Angiotensin II receptor expression in the conduction system and arterial duct of neonatal and adult rat hearts.

Paralleling the classic circulating system, recent evidence has demonstrated the presence of a cardiac renin-angiotensin system, as well as the synthesis of angiotensin II in the heart. Two receptors for angiotensin II have been identified and classified as AT1 and AT2. The proportions of these receptor subtypes vary with the tissues, species and stage of development. From the results of other studies, it might be generalized that the expression of angiotensin II receptors and the proportion of AT2 receptor subtype are much higher in fetal and neonatal tissues than in the same tissues from an adult. The aim of this study was to specifically evaluate the AT1/AT2 ratio in the neonatal and adult conduction systems of rat hearts by means of quantitative autoradiogrphy. In the neonatal hearts, angiotensin II binding sites were highly concentrated in the vasculature, arterial duct, and conduction system, whereas their concentrations were barely detectable in the myocardium. Incubation with selective angiotensin II receptor ligands (losartan and CGP 42112) revealed that AT2 was the major subtype in vasculature (86 +/- 3%) and conduction system (73 +/- 4%). In the adult conduction system, the total expression of angiotensin II receptors was greatly reduced meanwhile the AT1 receptors represented the major proportion of the binding sites (80 +/- 3%). Our results demonstrated that the pattern of angiotensin II receptor expression in the conduction system of the rat heart is developmentally regulated. We suggest, as others have already, that the renin-angiotensin system plays a role during the early stage of cardiac development.

Regulation of protease nexin-1 and angiotensin II receptor subtype 1 expression: inverse relationship in experimental models of nerve injury.

The up-regulation of PN-1 following nerve lesion has been investigated in vitro in cultures of dorsal root ganglion (DRG) explants, sciatic nerve segments, and isolated Schwann cells. In the first culture model, Schwann cells associated with neuronal processes synthesized small amounts of PN-1. Injury of the neurites emerging from the DRGs led to enhanced levels of PN-1 in Schwann cells located distal to the lesion site where degeneration of neuronal processes took place. In cultured sciatic nerve segments, PN-1 synthesis increased with a time-course comparable to that in ganglion explants following lesion. In the third model, PN-1 levels gradually rose in isolated Schwann cells during the first 3-8 days in culture. Dissociation of Schwann cells from the sciatic nerve therefore causes an effect similar to nerve damage. Impairment of Schwann cells-neuron interactions was followed by a reduction in the expression levels of the angiotensin II (Ang II) receptor subtype AT1 in all three systems studied. Since the neuropeptide Ang II is able to repress PN-1 synthesis in cultured Schwann cells, loss of neuronal contact might decrease their responsiveness to Ang II, thus resulting in PN-1 up-regulation by default.

Dopamine antagonizes the actions of angiotensin II in renal brush-border membrane.

In the present study, we examined the effects of dopamine and angiotensin II (ANG II) in renal brush-border membrane (BBM). With isolated BBM vesicles, dopamine (> 10(-4) M) directly inhibited BBM 22Na+ uptake and activated phospholipase C (PLC). These effects were mimicked by DA1 agonist but not DA2 agonist and were prevented by DA1 antagonist but not DA2 antagonist, indicating the involvement of DA1 receptors. In contrast to dopamine, ANG II directly stimulated BBM 22Na+ uptake and activated BBM phospholipase A2 (PLA2). Neither dopamine nor ANG II altered BBM adenosine 3',5'-cyclic monophosphate content. In the presence of dopamine, ANG II failed to stimulate BBM Na+ uptake and PLA2. However, both DA1 and DA2 agonists similarly abrogated the actions of ANG II, and both DA1 and DA2 antagonists were required to restore ANG II actions in the presence of dopamine, indicating the involvement of both DA1 and DA2 receptors in the antagonistic effect of dopamine. Dopamine, as well as DA1 or DA2 agonists, also lowered 125I-ANG II BBM binding. In summary, these results show that, in renal BBM, dopamine impedes ANG II receptor binding and antagonizes the stimulatory effects of ANG II on Na+ uptake and PLA2. This occurred through both DA1 and DA2 receptors and independent of DA1 effects on BBM Na+ uptake or PLC.

Hypothalamic regulatory peptides and their receptors: cytochemical studies of their role in regulation at the adenohypophyseal level.

Hypothalamic regulatory peptides bind to specific receptors on target cells in the pituitary and control secretion. They in turn can be regulated at the pituitary level by steroid and peptide modulators. Affinity cytochemical techniques are important tools for the identification of specific target binding sites for these regulatory peptides. This presentation reviews the work in which potent, biotinylated ligands of gonadotropin releasing hormone (bio-GnRH), corticotropin releasing hormone (bio-CRH), and arginine vasopressin (bio-AVP) were applied to study the target cell responses. Bio-GnRH, bio-CRH, and bio-AVP bind to membrane receptors on specific anterior pituitary cells. Dual labeling for either gonadotropin or adrenocorticotropin (ACTH) antigens further identified the target cells. After 1-3 minutes, the label was in patches or capped on the surface. After 3 minutes, it was internalized in small vesicles and sent to receptosomes and vacuoles in the Golgi complex. Eventually the biotinylated peptides, or a metabolite, was found in the lysosomes (multivesicular bodies) and a subpopulation of secretory granules. The route and rate of uptake was similar to that described for the classical receptor-mediated endocytosis process. In contrast, intermediate lobe corticotropes internalized the bio-CRH in less than 1 minute. The route through the Golgi complex appeared to be bypassed. Instead the labeled peptide was in vesicles, on the membranes of scattered vacuoles, and in multivesicular bodies. Modulation of ligand binding by steroids showed that changes in receptor numbers correlated with changes in the number of cells that bound the ligand. In male rats, dihydrotestosterone reduced the percentage of GnRH-bound cells by 50%. Most of the reduction appeared in cells that stored luteinizing hormone (LH) antigens. In diestrous female rats, estradiol increased the percentage of bio-GnRH-bound cells. However, the steroid decreased the percentage of GnRH-bound cells in cells from proestrous rats. Glucocorticoids decreased the percentage of CRH-bound corticotropes in as little as 10 minutes. Potentiation of secretion by these ligands was correlated with increases in the percentage of ligand-bound cells. AVP pretreatment of corticotropes increased the percentage of cells that bound bio-CRH. It also increased the rate of receptor-mediated endocytosis of CRH and changed the route so that the Golgi complex was bypassed. This effect could be mimicked by activation of its second messengers (calcium and protein kinase C). Similarly, CRH pretreatment increased the percentage of corticotropes that bound AVP. Thyrotropin releasing hormone (TRH) pretreatment also increased the percentage of thyrotropes that bound AVP.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple forms of angiotensin II receptors in rat tissues.

Angiotensin II (AII) receptors were identified in rat tissue membranes by specific binding of 125I-labelled AII. Using an isoelectric focusing technique, two forms of the high-affinity AII receptor were identified in rat adrenal zona glomerulosa and liver membranes. These migrated to isoelectric points (pI) 6.8 and 6.7. Two low-affinity forms migrated to pI 6.5 and 6.3. The two high-affinity forms were in greatest abundance in the zona glomerulosa, while the low-affinity pI 6.5 isoform was predominant in liver membranes. In uterine membranes both low-affinity isoforms were observed, but there was only one of the high-affinity forms (pI 6.7). Concentrations of AII receptor isoforms were increased in the zona glomerulosa of sodium-deprived rats. Reduction of disulphide bridges with dithiothreitol (DTT) had different effects on the various AII receptor isoforms. Thus 1 mmol DTT/1 caused a twofold increase in 125I-labelled AII binding in zona glomerulosa membranes. DTT produced no appreciable differences in specific AII binding in uterine membranes, whereas there was a 50% reduction of binding in liver membranes. At 20 mmol/l, DTT greatly decreased AII binding in all tissues. The data suggest the existence of multiple forms of AII receptors which may have different functions.

Steroidogenic enzyme activities, morphology, and receptor studies of a testicular adrenal rest in a patient with congenital adrenal hyperplasia.

Steroid-secreting tumors of the testis have generally been considered to be of Leydig cell origin. Testicular tumors in patients with congenital adrenal hyperplasia have been thought to be adrenal rests, but no conclusive evidence supporting the hypothesis has been presented. We report a morphological and biochemical analysis of a patient with 21-hydroxylase deficiency who developed bilateral nodular hyperplasia of steroid-secreting tissue within the testis, despite suppression therapy with both exogenous glucocorticoids and testosterone. The tissue was formed of confluent nodules of homogenous cells. Electron microscopy showed the cells to have abundant smooth endoplasmic reticulum, well developed Golgi apparatus, and mitochondria with predominantly tubular cristae, features characteristic of steroid-secreting cells of adrenocortical origin. Crystals of Reinke were not observed. Functional studies in vivo showed a marked response to ACTH infusion, with 17-hydroxyprogesterone rising from 56 to 13,500 ng/mL, cortisol from less than 2 to 19 micrograms/dL, and testosterone from 369 to 629 ng/dL, with an attendant increase in testicular size and pain over 48 h. Receptor studies in vitro revealed no gonadotropin receptors, but abundant angiotensin-II receptors. Enzyme activity analysis in vitro showed undetectable 21-hydroxylase activity and an enzyme profile consistent with adrenocortical cells rather than Leydig cells. Based on these morphological and biochemical findings, we conclude that the nodular steroidogenic tissue that replaced this patient's testes was of adrenal origin. The study documents for the first time the development of adrenocortical tumors from adrenal rest tissue within the testis.

[Ultrastructure of the apical plasma membrane of the granular cells in the frog bladder during cobalt-ion decrease in the vasopressin effect]. / Ul'trastruktura apikal'noi plazmaticheskoi membrany granuliarnykh kletok mochevogo puzyria liagushki pri snizhenii éffekta vazopressina ionami kobal'ta.

Simultaneous studies were performed on changes in water permeability and on the ultrastructural organization of the frog urinary bladder epithelium in the presence of Co-ions under vasopressin-stimulated water flow. A possible inhibition of the vasopressin-stimulated water flows by Co-ions is supposed from the extracellular surface of the apical membrane of granular cells responsible for water permeability of this epithelium. Using the freeze-fracture technique for studying the apical membrane ultrastructure, it was shown that with the maximum water flow the square occupied by intramembrane particle aggregates was as much as 1.8% of the total square of membranes, to reduce to 0.3% with the smaller water flow, the average sizes of aggregates being 0.35 mkm and 0.08 mkm in both these cases, respectively. Application of 1 x 10(-3)-1 x 10(-4) M CoCl2 from the mucose part inhibits the vasopressin-stimulated water flow. In this case no aggregates are actually seen on the P-face of the apical membrane, the number of intramembrane particles of the E-face being similar to that when the water permeability was originally low. It is concluded that Co-ion may influence the structure and function of the apical plasma membrane from its extracellular surface.

Appearance and transient expression of oxytocin receptors in fetal, infant, and peripubertal rat brain studied by autoradiography and electrophysiology.

The development of oxytocin (OT) receptors in the rat brain and spinal cord was studied by in vitro light microscopic autoradiography and by electrophysiology. OT receptors were labeled using a monoiodinated OT antagonist in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the response of ongoing spike activity to the addition of OT was assessed in neurons located in the dorsal motor nucleus of the vagus nerve of the neonate. Specific binding was detected first at E14 in a region that later differentiated into the dorsal motor nucleus of the vagus nerve. Many other regions were progressively labeled between E20 and PN5. From PN5 to PN16, the distribution of binding sites remained essentially unchanged but differed markedly from that characteristic of the adult. The change-over from the "infant pattern" to the "adult pattern" occurred in 2 stages: the first change took place between PN16 and PN22, a time corresponding to the preweaning period; the second change occurred after PN35 and thus coincided with the onset of puberty. During the first transition period, binding was reduced or disappeared in several areas intensely labeled at earlier stages, in particular, in the cingulate cortex and the dorsal hippocampus. At the same time, binding sites appeared in the ventral hippocampus. At puberty, high densities of OT binding sites appeared in the ventromedial hypothalamic nucleus and the olfactory tubercle. Electrophysiological activity was recorded from vagal neurons in slices obtained from animals sacrificed at PN1-PN12. OT and a selective OT agonist reversibly increased the firing rate of these neurons in a concentration-dependent manner. The neuronal responsiveness was similar to that reported previously in the adult. These results suggest that OT binding sites detected by autoradiography in the developing rat brain represent, at least in some areas, functional neuronal receptors.