receptors.

Angiotensin Receptor Antagonists↗
PubMed1997

Melatonin receptors mediate contraction of a rat cerebral artery.

Melatonin receptors are expressed in the cerebral arteries of the rat which form the circle of Willis. We report here that melatonin induces contraction of in vitro preparations of pressurized rat posterior communicating artery, in a concentration-dependent manner. This action of melatonin is inhibited by S-20928, a specific melatonin receptor antagonist. Our results demonstrate that the contractile action of melatonin is mediated by its receptors in the cerebral artery, and suggest a role for melatonin in the regulation of cerebral circulation.

Animals↗
PubMed1997

Cellular localization and expression of the serotonin transporter in mouse brain.

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using 35S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [125I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.

Animals↗
PubMed1997

Angiotensin II AT1A receptor mRNA expression is induced by estrogen-progesterone in dopaminergic neurons of the female rat arcuate nucleus.

Brain angiotensin II (Ang II) inhibits pituitary prolactin release by an indirect mechanism requiring stimulation of dopamine formation and release. We report that [125I]Sar1-Ang II binding to AT1 receptors and AT1A receptor mRNA expression increase selectively in the dorsomedial arcuate nucleus of 17beta-estradiol-primed ovariectomized rats after treatment with progesterone. In hormone-treated rats, arcuate nucleus AT1A receptor mRNA expression is associated with tyrosine hydroxylase-positive neurons. No AT1A receptor mRNA was detected in tyrosine hydroxylase-positive cells of the arcuate nucleus of intact male rats. Conversely, in the anterior pituitary, where local or circulating Ang II stimulates prolactin release, [125I]Sar1-Ang II binding to AT1 receptors and AT1B receptor mRNA expression are decreased in 17beta-estradiol/progesterone-treated ovariectomized rats. Thus, AT1A receptors in the dorsal arcuate nucleus and AT1B receptors in the anterior pituitary are regulated inversely by estrogen/progesterone treatment, supporting the hypothesis of a dual role for brain and pituitary Ang II on prolactin release. The colocalization of AT1A receptor mRNA and tyrosine hydroxylase in neurons of the arcuate nucleus furthermore indicates that within this area central Ang II acts directly on dopaminergic neurons. These results support the hypothesis that central Ang II inhibits pituitary prolactin release indirectly via modulation of dopaminergic activity in the arcuate nucleus.

Angiotensin II↗
PubMed1997

Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus.

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [125I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [125I]Sar1-Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [125I]Sar1-Ang II binding was displaced by the AT1 receptor antagonist losartan, indicating the presence of AT1 receptors. In the paraventricular nucleus [125I]Sar1-Ang II binding was displaced by Ang II (Ki = 7.6 X 10(-9)) and losartan (Ki = 1.4 X 10(-7)) but also by the AT2 receptor ligand PD 123319 (Ki = 5.0 X 10(-7)). In addition, a low amount of AT2 receptor binding was detected in the paraventricular nucleus using [125I]CGP42112 as radioligand, and the binding was displaced by Ang II (Ki = 2.4 X 10(-9)), CGP42112 (Ki = 7.9 x 10(-10)), and PD123319 (Ki = 2.2 x 10(-7)). ACE, Ang II, and AT1 as well as AT2 receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.

Angiotensin II↗
PubMed1997

Increased endothelin ET(A) receptor expression in rat carotid arteries after balloon injury.

Endothelins are vasoactive peptides and are believed to act as vascular smooth muscle mitogens. Vascular injury results in medial smooth muscle migration and proliferation with the formation of a neointima. Using quantitative autoradiography, we examined the expression of endothelin receptor subtypes ET(A) and ET(B) in the rat carotid artery 2, 8, and 16 days after balloon-catheter injury. At two and eight days after balloon catheterization, ET(A) receptor expression was significantly increased in the media of the injured vessel when compared to that in the media of the intact vessel. The enhanced expression of receptors returned to normal levels by 16 days after the injury. Neointimal cells also expressed ET(A) receptors at a lower level than that expressed by the injured media 8 days after injury, and continued to express ET(A) receptors 16 days after the injury. ET(B) receptors were not detectable in the media or the neointima at any time after the injury. Our results suggest the ET(A) receptors may have a significant role in injury induced vascular smooth muscle proliferation and neointima formation.

Animals↗
PubMed1997

Selective chronic sodium or chloride depletion specifically modulates subfornical organ atrial natriuretic peptide receptor number in young rats.

1. We studied the effects of selective chronic sodium depletion of chloride depletion on atrial natriuretic peptide receptor number in the subfornical organ and paraventricular nucleus of young rats. 2. Sodium or chloride depletion decreased plasma levels of atrial natriuretic peptide, increased plasma renin activity, and induced extracellular fluid volume contraction. Chloride depletion induced more significant changes in extracellular fluid volume contraction than sodium depletion. 3. In the subfornical organ, atrial natriuretic peptide receptor number significantly decreased (30%) after sodium depletion, while chloride depletion induced a smaller, not statistically significant decrease. Conversely, atrial natriuretic peptide receptors located in the paraventricular nucleus of young rats were not significantly affected by sodium or chloride depletion. 4. Water deprivation reversed the decrease in atrial natriuretic peptide receptors produced by sodium depletion. Water-deprived sodium-depleted rats actually had higher numbers of atrial natriuretic peptide receptors in the subfornical organ than control rats. These changes were associated with severe extracellular fluid volume contraction and up regulation of brain vasopressin mRNA steady-state levels. Thus, the direction of change in the number of subfornical organ atrial natriuretic peptide receptors was dependent on the degree of extracellular fluid volume contraction. 5. Our results suggest that atrial natriuretic peptide receptors located in the subfornical organ, and not in the paraventricular nucleus, are selectively regulated by sodium depletion and extracellular fluid volume contraction.

Age Factors↗
PubMed1997

CGP-42112 partially activates human monocytes and reduces their stimulation by lipopolysaccharides.

CGP 42112, a high-affinity ligand for angiotensin II AT2 receptors, binds to rat macrophage/microglia lacking AT2 receptors. Here we report that CGP-42112 binds to human monocytes and exerts specific effects. Binding studies revealed a single site, highly specific for CGP-42112, not displaceable by angiotensin II, angiotensin fragments, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-4, IL-10, transforming growth factor-beta, or lipopolysaccharide (LPS). Incubation of purified human monocytes in serum-free medium with CGP-42112 enhanced, in a dose-dependent manner, cell attachment to fibronectin and collagen-coated dishes as well as matrix metalloproteinase-9 secretion. CGP-42112 did not promote cytokine secretion. In contrast, when added in the presence of low doses of LPS, CGP-42112 reduced the LPS-stimulated secretion of TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6 without affecting IL-10 and decreased the LPS-stimulated matrix metalloproteinase-9 activity. Additionally, CGP-42112 inhibited the increase in protein kinase A activity produced by LPS. Our results indicate that CGP-42112 may modulate monocyte activation through binding to a novel receptor.

Animals↗
PubMed1997

Water deprivation upregulates ANG II AT1 binding and mRNA in rat subfornical organ and anterior pituitary.

We studied angiotensin II (ANG II) receptor subtype expression in selected brain nuclei and pituitary gland after water deprivation by in vitro receptor autoradiography using 125I-labeled [Sar1]ANG II and by in situ hybridization using 35S-labeled AT1A, AT1B, and AT2 receptor-specific riboprobes. In control rats we found binding to AT1 receptors in the subfornical organ, paraventricular nucleus, median eminence, and anterior pituitary; AT1A mRNA expression in the subfornical organ and paraventricular nucleus; and AT1B mRNA expression in the anterior pituitary. No receptor mRNA was found in the median eminence. AT1 receptors and AT1A receptor mRNA levels were increased in the subfornical organ, and, in the anterior pituitary, AT1 receptors and AT1B receptor mRNA were increased, only after 5 days of water deprivation. No significant changes occurred after 1 or 3 days of water deprivation, and no regulation of ANG II receptor expression was detected in other brain areas. Our results show that prolonged water deprivation selectively regulates AT1 receptor expression and AT1A and AT1B receptor mRNA levels in the subfornical organ and anterior pituitary, respectively, supporting a role for these receptors during sustained dehydration.

Animals↗