Using GIS Mapping to Target Public Health Interventions: Examining Birth Outcomes Across GIS Techniques.

With advances in spatial analysis techniques, there has been a trend in recent public health research to assess the contribution of area-level factors to health disparity for a number of outcomes, including births. Although it is widely accepted that health disparity is best addressed by targeted, evidence-based and data-driven community efforts, and despite national and local focus in the U.S. to reduce infant mortality and improve maternal-child health, there is little work exploring how choice of scale and specific GIS visualization technique may alter the perception of analyses focused on health disparity in birth outcomes. Retrospective cohort study. Spatial analysis of individual-level vital records data for low birthweight and preterm births born to black women from 2007 to 2012 in one mid-sized Midwest city using different geographic information systems (GIS) visualization techniques [geocoded address records were aggregated at two levels of scale and additionally mapped using kernel density estimation (KDE)]. GIS analyses in this study support our hypothesis that choice of geographic scale (neighborhood or census tract) for aggregated birth data can alter programmatic decision-making. Results indicate that the relative merits of aggregated visualization or the use of KDE technique depend on the scale of intervention. The KDE map proved useful in targeting specific areas for interventions in cities with smaller populations and larger census tracts, where they allow for greater specificity in identifying intervention areas. When public health programmers seek to inform intervention placement in highly populated areas, however, aggregated data at the census tract level may be preferred, since it requires lower investments in terms of time and cartographic skill and, unlike neighborhood, census tracts are standardized in that they become smaller as the population density of an area increases.

Nutritional value of soybean meal produced from conventional, high-protein, or low-oligosaccharide varieties of soybeans and fed to broiler chicks.

Three experiments were conducted to determine the feeding value to broiler chicks of soybean meal (SBM) produced from high-protein (SBM-HP), low-oligosaccharide (SBM-LO), or conventional (SBM-CV) varieties of soybeans. The 3 SBM contained 54.9, 53.6, and 47.5% CP, respectively. The standardized digestibility (SDD) of amino acids (AA) in the 3 ingredients was measured using a precision-fed rooster assay with cecectomized Single Comb White Leghorn roosters. Results indicated that the SDD of AA was not different among the 3 sources of SBM, with the exception that the SDD of Lys in SBM-HP tended to be greater (P = 0.07) than that in SBM-CV. In the second experiment, a precision-fed rooster assay was used to measure the concentration of TME(n) in each source of SBM. Results indicated that the TME(n) in SBM-HP was greater (P < 0.001) than those in SBM-LO and SBM-CV (3,104 vs. 2,984 and 2,963 kcal/kg of DM). A 14-d growth performance experiment was also conducted using 120 Ross 308 male commercial broiler chicks (mean initial BW = 102.6 g) that were allotted to a completely randomized design. There were 5 chicks/pen and 8 replicate pens/diet. Three corn- and SBM-based diets were formulated based on the data for digestible AA and TME(n) that were measured in the previous experiments. Each source of SBM was used in 1 diet, but because of the greater concentrations of digestible AA in SBM-HP and SBM-LO than in SBM-CV, the inclusion of SBM-HP and SBM-LO were 31.21 and 32.60%, respectively, whereas an inclusion of 38.21% SBM-CV was used. There were no differences among the 3 diets for BW gain or feed efficiency, which indicated that the reduced inclusion rates of SBM-HP and SBM-LO compared with SBM-CV were not detrimental to broiler chick growth performance. It was concluded that, compared with SBM-CV, SBM-HP and SBM-LO are needed in lower concentrations in diets fed to broiler chicks because these 2 sources of SBM have a greater nutritional value than does SBM-CV.

Phosphorylation of the angiotensin II (AT1A) receptor carboxyl terminus: a role in receptor endocytosis.

The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337 Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar(1)Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.

Left ventricular mural thrombus in a patient with thrombocytosis and agnogenic myeloid metaplasia.

The two factors responsible for the development of left ventricular mural thrombi are endocardial injury secondary to old or recent anterior myocardial infarction and left ventricular dysfunction. Endothelial damage also is thought to be the initial event in the development of arterial thrombi. However, arterial thrombi may develop in patients with thrombocytosis secondary to myeloproliferative disorders in the absence of endothelial injury. A patient had thrombocytosis secondary to agnogenic myeloid metaplasia and a left ventricular mural thrombus developed in the absence of clinical or laboratory evidence of old or coronary angiogram and left ventricular function. To our knowledge, this is the first such case reported.

Evidence for a role of an intracardiac renin-angiotensin system in normal and failing hearts.

Although substantial evidence of a cardiac RAS has been obtained in the past decade, a number of important questions remain unanswered. These include identification and localization of the cell types responsible for production of the system's components as well as the regulation of synthesis, storage, and secretion pathways for each component. Future studies, which will utilize tools of molecular biology that have become recently available (for example, transgenic animal models), renin inhibitors, angiotensin receptor antagonists, and bradykinin antagonists, will help to elucidate specific roles of the cardiac RAS in normal and failing hearts.

Molecular signalling mechanisms controlling growth and function of cardiac fibroblasts.

Cardiac fibroblasts appear to be important in producing and maintaining the extracellular matrix (ECM) of the heart. The abnormal proliferation of cardiac fibroblasts and deposition of the ECM protein, collagen, associated with hypertension and myocardial infarction, may adversely affect the performance of the heart. Several groups of factors affect collagen gene expression and/or growth of cardiac fibroblasts. Angiotensin II, aldosterone and endothelins play a central role in the remodeling of the ECM in hypertension, and decrease collagenase activity and/or increase collagen synthesis in cultured cells. Regulatory peptides that are generally elevated at sites of injury, such as TGF-beta 1 and PDGF, increase collagen synthesis and/or stimulate mitogenesis. Mechanical stretch enhances collagen expression and cell proliferation, responses which could in part be due to integrin activation. Cytokines may stimulate or inhibit cell growth, the latter through prostaglandin formation. Angiotensin II is a principal determinant in vivo of cardiac fibroplasia and synthesis of the ECM proteins, collagen and fibronectin. Cardiac fibroblasts possess G-protein-coupled AT1 receptors for angiotensin II that couple to activation of multiple signalling pathways, including: phospholipase C-beta, with the subsequent release of Ca2+ from intracellular stores and activation of protein kinase C, mitogen-activated protein kinases, tyrosine kinases, phospholipase D, phosphatidic acid formation, and the STAT family of transcription factors. Cardiac fibroblasts respond to angiotensin II with hyperplastic/hypertrophic growth, and increased expression of collagen, fibronectin, and integrins. The mechanisms by which the AT1 receptor activates multiple signalling pathways are not known, although the receptor might interact at some level with both integrins and cytokine receptors. Different signalling pathways of the AT1 receptor may subserve different cellular responses, such as mitogenesis, ECM synthesis, or an inflammatory/stress response. Crosstalk among the signalling pathways of the AT1 receptor, and those of G-protein, cytokine, and growth-factor receptors, may determine the ultimate response of the cell.

Angiotensin-II-binding sites on hepatocyte nuclei.

Angiotensin-II (Ang II) stimulates gene expression and cell growth in several cell types. Studies that have shown localization of Ang II to nuclei of myocytes and hepatic nuclear Ang II binding suggest that these actions may be mediated by nuclear receptors. We characterized Ang II binding to rat liver nuclei, which were free of plasma membrane based on enzyme analysis and electron microscopy. At 18 C, specific binding of 0.1-0.3 nM [125I]Ang II to nuclei and nuclear envelopes reached equilibrium by 2 h. Unlabeled Ang II inhibited [125I]Ang II binding to nuclei with an IC50 of 1.4 +/- 0.2 nM (+/- SE; n = 6). In half of the nuclear preparations, a lower affinity site (IC50, 50.4 +/- 23.6 nM), which accounted for 7-32% of specific Ang II binding, was detected by Scatchard analysis. Results similar to these were obtained with nuclear envelopes. Other Ang peptides competed for binding in the rank order: Ang III (IC50, 2.1 nM) greater than Ang I (IC50, 33) greater than [Des-Phe8]Ang II (IC50, 362) greater than [Des-Asp1-Des-Arg2]Ang II (IC50, 736). Losartan (DuP 753), an AT1 receptor antagonist, inhibited binding (IC50, 10.9 +/- 0.9 nM), whereas the AT2 receptor antagonist PD123177 did not. The pH optimum for binding to nuclear envelopes was 7, with binding more sensitive to low (5 and 6) than high (8 and 9) pH. Nonhydrolyzable GTP analogs accelerated displacement of bound [125I]Ang II by 10(-5) M Ang II. Differences were noted in pH sensitivity, time course, binding affinity for Ang I, II, and III, and rate of dissociation between nuclei or nuclear envelopes and plasma membrane Ang II binding. These results suggest that nuclear envelopes have a G-protein-coupled Ang II-binding site, which belongs to the AT1 class of Ang II receptors, with properties different from the plasma membrane receptor.

Role of type 1 and type 2 angiotensin receptors in angiotensin II-induced cardiomyocyte hypertrophy.

We compared the ability of angiotensin II (Ang II) to induce hypertrophy of neonatal rat ventricular myocytes with that of endothelin-1. Over 72 hours, Ang II (1 mumol/L) increased the ratio of protein to DNA by less than 10%, whereas endothelin-1 (100 nmol/L) produced a 28% increase. The growth effects of either agonist occurred independently of chronotropic actions. Radioligand binding studies showed that myocytes have nearly 300-fold more receptors for endothelin-1 than Ang II, and type 1 and type 2 Ang II receptor subtypes (AT1 and AT2) are present in near equal proportions. Cotreatment with a 10-fold molar excess of AT2 antagonists (PD 123177 or CGP 42112) for 72 hours augmented the Ang II-induced increase in the protein-to-DNA ratio to levels nearly as high (23%) as those with endothelin-1 (28%). AT2 antagonists enhanced Ang II stimulation of protein synthesis, as indexed by [3H]leucine incorporation, whereas an AT1 antagonist blocked Ang II-induced incorporation. An AT2 antagonist also prevented Ang II-induced protein degradation. In conclusion, Ang II-induced myocyte growth is tempered because of low AT1 levels and an antigrowth effect of AT2. These findings have potential clinical significance in that regression of hypertension-induced cardiac hypertrophy by AT1 antagonists may be in part due to an unopposed antigrowth effect of Ang II mediated via AT2.

Angiotensin II-stimulated induction of sis-inducing factor is mediated by pertussis toxin-insensitive G(q) proteins in cardiac myocytes.

The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is stimulated by angiotensin II (Ang II) via the type 1 receptor after acute pressure overload in the heart. The purpose of this study was to determine whether activation of the JAK-STAT pathway by Ang II is dependent on G proteins. Ang II (100 nmol/L for 120 minutes) caused formation of sis-inducing factor (SIF) complexes and tyrosine phosphorylation of STAT proteins in neonatal rat ventricular myocytes. The percentage of change in Ang II-stimulated SIF induction was not affected by pertussis toxin (PTX) or GP antagonist-2A, compounds that inhibit activation of G(i) and G(o) proteins. In contrast, GP antagonist-2A, a peptide that selectively inhibits activation of G(q) proteins, completely abolished Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation. Pretreatment of cardiac myocytes with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) activity, decreased Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation in a dose-dependent manner. Chelation of intracellular Ca(2+) with BAPTA-AM did not alter Ang II-stimulated SIF induction. In contrast, pretreatment of cardiac myocytes with Ro-31-8220, a potent and specific inhibitor of protein kinase C (PKC), decreased Ang II-stimulated SIF induction in a dose-dependent manner. Ang II-stimulated SIF induction was abolished in cardiac myocytes after downregulation of PKC by treatment with PMA. From these data, we conclude that Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation is mediated by PTX-insensitive G proteins through a G(q)-PLC-PKC-mediated pathway in neonatal rat ventricular myocytes.

Alpha-thrombin stimulates sis-inducing factor-A DNA binding activity in rat aortic smooth muscle cells.

Exposure of rat aortic vascular smooth muscle cells to alpha-thrombin resulted in the appearance of sis-inducing factor-A (SIF-A)-like DNA binding activity. This response to alpha-thrombin was delayed (detectable at 1 hour) compared with the rapid activation (15 to 30 minutes) by platelet-derived growth factor and the cytokine interleukin-6. alpha-Thrombin-induced SIF-A was sensitive to treatment with the tyrosine kinase inhibitor genistein. The thrombin inhibitor hirudin prevented the alpha-thrombin-mediated SIF-A induction. Cycloheximide had no effect on the ability of alpha-thrombin to induce SIF-A, suggesting that induction does not require new protein synthesis. alpha-Thrombin-induced SIF-A could be resolved into two additional subcomplexes termed SIF-A, and SIF-As. Antibodies against Stat3 reacted with alpha-thrombin-induced SIF-Af, suggesting that Stat3 or a related protein is present in this subcomplex. Induction of SIF-A DNA binding activity may contribute to alpha-thrombin-mediated cellular responses, including wound healing, cell proliferation, and inflammation in the vasculature.

Ischemic cardiovascular complications concurrent with administration of captopril. A clinical note.

Administration of potent vasodepressor agents such as the angiotensin converting enzyme inhibitor, captopril, may precipitate myocardial ischemic events in patients with coronary artery disease, particularly if this treatment is preceded by a discontinuation of beta-blocking drugs such as propranolol. In one case studied, a patient experienced three episodes of angina pectoris under these conditions; in another, acute anterior myocardial infarction was suspect.

Cardiotrophin-1 increases angiotensinogen mRNA in rat cardiac myocytes through STAT3 : an autocrine loop for hypertrophy.

-Cardiotrophin-1, an interleukin-6-related cytokine, stimulates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and induces cardiac myocyte hypertrophy. In this study, we demonstrate that cardiotrophin-1 induces cardiac myocyte hypertrophy in part by upregulation of a local renin-angiotensin system through the JAK/STAT pathway. We found that cardiotrophin-1 increased angiotensinogen mRNA expression in cardiac myocytes via STAT3 activation. Tyrosine phosphorylation of STAT3 by cardiotrophin-1 treatment resulted in STAT3 homodimer binding to the St-domain in the angiotensinogen gene promoter, which lead to promoter activation in a transient transfection assay. Cardiotrophin-1-induced STAT3 tyrosine phosphorylation and binding to the St-domain were suppressed by AG490, a specific JAK2 inhibitor, which also attenuated cardiotrophin-1-stimulated angiotensinogen promoter activity. Cardiotrophin-1 did not activate the angiotensinogen gene promoter that contained a substitution mutation within the St-domain. Finally, losartan, an angiotensin II type 1 receptor antagonist, significantly attenuated cardiotrophin-1-induced hypertrophy of neonatal rat cardiac myocytes. Angiotensin II is known to induce cardiac myocyte hypertrophy by activating the G-protein-coupled angiotensin II type 1 receptor. Our results suggest that upregulation of angiotensinogen and angiotensin II production contribute to cardiotrophin-1-induced cardiac myocyte hypertrophy and emphasize an important interaction between G-protein-coupled and cytokine receptors.

Captopril modifies gene expression in hypertrophied and failing hearts of aged spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in alpha-myosin heavy chain (alpha-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-alpha1(III) collagen, and transforming growth factor beta1 (TGF-beta1) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normotensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in alpha-MHC, ANF, pro-alpha1(III) collagen, and TGF-beta1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-beta1 mRNA by 50% (P<.05) and elevated levels of alpha-MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding alpha-MHC, ANF, TGF-beta1, pro-alpha1(III) collagen, and renin-angiotensin system components.

Sensitive bioassay for the detection and quantification of angiotensin II in tissue culture medium.

We have developed a sensitive, high-throughput bioassay to quantify angiotensin II in culture medium. Using Chinese hamster ovary cells that stably express a transfected angiotensin II receptor as target cells, we demonstrated that an agonist-stimulated myelin basic protein kinase response can be used as a basis of quantitative bioassay for angiotensin II. The assay permits detection of as little as 10 pg of angiotensin II in 1 mL of medium and offers an excellent alternative to HPLC and radioimmunoassay. This approach may also be applicable for quantification of other peptide hormones or growth factors produced by cell cultures.

Paracrine actions of cardiac fibroblasts on cardiomyocytes: implications for the cardiac renin-angiotensin system.

Conditioned medium of cardiac fibroblasts was found to induce protein synthesis and signal transduction events rapidly, and to increase angiotensinogen messenger RNA (mRNA) levels in neonatal rat ventricular myocytes. Within 4 hours, fibroblast-conditioned medium (FCM) stimulated protein synthesis in cardiac myocytes, independent of the contractile state, and induced marked increases within 24 hours in total protein content. Endothelin- released by cardiac fibroblasts was not responsible for the stimulation of protein synthesis. FCM rapidly activated signal transduction events in cardiac myocytes associated with hypertrophic stimuli, including: (1) increased tyrosine phosphorylation of several prominent protein bands; (2) mitogen-activated protein kinases (ERK 1 and ERK 2); and (3) protein kinase C. Finally, FCM caused an increase at 8 hours in angiotensinogen mRNA levels of cardiac myocytes, whereas no effect was observed on mRNA levels for renin or the type 1 angiotensin II receptor (AT1). Our results suggest that cardiac fibroblasts produce a factor that rapidly activates cardiac myocyte growth through a membrane receptor that couples to conventional signal transduction pathways.

Enalapril maleate versus captopril. A comparison of the hormonal and antihypertensive effects.

24 hypertensive patients were randomised into 2 groups to compare the antihypertensive effects of enalapril and captopril over a 10-week period. In the hydrochlorothiazide run-in period, blood pressure was reduced from 171 +/- 4/109 +/- 1mm Hg to 160 +/- 4/103 +/- 1mm Hg (p less than 0.05). Angiotensin-converting enzyme (ACE) inhibition decreased blood pressure to 132 +/- 3/87 +/- 2mm Hg. Captopril decreased diastolic blood pressure significantly more after 3 hours than enalapril (-24 versus -17mm Hg, p less than 0.05). After 10 weeks of therapy, this antihypertensive response was maintained at 134 +/- 3/83 +/- 1mm Hg. There was no difference between the captopril and enalapril treated groups. Acute and chronic responses of plasma renin activity, plasma aldosterone and ACE were determined. There was an acute positive correlation between the rise in plasma renin activity and the fall in blood pressures with captopril but not with enalapril. With chronic treatment there was no difference in the ability of either of the 2 drugs to reduce blood pressure, inhibit ACE, reduce aldosterone or stimulate plasma renin activity.

Long-term survival in typical thanatophoric dysplasia type 1.

Thanatophoric dysplasia (TD), a severe skeletal dysplasia, is virtually always lethal neonatally, although a few previous reports have documented survival up to 4.75 years. We present a patient with survival beyond age 9 years and summarize his growth, development and medical history. The common Arg248Cys mutation in the extracellular region of fibroblast growth factor receptor 3 (FGFR3) was identified, eliminating the possibility that his long-term survival is attributable to an atypical mutation. This patient (and at least one other TD long-term survivor) have a rare skin disorder, acanthosis nigricans, which also occurs in Crouzon syndrome when caused by a FGFR3 mutation. Therefore, any molecular model of the origin of acanthosis nigricans secondary to FGFR3 mutations must account for the association of diverse mutations and these cutaneous effects.

Angiotensin II stimulation of left ventricular hypertrophy in adult rat heart. Mediation by the AT1 receptor.

Angiotensin II (AII) has been reported to have direct hypertrophic actions in mammalian and avian myocardium. In this study we determined whether AII had receptor-mediated effects on stimulating cardiac hypertrophy independent of mechanical stimuli (increased cardiac afterload) in adult rats. Angiotensin II was infused into Sprague-Dawley rats for 7 and 14 days. Following this infusion, left ventricular mass indexed to body weight (LV/BW) increased 18.6 and 17.3%, respectively, compared with control (saline infused) rats. Administration of the nonpeptide AII receptor antagonist Dup 753 prevented the increase in left ventricular hypertrophy. Blockade of converting enzyme with enalapril maleate and treatment with a vasodilator had no effect on the AII-induced hypertrophy. In this animal model, the cardiac hypertrophy appeared to be independent of cardiac afterload, because normalization of blood pressure with hydralazine did not prevent the AII-induced hypertrophy. These in vivo studies indicate that AII-induced cardiac hypertrophy is mediated through AT1 angiotensin receptors.

Protein kinase C in angiotensin II signalling in neonatal rat cardiac fibroblasts. Role in the mitogenic response.

The mitogenic effects of angiotensin II on cardiac fibroblasts are mediated by membrane receptors that are classified as AT1. These receptors are prototypical of the seven transmembrane group of receptors that couple, via G-proteins, to phospholipase C, thereby generating the endogenous activator of protein kinase C, diacylglycerol. Phorbol ester activators of protein kinase C exhibit growth-promoting effects in many cell types, suggesting that this enzyme may be responsible for the growth effects of angiotensin II on cardiac fibroblasts. Both kinase assays and Western analysis demonstrated that angiotensin II does induce translocation of protein kinase C to the detergent-soluble, membrane compartment of cardiac fibroblasts. Although translocation is commonly interpreted to mean activation of protein kinase C, in situ assays on permeabilized cells failed to detect increased enzymatic activity in response to angiotensin II. Nonetheless, this hormone did activate protein kinase C, leading to activation of mitogen-activated protein (MAP) kinases. However, a PKC-independent pathway for activation of MAP kinases exists as well. Downregulation and inhibitor studies indicated that protein kinase C is not critically involved in angiotensin II-induced thymidine incorporation into DNA. Furthermore, phorbol esters that activate protein kinase C do not elicit a mitogenic response in these cells. In conclusion, the mitogenic effects of angiotensin II on cardiac fibroblasts are not simply explained by activation of protein kinase C.

Amplification of angiotensin II signaling in cardiac myocytes by adenovirus-mediated overexpression of the AT1 receptor.

Low levels of AT1 receptor can make studying the growth-related signal transduction events mediated by this angiotensin II receptor in cardiac myocytes technically difficult. The purpose of the present study was to establish whether an adenovirus expression system could be used to increase the number of plasma membrane AT1 receptors in neonatal rat ventricular myocytes, thereby amplifying the signaling pathways activated by this receptor. Cardiac myocytes infected with adenovirus expressing the AT1 receptor exhibited increased ligand binding. The overexpressed receptor appeared to function like the endogenous receptor, in regard to agonist-induced internalization, as well as coupling to MAPK activation and protein tyrosine phosphorylation events. In addition, adenovirus-mediated overexpression of the AT1 receptor resulted in the amplification of angiotensin II intracellular signaling. In conclusion, adenovirus-mediated overexpression of angiotensin II receptors appears to be a useful strategy for studying the signal transduction events activated by this hormone in cardiac myocytes and for unraveling the molecular means by which this receptor type couples to a hypertrophic pattern of growth and gene expression.