Judith E. Kalinyak

Expression of AT2 receptors in the developing rat fetus.

Angiotensin II is known primarily for its effects on blood pressure and electrolyte homeostasis, but recent studies suggest that angiotensin II may play a role in the regulation of cellular growth. This study was undertaken to identify the angiotensin II receptor subtypes expressed during fetal and neonatal development and to characterize their cellular localization. Using an in situ receptor binding assay on sagittal frozen sections of fetal and neonatal rats, bound 125I-[Sar1,Ile8]-angiotensin II was visualized by film and emulsion autoradiography. Bound radioligand was detected by E11 (embryonic day 11) and maximal binding occurred by E19-21. Radioligand binding remained unaltered 30 min after birth, whereas a noticeable and stable decrease was observed 12 h postparturition. The highly abundant angiotensin II receptors were shown to be AT2 by the marked reduction in radioligand binding achieved with PD123177 (10(-7)M), a specific AT2 receptor antagonist, whereas DuP 753 (10(-5)M), an AT1 receptor antagonist, had little effect. Emulsion autoradiography showed radioligand binding in the undifferentiated mesenchyme of the submucosal layers of the intestine and stomach, connective tissue and choroid surrounding the retina, subdermal mesenchyme adjacent to developing cartilage, diaphragm, and tongue. Residual AT2 receptors were found on the dorsal subdermal region of the tongue 72 h after birth. AT1 receptors were detected in the placenta at E13 and in the aorta, kidney, lung, liver, and adrenal gland at E19-21, consistent with an adult distribution. The transient expression of AT2 receptors in the mesenchyme of the fetus suggests a role of angiotensin II in fetal development.

Angiotensin II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-β1 and endothelin-1 from fibroblasts

OBJECTIVE We sought to determine whether angiotensin II (Ang II) promotes hypertrophy of cardiac directly or via paracrine mechanisms mediated by cardiac fibroblasts. METHODS We studied neonatal rat cardiac myocytes and fibroblasts in culture as a model system. Paracrine effects of Ang II were identified using conditioned medium and co-culture experiments. RESULTS Ang II type 1 (AT1) receptors responsible for myocyte growth localized to fibroblasts in radioligand binding, emulsion autoradiography, Western analysis, and immunofluorescence staining experiments. The bulk of AT1 receptor binding in myocyte cultures (1343 +/- 472 sites/cell) was to Ang II receptors on contaminating fibroblasts (9747 +/- 2126 sites/cell). Ang II induced significant paracrine trophic effects on myocytes in conditioned medium (40% increase in protein synthesis over control) and co-culture (4-fold increase over control) experiments. TGF-beta 1 and endothelin-1 were paracrine mediators of hypertrophy in neutralization experiments. CONCLUSIONS Ang II stimulates cardiac myocyte hypertrophy via paracrine release of TGF-beta 1 and endothelin-1 from cardiac fibroblasts in a neonatal rat cell culture model.

Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat.

The biological action of glucocorticoids is dependent upon tissue-specific levels of the glucocorticoid receptor (GR). During stress, the hypothalamic-pituitary-adrenal axis is stimulated, and high levels of glucocorticoids circulate. This axis is modulated by negative feedback by glucocorticoids, which inhibit hypothalamic and pituitary hormone secretion and downregulate GR gene expression. To study the developmental tissue-specific regulation of the GR, we measured the relative concentration of GR mRNA in fetal, neonatal, adult, and aged rats and examined the effects of dexamethasone on GR gene expression. Three different tissue-specific developmental patterns of GR mRNA accumulation were found. In addition, there was an age-dependent tissue-specific pattern in the feedback regulation of GR mRNA by glucocorticoids. In the fetus and neonate, GR mRNA abundance was not regulated by circulating glucocorticoids. The adult pattern of glucocorticoid feedback inhibition of GR mRNA expression appeared between 2 and 7 d of life in liver, and after 7 but before 14 d of age in brain. The GR was biologically active in the 2-d-old neonate, however, since dexamethasone enhanced gene expression of angiotensinogen, which is another glucocorticoid responsive gene. These data demonstrate that the GR gene is regulated by both developmental and tissue-specific factors, and provide another molecular basis for ontogenic variations in the hypothalamic-pituitary-adrenal response to stress.

Dietary Sodium Intake Modulates Pituitary Proopiomelanocortin mRNA Abundance

The pituitary prohormone proopiomelanocortin gives rise to melanocortins of alpha, beta, and gamma primary structure in addition to corticotropin. Melanocortins have a variety of actions in mammals, and each is natriuretic. In particular, gamma-melanocyte-stimulating hormone has been shown to mediate reflex natriuresis after acute unilateral nephrectomy. We examined whether this peptide could play a role in longer term adjustments in sodium balance by measuring plasma gamma-melanocyte-stimulating hormone and corticotropin concentrations, as well as pituitary proopiomelanocortin mRNA abundance, in Sprague-Dawley rats ingesting either a low (0.07% NaCl) or high (7.5% NaCl) sodium diet. One week after the high sodium diet, plasma gamma-melanocyte-stimulating hormone concentration was double the value seen in rats on the low sodium diet (158 +/- 5 [SE] versus 76 +/- 9 fmol/mL, P < .001), a change that was accompanied by a fivefold increase in plasma atrial natriuretic peptide concentration but no change in plasma corticotropin. Whole pituitary proopiomelanocortin mRNA abundance, measured with a probe to exon 3 of the rat proopiomelanocortin gene, was significantly increased after 1 week of the high sodium diet compared with the low sodium diet and increased further at 2 and 3 weeks. This increase occurred primarily in the neurointermediate lobe as demonstrated by in situ hybridization; the content of gamma-melanocyte-stimulating hormone immunoreactivity was also increased in this lobe, but not the anterior lobe, after 1 week of the high sodium diet. These results demonstrate that high dietary sodium intake increases neurointermediate lobe proopiomelanocortin mRNA abundance compared with a very low sodium diet and also suggest that proopiomelanocortin is preferentially processed into gamma-melanocyte-stimulating hormone rather than corticotropin. These observations consequently raise the possibility of a role for this peptide hormone system in the adjustments to a high salt diet.

Tissue-Specific Regulation of Insulin Receptor mRNA Levels in Rats With STZ-Induced Diabetes Mellitus

In rats with STZ-induced diabetes mellitus, a reduction in insulin secretion is associated with increased insulin binding in the liver, muscle, fat, and kidney, but not in the brain. To test the hypothesis that tissue-specific modulation of insulin receptors (IRs) in STZ-induced diabetes occurs at the level of mRNA, IR mRNA levels were measured in the liver, kidney, and brain of Sprague-Dawley rats 15 days after intravenous administration of STZ (60 mg/kg body weight) and compared with those of control rats. Diabetic rats were either left untreated or given differing insulin regimens that were designed to achieve varying degrees of metabolic control. IR mRNA levels were measured by slot blot hybridization with a 32P-labeled rlR probe and standardized by 28S ribosomal RNA determination. Hepatic IR mRNA levels were increased significantly in both untreated diabetic rats and in those that received low-dose (2 U/day) insulin therapy. In contrast, hepatic IR mRNA levels did not differ significantly from controls in those that received moderate doses of insulin (3–8 U/day) and were significantly less than controls in those that received the highest doses (6–10 U/day). Renal IR mRNA levels also were increased significantly in the untreated diabetic rats but not in those that received low- or moderate-dose insulin therapy, and were significantly less than controls in those that received the highest doses. A highly significant negative correlation was observed between the level of hepatic (r = −0.84, P <0.001) and renal (r = −0.64, P < 0.001) IR mRNA, and the plasma concentration of insulin obtained at the time of death. No significant difference was observed in brain IR mRNA levels between untreated diabetic and control rats. Thus, in rats with insulin deficiency, modulation of insulin binding in the liver and kidney can be attributed, at least in part, to a change in steady-state IR mRNA levels.

Dietary sodium modulates mRNA abundance of enzymes involved in pituitary processing of proopiomelanocortin.

The messenger RNA abundance of proopiomelanocortin (POMC) is increased in neurointermediate lobe (NIL) of rat pituitary when ingesting a high sodium diet (8%; HSD), as is the plasma concentration of the natriuretic peptide γ-melanocyte stimulating hormone (γ-MSH) derived from it. We examined whether the HSD also increases the mRNA abundance in rat NIL of proconvertases 1 and 2 (PC1, PC2), enzymes involved in the processing of POMC into γ-MSH. PC1 mRNA increased by 40% after two weeks of the HSD and by 84% after three weeks. PC2 mRNA increased by 40% after two weeks and by more than 3 fold after three weeks. These results for PC2 were confined to NIL as shown by in situ hybridization at one and two weeks, and were accompanied by a significant increase in NIL PC2 protein after three weeks of the HSD as measured by immunoblotting. The increases in PC1 and PC2 mRNA abundance were paralleled by an increase in POMC mRNA level in NIL. Plasma γ-MSH immunoreactivity averaged 35.1±3.3 fmol/ml in rats on the LSD, but increased to 70.9±4.8 fmol/ml after 3 weeks of the HSD (p < 0.002 vs LSD). These results confirm that the HSD increases the plasma concentration of γ-MSH, consistent with a role for it as a circulating natriuretic peptide. The increased NIL expression of PC1 and PC2 in parallel with POMC in response to the HSD suggests that these changes are part of the coordinated response to states of sodium surfeit.

Expression of AT2 receptors in rat fetal subdermal cells.

Using an in situ receptor binding assay with emulsion autoradiography, angiotensin II receptors have been localized to the subdermal/subcutaneous region of the E19 rat skin. Radioligand binding studies on membranes of epidermis, dermis, and subdermal tissues confirmed the localization of receptors to these regions and displacement of binding by PD123177 showed the receptor was AT2. Scatchard analysis of whole skin membrane binding studies showed the receptor had a Kd of 0.8 nM, with a Bmax of 2240 fM/mg protein. Fetal mesenchymal cells were placed in culture and angiotensin II binding which was minimal at 48 h increased by 30-50-fold after 96 h in culture with the majority of the receptors being AT2. Increasing concentrations of FBS caused a decrease in angiotensin II binding, while maintaining the same percentage of AT2 binding sites. Increasing the initial cell density at which the cells were plated dramatically increased the angiotensin II radioligand bound, while decreasing the percentage bound to AT2 receptors. These findings demonstrate the presence of both angiotensin II receptor subtypes in cultured skin mesenchymal cells. They also demonstrate that the culture conditions used can either modulate the expression of receptor subtypes or select for cells expressing a receptor phenotype. The lower number of AT2 binding sites in rapidly dividing cell cultures suggests that the AT2 receptor may not have a function in cell replication or growth.