Angiotensin and renin in rat and dog brain.

An enzyme with the characteristics of classical renin was isolated from brain extracts of nephrectomized dogs. This enzyme is thermolabile, nondialyzable, and forms a vasoconstrictive material when incubated with renin plasma substrate at pH 7. A short lasting pressor activity was also found in brain extracts of dogs and rats. This activity was due to a substance identified by chemical and pharmacological tests as angiotensin. Countercurrent distribution of brain extracts of rats showed that 38% of the pressor activity corresponded to angiotensin II and the remainder to angiotensin I. A remarkable correlation was found between angiotensin and norepinphrine concentrations in different portions of the encephalon of the dog.

Angiotensin regulates release and synthesis of serotonin in brain.

Angiotensin II released serotonin from neuron terminals and accelerated synthesis of the serotonin. This increase in synthesis depended on the activation of tryptophan hydroxylase. A biphasic effect was observed: at high doses the stimulatory effect depended on conversion of angiotensin II to angiotensin III. At low doses an inhibitory effect was found, possible dependent on an angiotensin II metabolite. These actions represent a subtle regulation of the open-loop serotonin system.

TNF-alpha enhances estrogen-induced cell proliferation of estrogen-dependent breast tumor cells through a complex containing nuclear factor-kappa B.

Breast tumors are usually classified according to their response to estrogens as hormone-dependent or -independent. In this work, we investigated the role of the proinflammatory cytokine TNF-alpha on the estrogen-receptor-positive T47D breast ductal tumor cells. We have found that TNF-alpha exerts a mitogenic effect, inducing cyclin D1 expression and activation of the transcription factor NF-kappaB. Importantly, activation of NF-kappaB was required for estrogen-induced proliferation and cyclin D1 expression. TNF-alpha enhanced the estrogen response by increasing the levels and availability of NF-kappaB. Chromatin immunoprecipitation analysis suggested that the action of estrogens is mediated by a protein complex that contains the activated estrogen receptor, the nuclear receptor coactivator RAC3 and a member of the NF-kappaB family. Finally, our results demonstrate that activation of this transcription factor could be one of the key signals for estrogen-mediated response.

Molecular and functional evidence for in vitro cytokine enhancement of human and murine target cell sensitivity to glucocorticoids. TNF-alpha priming increases glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis.

Cytokine-induced glucocorticoid secretion and glucocorticoid inhibition of cytokine synthesis and pleiotropic actions act as important safeguards in preventing cytokine overreaction. We found that TNF-alpha increased glucocorticoid-induced transcriptional activity of the glucocorticoid receptor (GR) via the glucocorticoid response elements (GRE) in L-929 mouse fibroblasts transfected with a glucocorticoid-inducible reporter plasmid. In addition, TNF-alpha also enhanced GR number. The TNF-alpha effect on transcriptional activity was absent in other cell lines that express TNF-alpha receptors but not GRs, and became manifest when a GR expression vector was cotransfected, indicating that TNF-alpha, independent of any effect it may have on GR number, has a stimulatory effect on the glucocorticoid-induced transcriptional activity of the GR. Moreover, TNF-alpha increased GR binding to GRE. As a functional biological correlate of this mechanism, priming of L-929 cells with a low (noncytotoxic) dose of TNF-alpha significantly increased the sensitivity to glucocorticoid inhibition of TNF-alpha-induced cytotoxicity/apoptosis. TNF-alpha and IL-1 beta had the same stimulatory action on glucocorticoid-induced transcriptional activity of the GR via the GRE, in different types of cytokine/glucocorticoid target cells (glioma, pituitary, epithelioid). The phenomenon may therefore reflect a general molecular mechanism whereby cytokines modulate the transcriptional activity of the GR, thus potentiating the counterregulation by glucocorticoids at the level of their target cells.

Adrenal proenkephalin-derived peptides during postnatal development in spontaneously hypertensive rats.

Adrenal enkephalin and enkephalin-containing peptides were studied during postnatal development in normotensive (WKY) and spontaneously hypertensive rats (SHR). The effect of chronic treatment with the ganglionic blocker chlorisondamine (5 mg/kg) was also assessed. Free enkephalin immunoreactivity and total enkephalin immunoreactivity, as determined by enzymatic digestion of large enkephalin containing fragments, were quantitated in the adrenal glands at 11 days and 7, 16, and 24 weeks of age. Both total and free metenkephalin were significantly diminished in the adrenal of SHR when compared to WKY at all ages tested. The analysis of the chromatographic profile showed that SHR displayed reduced levels of high and low molecular weight materials at 11 days and 16 weeks of age; however intermediate compounds were high in the glands of these animals. Similar increased values for free met-enkephalin were found in adrenals of WKY and SHR after ganglionic blocker treatment, which means that the relative increase was larger in SHR than WKY; while for total enkephalin the relative increase and the concentration reached in SHR was about half of those presented in WKY. These and other results presented suggest that the basic alteration of the adrenal proenkephalin system of SHR may be due to a genetic reduction of proenkephalin levels. Otherwise, the free enkephalin decrease could be related to changes in nervous input to the adrenal gland.

Differential posttranslational processing of proenkephalin in rat bone marrow and spleen mononuclear cells: evidence for synenkephalin cleavage.

Proenkephalin (PENK) messenger RNA was reported to be present in bone marrow mononuclear cells (BMMC) and spleen mononuclear cells (SMC). Nevertheless, the pattern of PENK products in normal cells of the rat immune system, which is important for defining the physiological role of PENK gene expression, has not been well established. In this work we have characterized the processing of the opioid portion (met-enkephalin-containing peptides) and nonopioid portion (synenkephalin-derived peptides) of PENK in rat BMMC and SMC. Met-enkephalin-containing peptides were detected in mononuclear cells of both hematopoietic tissues. In BMMC, free immunoreactive (IR)-met-enkephalin corresponded only to the 15% of total met-enkephalin-IR, whereas in SMC it represented the 66.5%. Gel filtration chromatography showed that BMMC contained partially processed PENK-derived peptides of high and intermediate molecular weight, whereas SMC displayed fully processed products containing met-enkephalin and/or the carboxyterminal portion of synenkephalin. HPLC purification of low molecular weight products showed that free IR-met-enkephalin in SMC mainly corresponded to met-enkephalin and oxidized met-enkephalin. In addition we have characterized in SMC three peptides lower than 3.0 kilodalton containing the C-terminal sequence of synenkephalin. These peptides were purified by gel filtration, affinity chromatography, ion exchange chromatography, and HPLC. These results show that PENK was processed in mononuclear cells of the primary (bone marrow) and secondary (spleen) organs of the rat hematopoietic system, as occurs in neural and endocrine tissues. Nevertheless, the precursor was cleaved only in the latter tissue to low molecular weight peptides. Furthermore we demonstrated that synenkephalin (proenkephalin 1-70) in SMC was processed to low molecular weight peptides containing the C-terminus free. This last result suggests that a dibasic Lys-Lys and monobasic (Lys) sites were cleaved.

Thyrotropin-releasing hormone hyperactivity in the preoptic area of spontaneously hypertensive rats.

Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation through the activation of different neurotransmitter systems at distinct extrahypothalamic sites. To study possible alterations in the TRH system in the hypertensive state, we measured TRH concentration in cerebrospinal fluid and TRH content of the preoptic area in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) by radioimmunoassay. In addition, we also measured the density of the TRH receptor in this area by a rapid filtration technique using [3H]methyl-TRH. We found a significant increase in both the TRH content (634 +/- 61 versus 350 +/- 26 pg/mg protein, SHR versus WKY; P < .01, n = 5) and density of TRH receptors without changes in affinity (Bmax, 5.0 +/- 0.1 versus 3.3 +/- 0.1 fmol/mg protein, P < .01, n = 4). An increase in TRH concentration was also found in the cerebrospinal fluid of SHR (30 +/- 3 versus 21 +/- 2 pg/mL, P < .01, n = 5), suggesting increased TRH release in the central nervous system. Northern blot analysis indicated a threefold augmented abundance of TRH precursor mRNA in the preoptic area of SHR. A polyclonal antibody raised against TRH injected peripherally or intracerebroventricularly lowered arterial blood pressure in SHR but not in WKY. In addition, long-term treatment with enalapril (5 mg/kg twice daily), which was effective in inhibiting serum angiotensin-converting enzyme activity by more than 50%, decreased arterial blood pressure and preoptic area TRH content of SHR, whereas another vasodilator, diltiazem (10 mg/kg every 8 hours), failed to produce a similar change.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential association of endogenous proenkephalin-derived peptides with membranes of microsomes from rat striatum, adrenal medulla and heart ventricle.

Proenkephalin-derived peptides, in common with other prohormones, are associated with membranes of microsomes and secretory granules in the bovine adrenal medulla. Post-translational processing of the precursor molecule varies depending upon the tissue. The relationship between post-translational events in different tissues was examined by studying the membrane association of endogenous proenkephalin-derived peptides in the crude microsomal fraction of rat adrenal medulla, brain striatum and heart ventricle. [Met]-Enkephalin and synenkephalin (proenkephalin(1-70)) immunoreactivities were quantified by radioimmunoassay after sequential enzymatic digestion with trypsin and carboxypeptidase B. Between 60 and 75% of total immunoreactive peptides present in intact microsomes of the three tissues were associated with membranes and specifically released with 2 M KSCN (pH 7.4). Analysis of the chromatographic profile of materials present in the soluble and associated fractions produced the following results. In the three tissues the materials associated with microsomal membranes corresponded to peptides larger than 3-5 kDa and displayed synenkephalin and [Met]-enkephalin immunoreactivity. Adrenal and heart microsomes showed a continuous pattern of membrane-associated proenkephalin-derived peptides of high, intermediate and low molecular weights containing the synenkephalin and [Met]-enkephalin sequences. These tissues, however, presented quantitative differences, as the highest concentrations belonged to materials larger and smaller than 12.5 kDa in adrenal and heart microsomes respectively. On the other hand, brain striatal microsomes displayed a discontinuous pattern of associated materials, with the absence of some products of high and intermediate molecular weight. Only in the soluble fraction of striatal microsomes were peptides detected of high and intermediate molecular weight containing the [Met]-enkephalin but not the synenkephalin sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurogenic hypertension after depletion of norepinephrine in anterior hypothalamus induced by 6-hydroxydopamine administration into the ventral pons: role of serotonin.

Destruction of the ventral noradrenergic pathway elicited by administration of 6-hydroxydopamine (6-OHDA, 5 micrograms into each side of the ventral pons) reduced the content of norepinephrine (NE) in the anterior hypothalamus (-80%) and induced an increase in arterial blood pressure (ABP) and in heart rate. These hypertensive rats, showed hypersensitivity to the hypotensive effect of NE (0.5-2 micrograms) and clonidine (0.75-1.5 micrograms) administered into the anterior hypothalamic preoptic (AH/PO) region. Methysergide (1-2 micrograms) and, to a lesser extent, ketanserin (1-2 micrograms) administered into the anterior hypothalamic preoptic region also reduced the arterial blood pressure in these rats treated with 6-OHDA. Bilateral administration of 5,7-dihydroxytryptamine (5,7-DHT, 8 micrograms) into the median forebrain bundle decreased the content of serotonin (5-HT) in the hypothalamus (-85%) without change in arterial blood pressure but largely prevented the development of hypertension after treatment with 6-OHDA in the ventral pons. These results suggest that neurogenic hypertension is produced after the removal of NE tonic depressor activity in the anterior hypothalamus and that serotonergic mechanisms play a major role in the development of the increased arterial blood pressure in this preparation.

Serotonin mediates cardiovascular responses to acetylcholine, bradykinin, angiotensin II and norepinephrine in the lateral septal area of the rat brain.

The infusion of acetylcholine, bradykinin, angiotensin II, norepinephrine and serotonin into the lateral septal area produced a dose-dependent increase of arterial blood pressure and heart rate. A pattern of inhibition of these cardiovascular responses, produced by pretreatment of the lateral septal area with phentolamine, 6-hydroxydopamine, methysergide and 5,7-dihydroxytryptamine was disclosed. These results suggest that the effects of acetylcholine, bradykinin and partially of angiotensin II, depend on the release of norepinephrine and the actions of this neurotransmitter in turn depend on the integrity of the serotonergic system in the lateral septal area.

Interaction between acetylcholine and bradykinin in the lateral septal area of the rat brain: involvement of muscarinic receptors in cardiovascular responses.

The lateral septal area was used as a model to study the interaction between acetylcholine (Ach) and bradykinin on arterial blood pressure, since both mediators are present in this region. In the lateral septal area, the administration of the peptide or Ach produced a long-lasting, sympathetic-mediated increase of arterial blood pressure which was blocked by atropine. Pretreatment of the lateral septal area with hemicholinium-3, which depletes stores of acetylcholine, partially blocked the pressor effect of bradykinin but not that of Ach. Captopril--an inhibitor of kininase II--enhanced the pressor effects of bradykinin and Ach. Synaptosomal studies showed that bradykinin increased sodium-dependent, high-affinity uptake of choline and the conversion of [3H]choline to [3H]acetylcholine. Competition experiments using the highly specific muscarinic antagonist [3H]quinuclidinyl benzilate, demonstrated that bradykinin displaced the muscarinic antagonist from its receptor-ligand complexes. These results suggest that in the lateral septal area acetylcholine and bradykinin interact in a positive feed-back which amplifies pressor responses.

Muscarinic M1 receptors in the lateral septal area mediate cardiovascular responses to cholinergic agonists and bradykinin: supersensitivity induced by chronic treatment with atropine.

The infusion of pilocarpine, acetylcholine, bradykinin and the selective M1 muscarinic agonist McNeil-A-343 into the lateral septal area produced a dose-dependent increase of arterial blood pressure and heart rate. The M1 muscarinic agonist carbamylcholine that causes a rise in arterial blood pressure when injected into the anterior lateral ventricles did not produce any cardiovascular effects when infused into the lateral septal area. Chronic treatment with atropine induced supersensitivity to the muscarinic agonists and a significant increase in the number of muscarinic receptors. In this study bradykinin failed to produce any significant change in cardiovascular activity. Pirenzepine, a M1 muscarinic blocking agent, inhibited completely the effect of both muscarinic agonists and bradykinin on cardiovascular activity. In fact, in vitro studies shows that the displacement of the binding of [3H]QNB by pirenzepine is compatible with the presence of the M1 subtype of muscarinic receptor in the lateral septal area, where it may play a major role on cardiovascular regulation.

Cholinergic hyperactivity in the lateral septal area of spontaneously hypertensive rats: depressor effect of hemicholinium-3 and pirenzepine.

In the lateral septal area of spontaneously hypertensive rats, but not in Wistar-Kyoto rats, the selective M1 antagonist, pirenzepine, and the depletion of acetylcholine storage, by hemicholinium-3 (HC-3), decreased blood pressure. The selective M1 agonist McNeil-A-343, produced a pressor response only after treatment of the lateral septal area with HC-3 in spontaneously hypertensive rats. Carbachol, at doses that mainly affect M2 muscarinic receptors, caused no cardiovascular changes in either strain, pointing to the main intervention of the M1 subtype of muscarinic receptor in the hypertensive condition. In addition, increases in the density of binding sites for [3H]QNB and in Vmax of sodium-dependent, HC-3-inhibitable, high affinity uptake of choline were demonstrated, without significant changes of the activity of choline acetyltransferase in the lateral septal area of spontaneously hypertensive rats. These results suggest that a hyperactivity of the cholinergic system of this area could play a role in the development and/or maintenance of hypertension in spontaneously hypertensive rats.

Angiotensin II and the transcription factor Rel/NF-kappaB link environmental water shortage with memory improvement.

One of the essential requirements even in the most ancient life forms is to be able to preserve body fluid medium. In line with such requirement, animals need to perform different behaviors to cope with water shortages. As angiotensin II (ANGII) is involved on a widespread range of functions in vertebrates, including memory modulation, an integrative role, in response to an environmental water shortage, has been envisioned. Previous work on the semi-terrestrial and brackish-water crab Chasmagnathus granulatus showed that endogenous ANGII enhanced an associative long-term memory and, in addition, that high salinity environment induces both an increase of brain ANGII levels and memory improvement. Here, we show that in the crab Chasmagnathus air exposure transiently increases blood sodium concentration, significantly increases brain ANGII immunoreactivity, and has a facilitatory effect on memory that is abolished by a non-selective ANGII receptor antagonist, saralasin. Furthermore, Rel/NF-kappaB, a transcription factor activated by ANGII in mammals and during memory consolidation in Chasmagnathus brain, is induced in the crab's brain by air exposure. Moreover, nuclear brain NF-kappaB is activated by ANGII, and this effect is reversed by saralasin. Our results constitute the first demonstration in an invertebrate that cognitive functions are modulated by an environmental stimulus through a neuropeptide and give evolutionary support to the role of angiotensins in memory processes. Moreover, these results suggest that angiotensinergic system is preserved across evolution not only in its structure and molecular mechanisms, but also in its capability of coordinating specific adaptative responses.

Interaction of angiotensin II with the cholinergic and noradrenergic systems in the rat pineal gland: regulation of indole metabolism.

The pineal gland, angiotensin, and noradrenergic and cholinergic systems are involved in the regulation of tissue indole metabolism. Angiotensin II increased noradrenaline release and the production of hydroxy- and methoxyindoles by pineal slices. Electrical field stimulation (EFS) of pineal slices released angiotensin II and reproduced many of the actions of exogenous angiotensin II on serotonin and melatonin biosynthesis and release. Both sarcosine-isoleucine-angiotensin II ([Sar, Ile]-ANG II) and atropine blocked, and nadolol increased, the effect of EFS and exogenous angiotensin II on serotonin production. Nadolol blocked both the EFS-induced and the angiotensin II-induced production of melatonin. Atropine and [Sar, Ile]-ANG II did not modify melatonin biosynthesis in electrically stimulated slices, but the muscarinic receptor antagonist increased the stimulatory effect of angiotensin II. These data showed that EFS released angiotensin II and noradrenaline from pineal slices and that a close functional connection exists between the peptide and acetylcholine. The stimulation of serotonin biosynthesis and release by these two neurotransmitters was negatively regulated by noradrenaline acting through beta-adrenergic receptors.

Muscarinic effects on the hydroxy- and methoxyindole pathway in the rat pineal gland.

[3H]Quinuclidinyl benzylate ([3H]QNB)-binding sites, showing similar properties to cholinergic muscarinic receptors in other tissues, were disclosed in the rat pineal gland. Functionality of these receptors was demonstrated, as in-vitro muscarinic activation by pilocarpine increased the pineal metabolic production of the hydroxyindole derivatives 5-hydroxytryptophan and serotonin, with a slight effect on melatonin biosynthesis. Electric-field stimulation of pineal slices caused similar metabolic effects. These effects were inhibited by muscarinic blockade with atropine and enhanced by neostigmine inactivation of acetylcholinesterase. These results suggest that acetylcholine is the neurotransmitter involved. Cholinergic activity may, therefore, regulate indole metabolism in the pineal gland.

Interaction between thyrotrophin-releasing hormone and the muscarinic cholinergic system in rat brain.

TRH increases the pressor response to acetylcholine through an increment in muscarinic receptors. As chronic atropinization produces a similar effect, we hypothesized that both phenomena may be related. The effect of chronic atropine treatment on the TRH content of several brain areas in Wistar rats was studied. Atropine produced significant increases in TRH content in the preoptic and septal areas, while decreases were observed in the hypothalamus and hypophysis. The concentration of TRH in cerebrospinal fluid rose significantly in atropine-treated rats compared with controls. A similar effect was observed with eserine, an acetylcholinesterase inhibitor. Finally, perfusion of brain preoptic area slices from normal rats with Krebs-Ringer solution in the presence of pilocarpine increased basal TRH release significantly and this effect was blocked by atropine. These results are compatible with a muscarinic control on the activity of the central TRH system.

Thyrotropin-releasing hormone increases the number of muscarinic receptors in the lateral septal area of the rat brain.

Stereotactic injection of acetylcholine (0.5-2 micrograms) into the lateral septal region of the rat brain produces a long-lasting sympathetic-mediated increase of the arterial blood pressure. This effect is mediated by muscarinic receptors since 1 microgram atropine abolishes this response. In this same brain region, TRH (0.5-4 micrograms) did not elicit any significant change in the arterial blood pressure, but potentiated the effect of acetylcholine. This phenomenon is apparently due to an increase of the number of muscarinic receptors in the lateral septal area of the rat brain.

Circadian rhythm and neural regulation of rat pineal angiotensin converting enzyme.

Angiotensin converting enzyme was detectable in rat pineal gland and exhibited a circadian rhythm in activity with maximum at the end of the light phase of daily photoperiod. Superior cervical ganglionectomy (SCGx) or exposure to light for 6 days increased enzyme activity and obliterated morning-evening differences, whereas injection of the beta-agonist isoproterenol depressed the high values observed in SCGx animals. These results indicate that angiotensin converting enzyme in the pineal gland is under negative control by the norepinephrine released from pineal sympathetic nerves.

Synenkephalin processing in embryonic rat brain.

Synenkephalin (proenkephalin 1-70) is produced and secreted as an intact molecule or as a part of precursors in the adult brain and adrenal medulla, respectively. However, it is cleaved to low molecular weight peptides in proliferating immune cells. Considering that the pre-proenkephalin gene is expressed in the embryonic rat brain during the cell proliferation stage, we studied the processing of synenkephalin in embryonic rat brains (E18) and compared it with the processing in adult rat brains. IR-synenkephalin was measured by RIA using a C-terminally directed antiserum. Adult rat brains contained higher concentrations of immunoreactive (IR)-synenkephalin (2,612 + 264) than embryonic rat brain (1,361 + 100) (results in fmol/mg proteins, n = 5). Gel filtration chromatography (Sephadex G-50) showed that in the extracts of adult rat brain, 50% of the IR-synenkephalin eluted in the position of the authentic peptide (8 kDa) and the rest of the immunoreactivity corresponded to partially processed peptides of 4.0 and 2.5 kDa. In embryonic rat brains synenkephalin was processed to intermediate peptides of 2.5, 1.7 and mainly to a low molecular weight peptide of 1.0 kDa. The concentration of this last peptide, which was further characterized by affinity column and HPLC, represented 45% of the total immunoreactivity. IR-met-enkephalin in embryonic rat brains (analyzed before and after enzymatic digestion with trypsin and carboxypeptidase B) corresponded principally to non-processed or partially processed products. However, these were cleaved to free met-enkephalin in adult rat brains.(ABSTRACT TRUNCATED AT 250 WORDS)