Angiotensin II receptor pharmacology and AT1-receptor blockers.

Angiotensin II (Ang II) has diverse physiological actions leading, for example, to increases in extracellular volume, peripheral vascular resistance and blood pressure, and has also been implicated in the regulation of cell growth and differentiation. Molecular cloning and pharmacological studies have defined two major classes of Ang II receptors, designated as AT1 and AT2. Most effects of Ang II are mediated by AT1 receptors. Much less is known about the physiological role of AT2 receptors. Recent evidence suggests involvement of AT2 receptors in development, cell differentiation, apoptosis and regeneration in various tissues. AT1 and AT2 receptors have been shown to exert counteracting effects on cellular growth and differentiation, vascular tone and the release of arginine vasopressin (AVP). In each condition the AT2 receptor appears to down-modulate actions mediated by the AT1 receptor, resulting in decreased cellular proliferation, decreased levels of serum AVP levels or decreased vasoconstrictor responses. In addition, in neuronal cell lines, the AT2 receptor reportedly exerts antiproliferative effects and promotes neurite outgrowth, an effect accompanied by significant changes in the gene expression pattern of growth- and differentiation-related genes.

Receptors and their classification: focus on angiotensin II and the AT2 receptor.

Angiotensin II mediates its effects through angiotensin receptors. The use of specific angiotensin receptor ligands and the cloning of these receptors allows their classification. So far, the AT1, AT2 and atypical angiotensin II receptors are recognised. The AT1 receptor is responsible for the classical effects of the renin-angiotensin system such as vasoconstriction, renal salt and water retention, central osmo-control and stimulation of cell growth. The function of the AT2 receptor is far from clear but this receptor appears to be important in fetal development, cell growth inhibition and differentiation processes. This review describes the angiotensin receptors and focuses on the possible functions of the AT2 receptor.

Angiotensin AT2 receptor degradation is prevented by ligand occupation.

A substantial increase in [125I]Sar1, Ile-Angiotensin II binding activity can be observed 24 hours after treatment of R3T3 cells with AT2 receptor agonists and antagonists. An increase in the radioligand binding activity, although less profound, can also be observed 6 hours after AT2 receptor ligand treatment, on fetal human kidney cells expressing a recombinant human AT2 receptor. However, the increase in radioligand binding activity cannot be detected unless the ligands are removed from the cell surface by an acid-glycine (pH 3) wash, just prior to the binding assay. Interestingly, an acid-glycine wash 24 hours prior to the binding assay causes a dramatic decrease in the radioligand binding activity on untreated R3T3 cells. This decrease, which was prevented by angiotensin II treatment, suggests the existence of an unknown endogenous factor which, like the AT2 receptor ligands, seems to prevent AT2 receptor degradation.

Oxytocin pathways mediate the cardiovascular and behavioral responses to substance P in the rat brain.

Stimulation of brain periventricular and hypothalamic substance P receptors induces a pressor response and tachycardia associated with mesenteric and renal vasoconstriction and hindlimb vasodilation resembling thus the classical defense reaction. This cardiovascular response is brought about by the activation of the sympathoadrenal system and is accompanied by grooming behavior. To address the role of oxytocinergic pathways in the brain in the mediation of these responses, we investigated the effects of central pretreatment of rats with oxytocin antisense, mixed base, and sense oligodeoxynucleotides on mean arterial pressure, heart rate, and grooming behavior induced by intracerebroventricular injections of substance P (50 pmol). Central pretreatment of conscious rats with the oxytocin antisense oligodeoxynucleotide (intracerebroventricular injections, 8 and 4 hours before administration of substance P) attenuated the mean arterial pressure (by 55%) and heart rate responses (by 58%) as well as grooming behavior induced by the peptide. A complete recovery of all substance P-induced responses was observed 28 hours after antisense oligodeoxynucleotide pretreatment. Intracerebroventricular pretreatment of rats with mixed base and sense oligodeoxynucleotides did not affect the cardiovascular and behavioral responses to substance P. The signal for oxytocin mRNA in the paraventricular nucleus was reduced only in rats pretreated with the antisense oligodeoxynucleotide. These results demonstrate that oxytocin neurons in the paraventricular nucleus, which innervate the cardiovascular centers in the hindbrain and the spinal cord, mediate the increases in blood pressure and heart rate induced by stimulation of substance P receptors in the forebrain. These neurons may also transmit signals, which are generated by substance P in the hypothalamus and are responsible for the sympathoadrenal activation in response to stress.

The angiotensin AT2 receptor down-regulates neurofilament M in PC12W cells.

The angiotensin (ANG II) AT2 receptor mediates antiproliferative effects and induces neurite outgrowth in PC12W cells. To further investigate the molecular events following AT2 receptor stimulation in these cells, we determined the expression pattern of the middle-sized neurofilament subunit (NF-M) using Western and Northern blot analysis and reverse-transcription polymerase chain reaction. On both, the protein and the mRNA level, ANG II via AT2 receptors not only counteracted nerve growth factor (NGF)-mediated NF-M up-regulation but also reduced NF-M levels in the absence of NGF by maximally 72%. The ANG II-induced effects were completely abolished by pretreatment with the AT2 receptor antagonist, PD123177. In view of previous findings of decreased NF levels in regenerating neurons and in neuronal cultures undergoing apoptosis, our observation suggests a new role of AT2 receptors in either of these processes.

Extrarenal aspects of angiotensin II function.

The cloning of angiotensin II (Ang II) receptor genes and the availability of specific receptor ligands allows characterization of Ang II receptor-mediated actions. Most of the well-known Ang II effects such as vasoconstriction, drinking response and cell proliferation are mediated through the AT1 receptor. Little is known about the physiological effect of the AT2 receptor, though there are some reports describing the involvement of the AT2 receptor in blood pressure regulation. Recent data demonstrate that the AT2-mediated actions are inhibitory to AT1- and mitogen-induced growth effects, indicating a balancing mechanism for Ang II-controlled mechanisms. It has also been demonstrated that AT2 receptor inactivation induces endothelial cell proliferation in the presence of Ang II. Additionally, AT2 receptor activation enhances nerve growth factor-induced differentiation of PCI2W cells and a role in apoptotic changes has also been reported. Based on recent findings, this article focuses on the role of Ang II in growth and differentiation processes with respect to the AT2 receptor in these events.

Renal expression of two rat kallikrein genes under diabetic conditions.

OBJECTIVE: We have reported that bradykinin (BK) excretion is increased in severely diabetic rats, independent of the activity of the main renal kinin-forming enzyme, true kallikrein (KLK). To further investigate the relationship between renal BK excretion and renal KLK in diabetes we studied the regulation of the renal kallikrein-like gene, rat kallikrein 7 (rKLK7), as well as of the KLK encoding gene, rKLK1, in streptozotocin-induced (STZ) diabetic rats. METHODS: Experiments were performed in STZ-induced diabetic male Wistar rats and their non-diabetic controls (n = 7 each group). Twelve weeks after STZ injection, urinary KLK activity, glomerular filtration rate and total protein excretion were determined. After extraction of total renal cortical RNA, specific oligonucleotides were used to generate a reverse transcription-polymerase chain reaction (RT-PCR) products of renal cortical rKLK1 and rKLK7 messenger (m)RNA. Southern blot analysis of these RT-PCR products were hybridized with appropriate gene-specific oligonucleotide probes. RESULTS: After 12 weeks, the rats showed hyperglycemia, proteinuria and a reduced glomerular filtration rate. Renal kininogenase was reduced, as indicated by a reduction in the expression of rKLK1, as well as of the KLK-related gene, rKLK7. CONCLUSIONS: Our data show that the expression of the two principal renal KLK genes is downregulated in the renal cortex of STZ-diabetic rats. We suggest that under severe diabetic conditions the rise in urinary BK excretion is not related to activation of the renal kinin-forming enzyme system.

Angiotensin receptors.

INTRODUCTION: The octapeptide angiotensin II, the potent effector molecule of the renin-angiotensin system, has been implicated in the pathology of hypertension, in cardiovascular diseases like cardiac left ventricular hypertrophy and in structural alterations of the heart such as post-infarct remodelling. ANGIOTENSIN RECEPTORS: The development of highly selective angiotensin II receptor ligands allowed the identification of angiotensin II receptor subtypes, designated AT1, AT2, AT3 and AT4. Most of the known effects of angiotensin II can be attributed to the AT1 receptor (e.g. vasoconstriction, aldosterone and vasopressin release and proliferative effects on vascular smooth muscle and other cells). The AT1 receptor is coupled to G-proteins and engages classical intracellular second messenger systems, for example activation of phospholipase C or inhibition of adenylate cyclase. In contrast, the function and the signal transduction pathways of the AT2 receptor, which exhibits only a 32-34% homology to the AT1 receptor, are so far not fully understood. Coupling of the AT2 receptor to phosphatases and inhibitory actions on AT1 receptor- and growth factor-mediated proliferation in endothelial and other cells as well as induction of neuronal outgrowth in PC12w cells have been demonstrated. Due to its wide distribution in fetal tissues including the central nervous system and its transient reappearance in the adult organism under pathological conditions (for instance after myocardial infarction) the AT2 receptor has been associated with cell differentiation and regeneration. RECEPTOR ANTAGONISTS: The application of orally active AT1 receptor antagonists as antihypertensive drugs has, compared to angiotensin converting enzyme inhibitors, the potential advantage of a more specific renin-angiotensin system inhibition. It is conceivable that the AT2 receptor, left unopposed by AT1 receptor antagonists, contributes to some of the actions of these drugs.

Temporal changes in insulin-like growth factor I, c-fos, and c-jun gene expression during hyperplastic kidney growth in weanling rats.

We have previously determined that compensatory renal growth (CRG) during the initial 24-48 h after uninephrectomy (UNX) is GH independent in weanling animals, but associated with significant increases in insulin-like growth factor I (IGF-I) and IGF-I receptor gene expression. The purpose of the present study was to determine the temporal sequence of molecular and cellular events that occur at various time points (1, 6, 12, 18, 24, 48, and 72 h post-UNX) during this early period of accelerated renal growth in the weanling (21- to 25-day-old) rat. Rapid and sustained increases in steady state renal IGF-I receptor and IGF-I messenger RNA (mRNA) were observed at 1 and 6 h, respectively, and remained elevated in the remnant kidneys until 72 h post-UNX. The mRNAs for the early response genes, c-fos and c-jun, were not induced in the remnant kidneys from weanling rats until between 12-18 h, but were also sustained through 48 h post-UNX. Increases in remnant kidney DNA content and [3H]thymidine incorporation also occurred from 18-48 h post-UNX and returned to baseline levels by 72 h post-UNX, indicating that the hyperplastic response in the weanling remnant kidney occurs over a discrete period early after UNX. Neither IGF-I nor early response genes were elevated in kidneys from adult animals, which exhibited only hypertrophic renal growth at those early time points after UNX. These findings suggest that early CRG in the weanling rat is associated with rapid increases in IGF-I mRNA followed by a rise in c-fos and c-jun gene expression and a mitogenic response. Furthermore, when the mRNA levels of IGF-I and early response genes returned to baseline levels, mitogenic growth stopped, and slower prolonged hypertrophic renal growth ensued.

Distribution of glucocorticoid receptor mRNA in the rat spinal cord.

Using in situ hybridization (ISH), we studied the distribution of rat glucocorticoid receptors (GR) mRNA in rat spinal cord. mRNA encoding for GR was abundant throughout the white matter and a clear pattern of distribution was detected within the grey matter. In the grey matter mRNA was primarily localized in the ventral horn, where motoneurones were strongly labelled. In the dorsal horn, the distribution appears more diffuse but the superficial layers (I and II) clearly exhibited a shigher signal. We conclude that, in rat spinal cord, GR are present in both glial and neuronal cells. In particular, both somatosensory and motor pathways contain GR.

Structure of the rat gene encoding the mitochondrial benzodiazepine receptor.

The gene encoding the rat mitochondrial benzodiazepine receptor (MBR) was cloned and characterized. Hybridization of a previously cloned cDNA for MBR to genomic Southern blots indicated that the gene was probably present at one copy per haploid genome. Rapid amplification of cDNA ends with rat adrenal RNA was used to obtain 47 nt of additional sequence upstream from our previously cloned MBR cDNA proving to be a crucial step in cloning the first exon of this gene. The MBR gene is comprised of four exons spanning approx. 10 kb. The first intron, contained within a 8-kb stretch of this gene, is located within the 5'-untranslated sequence, whereas the remaining two introns are much shorter (641 and 854 bp) and interrupt the coding sequence. The third intron contains sequences homologous to rodent B1 repetitive elements and a novel sequence closely resembling part of a repetitive element belonging to the Alu family in humans. The transcription start point was mapped by S1 nuclease protection assays suggesting that the first exon is just 56 bp in length. The sequence upstream from this region contains three GC boxes but lacks other known consensus recognition sites for sequence-specific transcription factors.

Rat hypothalamic proopiomelanocortin messenger RNA is unaffected by adrenalectomy.

The negative feedback control of hypothalamic cortocotrophin releasing factor (CRF) and anterior pituitary proopiomelanocortin (POMC) by corticosteroids is well understood. However, less is known about the mechanisms that regulate POMC gene expression in the arcuate nuclei in the medial basal hypothalamus (MBH). Using a sensitive and specific S1 endonuclease protection assay, we have examined the effect of adrenalectomy on POMC mRNA in the rat MBH and pituitary. Our results show that adrenalectomy does not change POMC mRNA levels in the MBH at 7 or 14 days post surgery. The neurointermediate lobe of the pituitary was similarly unaffected by adrenalectomy, while in the anterior lobe, POMC mRNA increased 7-10 fold at both time points, effects that were prevented by dexamethasone treatment. We conclude that while POMC mRNA in the anterior lobe of the pituitary is regulated by plasma glucocorticoids, in the MBH and neurointermediate lobe, it is not.

Stimulus-evoked release of tritiated monoamines from rat periaqueductal gray slices in vitro and its receptor-mediated modulation.

The periaqueductal gray is a brain region of considerable interest. It is innervated by monoamine-containing neurons as well as by a variety of peptidergic fiber systems, and it participates in the regulation of various functions. Virtually nothing is known about monoamine release in the periaqueductal gray and its receptor-mediated modulation. We therefore studied the release of radioactivity from periaqueductal gray slices preloaded with tritriated monoamines, using an in vitro superfusion method. The release of radioactivity from superfused periaqueductal gray slices after preloading of the tissue with [3H]noradrenaline increased upon electrical stimulation in a frequency-dependent manner. The stimulus-evoked release of radioactivity was Ca(2+)-dependent. Clonidine reduced and yohimbine enhanced the release. The inhibition curve for the effect of clonidine was shifted to the right in the presence of 10(-6) M yohimbine. While phenylephrine, isoprenaline, SK & F 38393, quinpirole, carbachol, [Arg8]vasopressin, alpha-MSH and ACTH-(1-24), at a concentration of 10(-6) M, did not influence the electrically evoked release of radioactivity, [Leu5]enkephalin reduced it. The selective mu-opioid receptor agonists [D-Ala2,NMePhe4,Gly-ol5]enkephalin and [D-Arg2,Lys4]-demorphin-(1----4)-amide reduced the release of radioactivity, whereas the selective delta opioid receptor agonist [D-Pen2,D-Pen5] enkephalin and the selective kappa opioid receptor agonist U-69593 had no effect. In the presence of naloxone, which by itself had no effect on the release of radioactivity, the effect of [D-Arg2,Lys4]dermorphin-(1-4)-amide was abolished. These results show that the release of noradrenaline from periaqueductal gray slices is via a Ca(2+)-dependent exocytotic process, and that it is modulated through alpha 2-adrenoceptors as well as via mu-opioid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)