Regulation of preprotachykinin-A mRNA in genetic hypertensive and normotensive rats

It is well-known that central administration of tachykinins (Tks) inhibit salt intake in rats. Recent studies have shown that conditions that arouse salt appetite, such as adrenalectomy and sodium depletion, induce a decrease in preprotachykinin-A (PPT-A) mRNA in discrete regions of the rat brain, suggesting that reduced levels of PPT-A mRNA in the brain may have a permissive role on the expression of salt appetite. It has also been shown that spontaneously hypertensive rats (SHR) show higher avidity for salty solutions than their normotensive control Wistar-Kyoto (WKY) rats. In this regard, the present study tested whether SHR and WKY rats differ in expression of the gene coding for PPT-A, the precursor for Tks peptides. Using semi-quantitative in situ hybridization histochemistry, we examined the level of PPT-A mRNA in discrete rat brain regions of SHR and WKY rats under no treatment, after 1 or 3 days of Na+ depletion. Levels of PPT-A mRNA were analysed in the olfactory tubercle (Tu), in the lateral olfactory tubercle (LOT), in the dorsal and ventral caudate putamen (d/v CPu), in the medial preoptic area (mPOA), in the bed nucleus of the stria terminalis (BNST), in the habenula (Hb) and in the postero-dorsal part of the amygdala (MePD). Semi-quantitative analysis of silver grains revealed a 27.5% lower expression of the PPT-A mRNA levels in SHR opposite to WKY rats under no treatment in v-CPu, mPOA, BNST and Hb. 1 day of Na+ depletion reduced PPT-A mRNA levels when opposite to Na+-repleted animals in Tu and mPOA in both SHR and WKY rats. On the other hand, when comparing SHR and WKY rats after 1 day of Na+ depletion, a 26% lower level of PPT-A mRNA was detected in Tu and d-CPu of SHR opposite to WKY rats whereas a 14% and an 18% lower level was detected in v-CPu and Hb, respectively. A lower expression of PPT-A mRNA in SHR compared to WKY rats was also found in BNST and MePD, although no statistical significance was detected in these two brain areas. In the last experiment, 3 days of Na+ depletion reduced PPT-A mRNA levels in mPOA while negligibly increased mRNA levels in d-CPu and v-CPu, in BNST, Hb and MePD, both in SHR and WKY rats. Conversely, when making comparisons between the two strains, a 35% lower level of PPT-A mRNA in SHR with respect to WKY rats was found after 3 days of Na+ depletion in d-CPu, v-CPu and mPOA. A lower gene expression, even though not statistically significant, was found in Tu, LOT, MePD. These findings show a consistent difference of PPT-A mRNA levels in discrete regions of the SHR brain opposite to WKY rats and confirm that 1 day of Na+ depletion reduces PPT-A mRNA in discrete brain regions. Since SHR are notoriously more salt-avid than WKY rats and Tks are potent inhibitors of sodium intake, the down-regulation of PPT-A mRNA may contribute to the higher natriophilia and, therefore, to the etiology of the hypertensive disease.     

Retrovirus-mediated transfer of an angiotensin type I receptor (AT1-R) antisense sequence decreases AT1-Rs and angiotensin II action in astroglial and neuronal cells in primary cultures from the brain

The AT1-R has been implicated in many cellular and physiological actions of angiotensin II (AII) in the brain. A retrovirus vector (LNSV) containing an AT1B-R antisense sequence (AT1B-AS) (termed LNSV-AT1B-AS) was constructed and used to determine the feasibility of using viral-mediated gene transfer to control AT1-Rs and AII actions in astroglial and neuronal cells in primary cultures from rat brain. Briefly, a 1.26-kb antisense sequence corresponding to nt -132 to +1128 of AT1-R cDNA was cloned into the LNSV vector, the vector was transfected into PA317 cells, and transfected cells were selected in G418. Incubation of brain cells with culture medium containing LNSV-AT1B-AS viral particles showed that AT1B-AS was integrated into the genome and transcribed in brain cells. This was associated with a significant decrease in AT1-Rs and in the AII-stimulated increase of c-fos mRNA, a measure of AT1-R function. These observations show that the AT1B-AS gene can be transferred into astroglial cells in culture by LNSV and that such a transfer inhibits AT1-Rs and the AII stimulation of cellular activities. In addition, the usefulness of this approach to study AII-dependent pathophysiology in primary neuronal cultures from brain, in particular, is established.     

Transformation of the methylotrophic actinomycete Amycolatopis methanolica with plasmid DNA: stimulatory effect of a pMEA300-encoded gene

The concentration of endothelin-1 (ET-1) in the brain regions, heart, and throacic aorta of 1-, 4-, 6- and 8-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined using radioimmunoassay. ET-1-like immunoreactivity in the brain regions of 1-week-old WKY and SHR rats was lower compared to older (6 and 8 weeks) rats. ET-1 levels in the central nervous system gradually increased with age in both SHR and WKY rats. However, the concentration of ET-1 in 8-week-old rats was lower in the brain regions of SHR compared to WKY rats. The concentration of ET-1 in the thoracic aorta of SHR (224 +/- 43 pg/g tissue) rats was lower than that of WKY (452 +/- 11 pg/g tissue) rats at 1 week of age. However, ET-1 levels gradually increased with age in SHR rats. By 8 weeks of age, levels of ET-1 in SHR (623 +/- 33 pg/g tissue) rats were higher compared to WKY (439 +/- 62 pg/g tissue) rats. In the heart, ET-1 levels were similar in WKY and SHR rats at 4 weeks of age, but at 8 weeks of age ET-1 levels were higher in SHR rats (364 +/- 33 pg/g tissue) compared to WKY rats (260 +/- 31 pg/g tissue). It appears that at 8 weeks of age when hypertension is fully expressed in rats, ET-1 levels are lower in the central nervous system and are higher in the thoracic aorta and heart of SHR compared to WKY rats.     

Increased oxidative stress in renal proximal tubules of the spontaneously hypertensive rat: a mechanism for defective dopamine D1A receptor/G-protein coupling

AIM: Defective dopamine D1A dopamine receptor/G-protein coupling has been demonstrated in renal proximal tubules of the spontaneously hypertensive rat (SHR). In the present study, we aimed to analyze the underlying mechanisms through which such defects are introduced into the D1A receptor protein of SHR. MATERIALS AND METHODS: The oxidative state of SHR proximal tubules was analyzed by measuring lipid peroxidation. D1A receptor/G-protein coupling was measured following the induction of oxidative stress in normotensive Wistar-Kyoto (WKY) rats. RESULTS: For the first time, an increased state of oxidative stress was demonstrated in SHR proximal tubules compared with those of normotensive controls, WKY and Sprague-Dawley rats. Lipid peroxidation levels in SHR were significantly higher by 66 and 79%, relative to WKY or Sprague-Dawley rats, respectively. Hydrogen peroxide treatment of proximal tubules from SHR, WKY and Sprague-Dawley rats induced an additional increase in lipid peroxidation in a dose-dependent manner, although the percentage induction was lower in SHR than in WKY and Sprague-Dawley rats. This induction of lipid peroxidation in WKY rats resulted in a loss of D1A/G-protein coupling, with no decrease in receptor protein. Treatment of WKY rat proximal tubules with an antioxidant, ascorbic acid, or a reducing agent, dithiothreitol, induced D1A receptor/G-protein coupling. CONCLUSIONS: These data indicate that D1A receptor/G-protein coupling is modulated by changes in redox states. Therefore, the D1A receptor/G-protein coupling in SHR may have been damaged by reactive oxygen species released as a result of the elevated oxidative stress seen in the proximal tubules.     

Nitric oxide-mediated changes in vascular reactivity in pregnancy in spontaneously hypertensive rats

1. To examine the mechanisms which may account for pregnancy-induced vasodilatation in spontaneously hypertensive rats (SHR), we have investigated the changes in vascular reactivity and the effects of endothelial nitric oxide (NO) inhibition in the in situ blood-perfused, mesenteric resistance vessels of 18-20 day pregnant SHR. The effects of NG-nitro-L-arginine (L-NOARG) were compared in pregnant and nonpregnant SHR and gestation matched normotensive Wistar-Kyoto (WKY) rats. 2. Intra-arterial mean blood pressures (MBP) were similar in pregnant and nonpregnant SHR. Basal perfusion pressures (BPP) were decreased in pregnant compared with nonpregnant SHR. Pregnant WKY had lower MBP and BPP than either pregnant or nonpregnant SHR. 3. Vasoconstrictor responses to electrical stimulation (ES) and intra-arterial noradrenaline (NA) were decreased in pregnant compared with nonpregnant SHR. These responses were still greater in pregnant SHR when compared with pregnant WKY. Vascular reactivity to angiotensin II (AII) in pregnant SHR was reduced to a similar level to that in pregnant WKY. 4. L-NOARG (5 mg kg-1, i.v.), an inhibitor of nitric oxide synthase, increased MBP and BPP in all groups. After L-NOARG, BPP were equalized between pregnant and nonpregnant SHR. Pregnant WKY still showed lower MBP and BPP than SHR groups. 5. L-NOARG potentiated vascular responses to ES, NA and AII in all groups. The blunted vascular responses to NA and ES were normalized and the reactivity to AII was only partially reversed in pregnant SHR compared with nonpregnant SHR. Pregnant WKY still had much lower vascular responses to ES and NA than either pregnant or nonpregnant SHR. L-NOARG enhanced vascular responses to All to a greater extent in pregnant SHR than in pregnant WKY.6. These results demonstrate that blunted responses to NA and ES were NO-dependent, while diminished reactivity to AII was only partially dependent on NO in the in situ blood perfused mesenteric resistance vessels of pregnant SHR.7. The present results in pregnant SHR differ from our previous finding with pregnant normotensive WKY, in which blunted responses to NA, but not to ES, were equalized by L-NOARG. Pregnancy induced vasodilatation in hypertensive rats appears to be more dependent on endothelial NO than in normotensive WKY. A defect of the endothelial NO generating pathway which promotes vasodilatation in pregnancy may contribute to the predisposition of women with essential hypertension to develop pre-eclampsia.     

Differential expression and autoradiographic localization of atrial natriuretic peptide receptor in spontaneously hypertensive and normotensive rat testes: diminution of testosterone in hypertension

Previous studies have shown that the diuretic hormone atrial natriuretic peptide (ANP) also regulates the steroidogenic responsiveness in isolated Leydig cells from mouse and rat testes. In the present study, we examined the distribution of specific receptors for ANP and C-type natriuretic peptide (CNP) in the testicular compartments of 12-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). We used an in vitro autoradiographic procedure on slide-mounted frozen testicular sections to localize the receptors of the natriuretic peptide hormone family using 125I-ANP and 125I-CNP as radioligands. A high level of specific 125I-ANP binding sites was localized largely in the Leydig cells of the interstitial compartment; other testicular cells were not significantly labeled. On the other hand, no significant difference was observed in 125I-CNP binding sites in the testicular cells of SHR and WKY. Semiquantitative analysis of the binding sites indicated that the density of 125I-ANP receptor binding in Leydig cells of WKY testis was ninefold higher than in those of SHR testis. A moderate level of 125I-ANP binding was also observed in seminiferous tubules, particularly in the spermatids of both SHR and WKY. 125I-ANP binding in WKY spermatids was approximately 2.5-fold higher than in SHR spermatids. Northern blot analysis showed that mRNA specific for guanylyl cyclase type A (Npra) was expressed at approximately twofold higher levels in WKY than in SHR testis. ANP (1 x 10(-8) mol/L) stimulated fourfold to fivefold increased levels of testosterone production in isolated Leydig cells from normotensive WKY compared with those from SHR. These findings support a new physiological role of ANP in Leydig cells, in which a functional relationship seems to exist between testicular ANP receptor expression and testosterone production and the state of hypertension in SHR.     

Parathyroid hormone-related protein expression in vascular smooth muscle of spontaneously hypertensive rats: evidence for lack of response to angiotensin II

OBJECTIVE: We studied the expression of parathyroid hormone (PTH)-related protein in vascular smooth muscle cells of spontaneously hypertensive rats (SHR) using Wistar-Kyoto (WKY) and Sprague-Dawley rats as normotensive controls. METHODS: Aortae from 4- and 18-week-old SHR versus age-matched WKY and Sprague-Dawley rats were excised to obtain total RNA or smooth muscle cells. The cells were subcultured in Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum, then serum-deprived for 72 h and stimulated with 0.1 micromol/I angiotensin II. PTH-related protein, c-myc and angiotensin II type qa receptor (AT1aR) messenger (m)RNA levels were measured by Northern blot, using total RNA extracted by phenol/chloroform. The effects of PTH-related protein(1-34)NH2 intravenous injections on arterial blood pressure and the heart rate were studied in anesthetized SHR and WKY rats. RESULTS: The Northern blots showed a significantly higher abundance of PTH-related protein mRNA in aortae of SHR versus WKY rats in the prehypertensive state but no significant difference in adult animals. In cultured aortic smooth muscle cells, angiotensin II induced a four- to sixfold increase in PTH-related protein mRNA levels in smooth muscle cells from normotensive animals, but failed to elicit a significant response in smooth muscle cells derived from SHR in either the prehypertensive or the hypertensive state. This lack of response to angiotensin II in SHR smooth muscle cells was not due to decreased expression or responsiveness of the AT1aR, since SHR smooth muscle cells had more AT1aR mRNA than Sprague-Dawley smooth muscle cells, and angiotensin II-induced activation of c-myc was faster and greater in smooth muscle cells derived from 4- or 18-week-old SHR than in Sprague-Dawley smooth muscle cells. In contrast, PTH-related protein(1-34)NH2 induced a long-lasting dose-dependent hypotensive and tachycardic response in both SHR and WKY rats, indicating that SHR retained responsiveness to the vasodilator. CONCLUSIONS: PTH-related protein gene expression in response to angiotensin II is impaired in SHR arteries. A deficiency in this potent local vasodilator may contribute to the development and/or maintenance of arterial hypertension in this model.     

Tissue-specific regulation of angiotensinogen gene expression in spontaneously hypertensive rats

Angiotensinogen is expressed in many tissues besides the liver. Recent studies have suggested that abnormalities in the regulation of angiotensinogen gene expression may be involved in the development of hypertension. However, little information is available concerning the functional significance of tissue angiotensinogen. In this study, we measured plasma angiotensinogen concentration by radioimmunoassay and examined the expression of tissue angiotensinogen by Northern blot analysis in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Although plasma angiotensinogen concentration in SHR was comparable to that in WKY at 6 weeks of age, it was increased significantly at 14 weeks of age in SHR and became higher than that in WKY. The levels of hepatic angiotensinogen mRNA were similar in SHR and WKY, and the levels of aortic, adrenal, and renal angiotensinogen mRNAs were lower in SHR than in WKY at both 6 and 14 weeks of age. Brain angiotensinogen expression in SHR was higher than in WKY at 6 weeks of age and was comparable to that in WKY at 14 weeks of age. On the other hand, cardiac and fat angiotensinogen mRNA levels were significantly increased at 14 weeks of age in SHR. These results demonstrate that the expression of tissue angiotensinogen is regulated differently in SHR and WKY and indicate that the development of hypertension is accompanied at least temporally with increases in plasma angiotensinogen concentration as well as cardiac and adipogenic angiotensinogen mRNA in SHR.     

Role of beta-adrenoceptor on renal interleukin-6 and tumor necrosis factor in spontaneously hypertensive rats

We have shown previously in the spontaneously hypertensive rat (SHR) kidney that interleukin-6 (IL-6) and tumor necrosis factor (TNF) mRNA levels were low under conditions of acute anaesthesia and surgical stress. The reasons for the suppression of IL-6 and TNF gene expression in the SHR were investigated by examining the influence of enhanced beta-adrenergic stimulation, high blood pressure, and renal function (renal blood flow, glomerular filtration rate, plasma creatinine levels) on renal IL-6 and TNF mRNAs. The experiments were performed by means of the following three studies; (1) SHR and Wistar rats at 4, 7, 9 week old were injected with lipopolysaccaride (LPS), and then a relationship between blood pressure levels and IL-6 and TNF mRNA levels were estimated, (2) isoproterenol and propranolol were administered into SHR and WKY rats, and the levels of IL-6 and TNF mRNA were compared, (3) under condition of anaesthesia and surgical stress, blood pressure and renal functions in SHR were measured, and then the relationships between these factors and IL-6 or TNF mRNA levels were analyzed. Renal IL-6 and TNF mRNAs in SHR remained low even though blood pressure increased with age and there was no significant correlation between IL-6 or TNF mRNA levels and values of blood pressure or renal function under anaesthesia and surgical stress. However, the inhibition of the IL-6 and TNF mRNAs in SHR was prevented by propranolol treatment. These results suggested that suppression of IL-6 and TNF mRNAs in the SHR kidney could be due to overactivity of beta-adrenergic influences which may importantly contribute to the development of hypertension.     

Isolation of an ovine genomic sequence containing the full-length angiotensin II type-1 receptor

We have isolated from a genomic library using PCR amplification an 1171 base sequence containing a putative ovine AT1-R protein coding sequence of 1080 bases. As expected the protein coding sequence is of greater than 99% homology to the partial protein coding sequence reported by Robillard et al, with only one base difference. Relative to other species, highest homology at the level of the cDNA protein coding sequence is to bovine (97.6%) and lowest homology to rat Type 1a (83.3%). The predicted protein amino acid sequence in turn encodes a protein with the properties of a seven alpha-helix transmembrane receptor (by TMPred) sharing closest homology (98.6%) to the bovine receptor and lowest to the rat Type 1a (90.2%). As expected from such a high degree of interspecies homology, amino acids identified by site-directed mutagenesis of the human or rat AT1A-R as involved in binding and action of AII are very highly conserved in the ovine sequence. In addition, both bovine and ovine AT1-R are known to exhibit lower affinity for DuP753 than human AT1-R, and in bovine AT1-R this has been suggested to coincide with the amino acid substitutions Ala->Thr (163) and Leu->Met (262) relative to the human sequence. Our ovine AT1-R cDNA sequence shares these same bovine substitutions.     

Angiotensin II type 2 receptor is upregulated in human heart with interstitial fibrosis, and cardiac fibroblasts are the major cell type for its expression

The expression pattern of angiotensin (Ang) II type 2 receptor (AT2-R) in the remodeling process of human left ventricles (LVs) remains poorly defined. We analyzed its expression at protein, mRNA, and cellular levels using autopsy, biopsy, or operation LV samples from patients with failing hearts caused by acute (AMI) or old (OMI) myocardial infarction and idiopathic dilated cardiomyopathy (DCM) and also examined functional biochemical responses of failing hearts to Ang II. In autopsy samples from the nonfailing heart group, the ratio of AT1-R and AT2-R was 59% and 41%, respectively. The expression of AT2-R was markedly increased in DCM hearts at protein (3.5-fold) and mRNA (3.1-fold) levels compared with AMI or OMI. AT1-R protein and mRNA levels in AMI hearts showed 1.5- and 2.1-fold increases, respectively, whereas in OMI and DCM hearts, AT1-R expression was significantly downregulated. AT1-R-mediated response in inositol phosphate production was significantly attenuated in LV homogenate from failing hearts compared with nonfailing hearts. AT2-R sites were highly localized in the interstitial region in either nonfailing or failing heart, whereas AT1-R was evenly distributed over myocardium at lower densities. Mitogen-activated protein kinase (MAPK) activation by Ang II was significantly decreased in fibroblast compartment from the failing hearts, and pretreatment with AT2-R antagonist caused an additional significant increase in Ang II-induced MAPK activity (36%). Cardiac hypertrophy suggested by atrial and brain natriuretic peptide levels was comparably increased in OMI and DCM, whereas accumulation of matrix proteins such as collagen type 1 and fibronectin was much more prominent in DCM than in OMI. These findings demonstrate that (1) AT2-R expression is upregulated in failing hearts, and fibroblasts present in the interstitial regions are the major cell type responsible for its expression, (2) AT2-R present in the fibroblasts exerts an inhibitory effect on Ang II-induced mitogen signals, and (3) AT1-R in atrial and LV tissues was downregulated during chronic heart failure, and AT1-R-mediated functional biochemical responsiveness was decreased in the failing hearts. Thus, the expression level of AT2-R is likely determined by the extent of interstitial fibrosis associated with heart failure, and the expression and function of AT1-R and AT2-R are differentially regulated in failing human hearts.     

Influence of dietary sodium restriction on angiotensin II receptors in rat adrenals

We studied the distribution of angiotensin II (AII) receptors type 1 (AT1) and type 2 (AT2) and the effects of a low sodium intake on these two subtypes of receptors in male rat adrenals. Binding studies on adrenal slices, on cell membranes and on cell suspensions were performed using [125I]AII and specific analogs for AT1 (Losartan) and AT2 (PD 123319) receptors. The distribution of AT1 was also studied by immunofluorescence. Complementary approaches were necessary to reach our goal. Indeed, by autoradiography on adrenal slices, [125I]AII was shown to bind to the zona glomerulosa (ZG) and to the medulla (M). When coincubated with [125I]AII, PD 123319 displaced [125I]AII from the medulla and from the ZG, indicating the presence of AT2 receptors in both zones. Losartan partially displaced [125I]AII from the ZG, indicating the presence of AT1 receptors in that zone. Furthermore, the labeling intensity of the medulla (AT2 receptors) was much stronger in adrenal sections from rats kept on a low sodium regimen than from controls. Immunofluorescence microscopy revealed that AT1 receptors were located mainly in the ZG of control rats. After sodium restriction, AT1 receptors appeared to be uniformly distributed within an enlarged ZG; furthermore AT1 receptor-positive cells were found to a limited degree in the zona fasciculata and possibly in the zona reticularis, and a greater number of these positive cells appeared in these zones under sodium restriction. Cell suspensions from rats fed a low sodium diet showed a 2.7- and 2.1-fold increase in total AII receptors in adrenal ZG and ZFR + M cells when compared with controls. Based on Losartan displacement, we calculated that [125I]AII bound to AT1 and to AT2 receptors was increased in both ZG and ZFR + M cell preparations under sodium restriction. Results of binding studies on cell membranes were also indicative of an increasing effect of sodium restriction on AT1 and AT2 receptors binding capacity. Furthermore, Northern blotting analysis revealed 3.0- and 2.5-fold increases in the level of AT1 receptor mRNA in the ZG and the ZFR + M of rats fed a low sodium diet as compared with those fed a normal diet. The low sodium intake resulted in a weaker increase (1.5-fold) in the level of AT2 receptor messenger RNA in the ZG, with no changes in the ZFR + M preparations. In conclusion, in this study complementary approaches were needed to determine the localization of AT1 and AT2 receptors in the rat adrenal, and to show the increasing effects of a low sodium regimen on the adrenal level of these receptors. Immunofluorescence studies revealed AT1 receptors mainly in the ZG and also in some cells of the inner adrenal cortex zones; in adrenals of rats kept on a low sodium diet the ZG was markedly enlarged, and an increased number of immunoreactive cells with AT1 receptors were observed throughout that zone; also more immunoreactive cells were present in the inner zones of the adrenal cortex. Furthermore in the adrenals of rats kept on a low sodium diet, we observed: 1) an increased number of AT1 and AT2 receptors in cell suspensions from the ZG, and in cell suspensions of the ZFR + M; 2) an increased level of AT1 and AT2 receptor mRNAs in the ZG; 3) an increased level of AT1 receptor mRNA, with no changes in the AT2 mRNA level in the ZFR + M. These results suggest a role for AT1 as well as for AT2 receptors in controlling adrenal function and differentiation under normal as well as under physiological stimulation of AII production following sodium restriction.     

Renal nerves and D1-dopamine receptor-mediated natriuresis

The resistance of the spontaneously hypertensive rat (SHR) kidney to the natriuretic effect of dopamine and D1 agonists may be due to increased renal nerve activity. Therefore, we compared the effects of the intrarenal arterial infusion of the D1 agonist, SKF 38383, into the denervated (DNX) kidney of saline-loaded-anesthetized SHR and its control, the Wistar-Kyoto (WKY) rat. In both WKY and SHR, DNX of the left kidney slightly decreased urine flow (UV) and absolute (UNaV) and fractional sodium excretion (FENa) in the innervated right kidney; neither vehicle nor D1 agonist infusion exerted any effect. In the left kidney, denervation increased UV, UNaV, and FENa to a similar degree in WKY and SHR (2-fold), without affecting renal blood flow, glomerular filtration rate, or blood pressure. In WKY but not in SHR, after DNX, the D1 agonist dose-dependently increased UV, UNaV, and FENa in the denervated kidney. We conclude that the decreased natriuretic effect of D1 agonists in the SHR is not due to increased renal nerve activity. These data support our previous studies implicating a defect of the D1 receptor or its regulation in the kidney in genetic hypertension.     

Renal vascular responses to angiotensin II in conscious spontaneously hypertensive and normotensive rats

It has been postulated that exaggerated renal sensitivity to angiotensin II may be involved in the development and maintenance of hypertension in the spontaneously hypertensive rat (SHR). The purpose of this study was to compare the renal vascular responses to short-term angiotensin II infusions (50 ng/kg/min, i.v.) in conscious SHRs and Wistar-Kyoto (WKY) rats. Renal cortical blood flow was measured in conscious rats by using quantitative renal perfusion imaging by magnetic resonance, and blood pressure was measured by an indwelling carotid catheter attached to a digital blood pressure analyzer. Renal vascular responses to angiotensin II were similar in control SHRs and WKY rats. Pretreatment with captopril to block endogenous production of angiotensin II significantly augmented the renal vascular response to exogenous angiotensin II in the SHRs but not in the WKY rats. The renal vascular responses to angiotensin II were significantly greater in captopril-pretreated SHRs than in WKY rats (cortical blood flow decreased by 1.66 +/- 0.13 ml/min/g cortex in WKY rats compared with 2.15 +/- 0.14 ml/min/g cortex in SHR; cortical vascular resistance increased by 10.5 +/- 1.4 mm Hg/ml/min/g cortex in WKY rats compared with 15.6 +/- 1.7 mm Hg/ml/min/g cortex in SHRs). Responses to angiotensin II were completely blocked in both strains by pretreatment with the angiotensin II AT1-receptor antagonist losartan. Results from this study in conscious rats confirm previous findings in anesthetized rats that (a) the short-term pressor and renal vascular responses to angiotensin II are mediated by the AT1 receptor in both SHRs and WKY rats, and (b) the renal vascular responses to angiotensin II are enhanced in SHRs compared with WKY rats when endogenous production of angiotensin II is inhibited by captopril pretreatment.     

Endogenous dopamine regulates phosphate reabsorption but not NaK-ATPase in spontaneously hypertensive rat kidneys

Dopamine's modulatory actions on signal transduction in the spontaneously hypertensive rat (SHR) proximal tubule are blunted; therefore, it was predicted that dopamine does not regulate phosphate (Pi) reabsorption in SHR. To test this hypothesis, dopamine production was inhibited with carbidopa (10 mg/kg ip) 18 h before and during clearance measurements of chronically denervated SHR and Wistar-Kyoto (WKY) rat kidneys. Dopamine excretion decreased 80% from SHR and 85% from WKY rats. Pi excretion decreased 60 to 67%. Plasma Pi and calcium, inulin clearance, and Na excretion did not change. Citrate excretion, which reflects proton secretion by proximal tubules, decreased 72% from WKY rats. Citrate excretion was significantly lower from SHR (5 +/- 10 pmol/min) than from WKY rats (73 +/- 11 pmol/min) and was not altered by carbidopa. Carbidopa, injected 18 and 1 h before kidneys were collected, increased NaK-ATPase in cortical basolateral membranes from WKY rats (27%) but not in membranes from SHR. After the incubation of renal cortical minceates for 15 min with L-DOPA (10(-5) M), there was no change in brush border membrane vesicle uptake of 32Pi, (3H)glucose, or (14C)citrate. Incubation with carbidopa (10(-4) M) increased 32Pi uptake by 11% (P < 0.001) and (3H)glucose uptake by 9% (P = 0.02). (14C)citrate uptake was not increased by carbidopa but was higher in SHR (977 +/- 2 pmol/10 s.mg) than in WKY rats (823 +/- 43 pmol/10 s.mg; P = 0.04). In summary, dopamine produced in WKY rat and SHR proximal tubules decreases Pi uptake by using a signaling process distinct from those that regulate NaK-ATPase and the antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct regulation of hypothalamic corticotropin-releasing-hormone neurons by angiotensin II

The possible role of angiotensin II (AII) in the control of the hypothalamic-pituitary-adrenal (HPA) axis was studied in the rat by examining the regulation and cellular localization of AII receptors in the paraventricular nucleus (PVN) of the hypothalamus and the effect of AII on corticotropin-releasing hormone (CRH) and vasopressin (VP) mRNA levels. In situ hybridization studies using cRNA 35S-labelled probes showed that while type 1 AII receptor (AT1) mRNA levels were high in the periventricular and parvicellular pars of the PVN, only very low levels were present in the magnocellular pars. A similar distribution of AT1 receptor binding in the periventricular, parvicellular and magnocellular divisions of the PVN was observed in autoradiographic studies in hypothalamic sections labelled with 125I[Sar1,Ile8]AII. In addition, AII receptor binding was clearly evident in nerve fibers adjacent to the PVN. Double-labelling hybridization using digoxigenin-labelled CRH, VP and oxytocin probes and 35S-labelled AT1 receptor cRNA probes showed AT1 receptor mRNA in cells stained for CRH mRNA, but not in VP or oxytocin cells. Four hours after a single intracerebroventricular (i.c.v.) injection of 50 ng AII in conscious rats, CRH mRNA levels in the PVN were increased by 43%, similar to the increases observed following acute stress by intraperitoneal (i.p.) injection of 1.5 M NaCl (76%). On the other hand, while i.p. hypertonic saline injection increased VP mRNA levels by 29% in the PVN and by 32% in the supraoptic nucleus, i.c.v. AII injection had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Up-regulation of angiotensin type 2 receptor mRNA by angiotensin II in rat cortical cells

The present experiment demonstrates that the exposure of angiotensin II (AII) produced an up-regulation of the AT2 receptor mRNA level in rat cortical cells. AII (10(-9)-10(-5) M) exerted a marked increase of AT2 receptor mRNA in a dose-dependent manner. The maximum increase was observed at 3 hr of AII stimulation and lasted 3 hr. The up-regulation of AT2 receptor mRNA was antagonized by PD123319, an AT2 receptor antagonist, but not by SC-52458, an AT1 receptor antagonist, thus suggesting that the increase in AT2 receptor mRNA is mediated via AT2 receptor. This increase is blocked by serine/threonine phosphatase inhibitor okadaic acid, but not by the phosphotyrosine phosphatase inhibitor sodium vanadate, thus suggesting the involvement of serine/threonine phosphatase in this process. Protein kinase C inhibitor, H-7 and calphostin C, did not inhibit the AII-induced up-regulation significantly. In addition, calcium ionophore, A23187 had no effect. These findings suggest that the AT2 receptor mRNA expression by AII is regulated by the activity of serine/threonine phosphatase in the cortical neurons. This observation is also the first example concerning the regulation of AT2 receptor within the brain.