Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina.

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Selective activation of angiotensin AT2 receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, which progressively leads to right-heart failure and death. A dys-regulated renin angiotensin system (RAS) has been implicated in the development and progression of PH. However, the role of the angiotensin AT2 receptor in PH has not been fully elucidated. We have taken advantage of a recently identified non-peptide AT2 receptor agonist, Compound 21 (C21), to investigate its effects on the well-established monocrotaline (MCT) rat model of PH. EXPERIMENTAL APPROACH: A single s.c. injection of MCT (50 mg·kg(-1) ) was used to induce PH in 8-week-old male Sprague Dawley rats. After 2 weeks of MCT administration, a subset of animals began receiving either 0.03 mg·kg(-1) C21, 3 mg·kg(-1) PD-123319 or 0.5 mg·kg(-1) A779 for an additional 2 weeks, after which right ventricular haemodynamic parameters were measured and tissues were collected for gene expression and histological analyses. KEY RESULTS: Initiation of C21 treatment significantly attenuated much of the pathophysiology associated with MCT-induced PH. Most notably, C21 reversed pulmonary fibrosis and prevented right ventricular fibrosis. These beneficial effects were associated with improvement in right heart function, decreased pulmonary vessel wall thickness, reduced pro-inflammatory cytokines and favourable modulation of the lung RAS. Conversely, co-administration of the AT2 receptor antagonist, PD-123319, or the Mas antagonist, A779, abolished the protective actions of C21. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that the AT2 receptor agonist, C21, may hold promise for patients with PH.

Gene therapy to control hypertension: current studies and future perspectives.

Hypertension is a complex pathophysiological state that leads to serious complications, including heart failure, coronary artery disease, and abnormal renal function. While traditional therapies can be effective in controlling the effects of hypertension, they offer no long-term cure and often lead to patient noncompliance, thereby diminishing their effectiveness. These reasons, coupled with the recent developments in gene transfer and somatic cell gene delivery, led researchers to explore alternative options that can produce long-term control of hypertension. Gene therapy offers the potential to yield lasting antihypertensive effects by influencing the genes associated with hypertension. In this review, we will discuss the merits of sense versus antisense strategies in controlling hypertension. We also discuss the advantages and disadvantages of both viral and nonviral vector types for the systemic delivery of genes for hypertension research. Results of our research group on the retrovirus-mediated delivery of the angiotensin type I receptor-antisense on the prevention of hypertension and related cardiovascular pathophysiology will be summarized. Finally, we discuss the future of this gene therapy approach in the reversal and long-term control of hypertension.

The future of hypertension therapy: sense, antisense, or nonsense?

Hypertension is a debilitating disease with significant socioeconomic and emotional impact. Despite recent success in the development of traditional pharmacotherapy for the management of hypertension, the incidence of this disease is on the rise and has reached epidemic proportions by all estimates. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau, and novel approaches for the treatment and control of hypertension must be explored. We have begun to investigate the possibility of treating and/or curing hypertension by using genetic means. In this review, we will provide evidence in favor of targeting of the renin-angiotensin system by antisense gene therapy as an effective strategy for the lifelong prevention of hypertension in the spontaneously hypertensive rat model. In addition, we will discuss the properties of an ideal vector for the systemic delivery of genes and the potential experimental hurdles that must be overcome to take this innovative approach to the next level of evaluation.

Genetic targeting for cardiovascular therapeutics: are we near the summit or just beginning the climb?

This article is based on an Experimental Biology symposium held in April 2001 and presents the current status of gene therapy for cardiovascular diseases in experimental studies and clinical trials. Evidence for the use of gene therapy to limit neointimal hyperplasia and confer myocardial protection was presented, and it was found that augmenting local nitric oxide (NO) production using gene transfer (GT) of NO synthase or interruption of cell cycle progression through a genetic transfer of cell cycle regulatory genes limited vascular smooth muscle hyperplasia in animal models and infra-inguinal bypass patients. The results of application of vascular endothelial growth factor (VEGF) GT strategies for therapeutic angiogenesis in critical limb and myocardial ischemia in pilot clinical trials was reviewed. In addition, experimental evidence was presented that genetic manipulation of peptide systems (i.e., the renin-angiotensin II system and the kallikrein-kinin system) was effective in the treatment of systemic cardiovascular diseases such as hypertension, heart failure, and renal failure. Although, as of yet, there are no well controlled human trials proving the clinical benefits of gene therapy for cardiovascular diseases, the data presented here in animal models and in human subjects show that genetic targeting is a promising and encouraging modality, not only for the treatment and long-term control of cardiovascular diseases, but for their prevention as well.

Attenuation of hypertension by systemic delivery of retroviral vector containing type I angiotensin II receptor antisense cDNA.

Despite recent strides in the traditional pharmacological therapies in the control and management of hypertension, a successful prevention and cure for this disease by conventional drug strategy remain at a standstill. We have begun to investigate the conceptual possibility of the use of gene therapy in the control of hypertension. In this article we describe an experimental protocol that provides proof of the principle that antisense (AS) inhibition of Type I angiotensin II receptor (AT(1)-R) could prevent development of hypertension on a long-term basis. A retrovirus-based vector has been used to deliver AT(1)R-AS with high efficiency that attenuates development of high blood pressure and hypertension-associated cardiac and vascular pathophysiology in the spontaneously hypertensive rat.

Angiotensin I-converting enzyme antisense gene therapy causes permanent antihypertensive effects in the SHR.

The renin-angiotensin system plays a critical role in the control of blood pressure (BP), and its hyperactivity is associated with the development and maintenance of hypertension. Although traditional pharmacological therapies targeted toward the inhibition of the renin-angiotensin system are effective in the control of this disease, they pose significant limitations. We used an antisense gene delivery strategy to circumvent these limitations and established that a single intracardiac administration of angiotensin type 1 receptor antisense (AT(1)R-AS) causes permanent prevention of hypertension in the spontaneously hypertensive rat (SHR), an animal model of primary human hypertension. Our objectives in this study were 2-fold: to determine (1) whether the targeting of angiotensin I-converting enzyme (ACE) mRNA by a similar antisense strategy would prevent the SHR from developing hypertension and (2) whether the antihypertensive phenotype is transmitted to the offspring from the antisense-treated parents. Administration of a retroviral vector containing ACE antisense (LNSV-ACE-AS) caused a modest yet significant attenuation of high BP ( approximately 15+/-2 mm Hg) exclusively in the SHR. This was associated with a complete prevention of cardiac and renovascular pathophysiological alterations that are characteristic of hypertension. Like their parents, the F(1) generation offspring of the LNSV-ACE-AS-treated SHR expressed lower BP, decreased cardiac hypertrophy, and normalization of renal arterial excitation-coupling compared with offspring derived from the LNSV-ACE-tS (truncated sense)-treated SHR. In addition, the endothelial dysfunction commonly observed in the SHR renal arterioles was significantly prevented in both parents and offspring of the LNSV-ACE-AS-treated SHR. Polymerase chain reaction followed by Southern analysis revealed that the ACE-AS was integrated into the SHR genome and transmitted to the offspring. These observations suggest that transmission of ACE-AS by retroviral vector may be responsible for the transference of normotensive phenotypes in the SHR offspring.

Angiotensin I-converting enzyme antisense prevents altered renal vascular reactivity, but not high blood pressure, in spontaneously hypertensive rats.

The renin-angiotensin system plays a critical role in the control of blood pressure, and its hyperactivity is associated with the development of human primary hypertension. Because low-dose angiotensin I-converting enzyme (ACE) inhibitors cause small reductions in blood pressure that are associated with the complete reversal of altered vascular pathophysiology, our objective in this study was to determine whether ACE antisense (ACE-AS) gene delivery prevents alterations in renal vascular physiology in the parents and F(1) offspring of AS-treated spontaneously hypertensive rats (SHR). A single bolus intracardiac injection of ACE-AS (2x10(8) colony-forming units) in SHR neonates caused a modest (18+/-3 mm Hg, n=7 to 9) lowering of blood pressure, which was maintained in the F(1) generation offspring (n=7 to 9). Alterations in renal vascular reactivity, electrophysiology, and [Ca(2+)](i) homeostasis are underlying mechanisms associated with the development and establishment of hypertension. Renal resistance arterioles from truncated ACE sense-treated SHR showed a significantly enhanced contractile response to KCl and phenylephrine (n=24 rings from 6 animals, P<0.01) and significantly attenuated acetylcholine-induced relaxations (n=24 rings from 6 animals, P<0.01) compared with arterioles from ACE-AS-treated SHR. In addition, compared with cells dissociated from arterioles of ACE-AS-treated SHR, cells from truncated ACE sense-treated animal vessels had a resting membrane potential that was 22+/-4 mV more depolarized (n=38, P<0.01), an enhanced L-type Ca(2+) current density (2.2+/-0.3 versus 1.2+/-0.2 pA/pF, n=23, P<0.01), a decreased Kv current density (16.2+/-1.3 versus 5.4+/-2.2 pA/pF, n=34, P<0.01), and increased Ang II-dependent changes in [Ca(2+)](i) (n=142, P<0.01). Similar effects of ACE-AS treatment were observed in the F(1) offspring. These results demonstrate that ACE-AS permanently prevents alterations in renal vascular pathophysiology in spite of the modest effect that ACE-AS had on high blood pressure in SHR.

Retrovirally mediated delivery of angiotensin II type 1 receptor antisense in vitro and in vivo.

In spite of excellent drugs that are available for the control of hypertension, the pharmacological approach has major disadvantages including compliance, side effects, and inability to cure the disease. In the present chapter we provide evidence that a gene therapy concept based on the inhibition of the RAS at a genetic level, with the use of an antisense to the AT1R, is an exciting and viable approach for long-term control of hypertension without the disadvantages inherent in pharmaceutical therapy. A retrovirus-based vector has been used to deliver AT1R-AS in Ang II target tissues both in vitro and in vivo. The transduction efficiency is high and leads to the attenuation of Ang II action in vitro and prevention of hypertension in the SH rat, a model for primary human hypertension. These studies have unveiled a new avenue in which a similar approach could be attempted in the reversal of hypertension in adult animals.

Sustained inhibition of angiotensin I-converting enzyme (ACE) expression and long-term antihypertensive action by virally mediated delivery of ACE antisense cDNA.

Angiotensin I-converting enzyme (ACE) inhibitors have been proven to be highly effective and are for the most part the drugs of choice in the treatment and control of hypertension, congestive heart failure, and left ventricular dysfunction. Despite this, questions regarding side effects and compliance with this traditional pharmacological strategy remain. In view of these observations, coupled with recent advances in gene-transfer technology, our objective in this study was to determine whether the expression of ACE could be controlled on a permanent basis at a genetic level. We argued that the introduction of ACE antisense to inhibit the enzyme would be a prerequisite in considering the antisense gene therapy for the control of hypertension and other related pathological states. Retroviral vectors (LNSV) containing ACE sense (LNSV-ACE-S) and ACE antisense (LNSV-ACE-AS) sequences were constructed and were used in rat pulmonary artery endothelial cells (RPAECs) to determine the feasibility of this approach. Infection of rat RPAECs with LNSV-ACE-S and LNSV-ACE-AS resulted in a robust expression of transcripts corresponding to ACE-S and ACE-AS, respectively, for the duration of these experiments, ie, 8 consecutive passages. The expression of ACE-AS but not of ACE-S was associated with a permanent decrease of approximately 70% to 75% in ACE expression and a 50% increase in the B(max) for the AT(1)s. Although angiotensin II caused a concentration-dependent stimulation of intracellular Ca(2+) levels in both ACE-S- and ACE-AS-expressing cells, the stimulation was significantly higher in ACE-AS-expressing RPAECs. In vivo experiments demonstrated a prolonged expression of ACE-AS transcripts in cardiovascularly relevant tissues of rats. This was associated with a long-term reduction in blood pressure by approximately 15 mm Hg, exclusively in the spontaneously hypertensive rat. These observations demonstrate that delivery of ACE-AS by retroviral vector results in a permanent inhibition of ACE and a long-term reduction in high blood pressure in the spontaneously hypertensive rat.

MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons that is independent of the MAP kinase pathway.

Prevention of renovascular and cardiac pathophysiological changes in hypertension by angiotensin II type 1 receptor antisense gene therapy.

Hypertension produces pathophysiological changes that are often responsible for the mortality associated with the disease. However, it is unclear whether normalizing blood pressure (BP) with conventional therapy is effective in reversing the pathophysiological damage. The duration and initiation of treatment, site of administration, and agent used all appear to influence the reversal of the pathophysiological alterations associated with hypertension. We have previously established that retrovirally mediated delivery of angiotensin II type 1 receptor antisense (AT1R-AS) attenuates the development of high BP in the spontaneously hypertensive (SH) rat model of human essential hypertension. Our objective was to determine whether this attenuation of high BP is associated with prevention of other pathophysiological changes induced by the hypertensive state. Intracardiac delivery of AT1R-AS in neonates prevented the development of hypertension in SH rats for at least 120 days. Contractile experiments demonstrated an impaired endothelium-dependent vascular relaxation (acetylcholine) and an enhanced contractile response to vasoactive agents (phenylephrine and KCl) in the SH rat renal vasculature. In addition, the voltage-dependent K+ current density, which is believed to contribute to smooth muscle resting membrane potential and basal tone, was decreased in renal resistance artery cells of the SH rat. AT1R-AS treatment prevented each of these renal vascular alterations. Finally, AT1R-AS delivery prevented the pathological alterations observed in the SH rat myocardium, including left ventricular hypertrophy, multifocal fibrosis, and perivascular fibrosis. These observations demonstrate that viral-mediated delivery of AT1R-AS attenuates the development of hypertension on a long term basis, and this is associated with prevention of pathophysiological changes in SH rats.

Molecular cloning of the cDNA coding sequence of IL-2 receptor-gamma (gammac) from human and murine forebrain: expression in the hippocampus in situ and by brain cells in vitro.

IL-2 has been implicated in various neurobiological processes of the mammalian CNS. To understand how IL-2 acts in the brain, our lab has sought to determine the molecular pharmacological characteristics of brain IL-2 receptors (IL-2R). The lymphocyte IL-2Rgamma, an essential subunit for IL-2 signaling, is also a common subunit (gammac) for multiple immune cytokine receptors (e.g., IL-4R, IL-7R, IL-9R, IL-15R). Having previously cloned the alpha and beta subunits of the IL-2R heterotrimer complex from normal murine forebrain, we examined the hypothesis that the brain IL-2Rgamma is derived from the same or a closely related gene coding sequence as that expressed by lymphocytes. In this study, we cloned and sequenced the full-length IL-2Rgamma coding region from saline-perfused mouse forebrain and from a human hippocampal library. The cDNA sequences of IL-2Rgamma from human and murine brain were 100% homologous to their lymphocyte sequences. Northern blot analysis showed that the mRNA transcripts in murine brain were the expected size, but the predominant transcript expressed in the brain was different than in the spleen. Compared to the spleen, very low levels of IL-2Rgamma were expressed in the forebrain. In the murine hippocampus, a region where a number of neurobiological actions of IL-2 have been reported, IL-2Rgamma mRNA was detected over the dentate gyrus and CA1-CA4 by in situ hybridization histochemistry. IL-2Rgamma was found to be constitutively expressed by murine HN33.dw hippocampal neuronal cells, murine NB41A3 neuroblastoma cells, astrocyte-enriched mixed glial cell cultures, and in SCID mouse forebrain. The human cortical neuronal cell lines, HCN-1A and HCN-2, did not express the IL-2Rgamma gene. These data suggest the possibility that, in addition to being essential in IL-2 signaling in brain, IL-2Rgamma could be a common subunit (gammac) for multiple cytokine receptors which may be operative in the mammalian CNS.

Attenuation of ANG II actions by adenovirus delivery of AT1 receptor antisense in neurons and SMC.

Both central and peripheral renin-angiotensin systems (RAS) are important in the development and establishment of hypertension. Thus, introducing genes relevant to RAS into neuronal and vascular smooth muscle (VSM) cells, two major targets for angiotensin (ANG) II action, is a prerequisite in considering a gene therapy approach for the control of ANG-dependent hypertension. In this study, we explored the use of adenoviral (Ad) vector to transfer AT1 receptor antisense cDNA (AT1R-AS) into neuronal and VSM cells with the anticipation of attenuation of ANG II-mediated cellular actions. Incubation of neurons and VSM cells with viral particles containing AT1R-AS (Ad-AT1R-AS) resulted in a robust expression of AT1R-AS in a majority (approximately 80%) of the cells. The expression was persistent for at least 28 days and was associated with decreases in the immunoreactive AT1 receptor protein and the maximal binding for AT1 receptor in a time- and dose-dependent manner in both cell types. ANG II stimulation of [3H]thymidine incorporation in VSM cells and norepinephrine transporter gene expression in neuronal cells were attenuated by Ad-AT1R-AS infection. Uninfected cells or cells infected with adenovirus particles containing a mutant AT1 receptor sense cDNA showed no effects on either AT1 receptor or on attenuation of ANG II's cellular affects. These observations show, for the first time, that adenovirus can be used to deliver AT1 receptor mutant sense and antisense cDNAs into two major ANG II target tissues. This consequently influences AT1 receptor-mediated cellular actions of ANG II.

AT1 receptor-mediated nuclear translocation of Raf-1 in brain neurons.

Angiotensin II (Ang II) interacts with the neuronal AT1 receptor subtype and initiates a cascade of signaling events involving activation of Ras-Raf-1-MAP kinase. Raf-1-dependent activation of mitogen-activated protein kinase (MAPK) is the key in the chronic norepinephrine neuromodulatory actions of Ang II and is associated with the translocation of MAPK into the nucleus. In view of these observations, this study was designed to determine if Ang II causes cellular redistribution of Raf-1 in neuronal cells. Most of Raf-1 was localized in the cytoplasmic compartment in neurons. Ang II treatment resulted in a time-dependent increase in the translocation of immunoreactive Raf-1 from the cytoplasm into the nucleus. A fourfold increase was observed in 15 min. The nuclear sequestration of Raf-1 was blocked by losartan, an AT1 receptor-specific antagonist, and not by PD123319, an AT2 receptor-specific antagonist. Confocal microscopic analysis of immunofluorescence data confirmed the nuclear translocation and further showed that Raf-1 was exclusively localized into the nucleolus. These observations demonstrate, for the first time, that Ang II stimulates Raf-1 targeting into the neuronal nucleus, and they suggest that this translocation may play a direct role in the transcriptional regulation of Ang II actions.

Losartan versus gene therapy: chronic control of high blood pressure in spontaneously hypertensive rats.

Interruption of the renin-angiotensin system by pharmacological manipulations attenuates high blood pressure (BP) in the spontaneously hypertensive rat (SHR). However, these agents, such as losartan, need to be administered daily to maintain effective BP control. Therefore, we have hypothesized that a genetic intervention in the expression of angiotensin type 1 receptor (AT1R) should attenuate development of hypertension on a long-term basis in SHR. A retroviral-mediated AT1R antisense cDNA gene delivery system (LNSV-AT1R-AS) was used to test this hypothesis and to compare its BP-lowering effects with those of losartan. Introduction of LNSV-AT1R-AS into 5-day-old Wistar-Kyoto rats and SHR resulted in a robust expression of AT1R antisense (AS) within 3 days and persisted for at least 30 days. This expression was associated with a selective attenuation of high BP in SHR by 25 to 30 mm Hg. Although basal lowering of BP was exclusive to SHR, the angiotensin II (Ang II) pressor response was significantly reduced in all LNSV-AT1R-AS-treated rats. The decreased response to Ang II was associated with a similar attenuation of Ang II-induced dipsogenic responses in both strains of rats. The BP-lowering effects of LNSV-AT1R-AS treatment and losartan treatment were similar and primarily observed in SHR. However, the antihypertensive effect lasted less than 24 hours in losartan-treated SHR compared with 90 days in LNSV-AT1R-AS-treated SHR. In addition, losartan was unable to further lower BP in LNSV-AT1R-AS-treated SHR. Collectively, these results suggest that both losartan and LNSV-AT1R-AS treatment produces an antihypertensive response selectively in SHR that is mediated by interruption of AT1R function. However, a single, acute genetic treatment with LNSV-AT1R-AS can result in long-term control of high BP at a similar level of effectiveness as losartan, without altering plasma Ang II levels.

Induced nitric oxide synthesis is dependent on induced alternatively spliced CAT-2 encoding L-arginine transport in brain astrocytes.

The inducible isoform II of nitric-oxide synthase (iNOS) was recently cloned from brain and identified in astroglial cells. Induced nitric oxide biosynthesis occurs in brain cells only if extracellular cerebrospinal fluid contains -arginine. This study demonstrates for the first time that induced iNOS activity is strictly dependent on concomitant induction of an alternatively spliced transcript of the cat-2 gene encoding high affinity -arginine transporter System y+ in cultured rat astrocytes. Inhibition profiles of radiolabeled -arginine and -leucine uptake identified the dominance of Na+-independent transport System y+ serving cationic amino acids, with insignificant activities of Systems y+L, bo,+, or Bo,+. A reverse transcription-polymerase chain reaction/sequencing/cloning strategy was used to identify a single 123-base nucleotide sequence coding the high affinity domain of alternatively spliced CAT-2 (not CAT-2a) in astrocytes activated by lipopolysaccharide/interferon-gamma. Using this sequence as a cDNA probe, it was determined that CAT-2 mRNA, iNOS mRNA, and System y+ activity were concomitantly and strongly induced in astrocytes. Constitutive CAT-1 mRNA was weakly present in neurons and astrocytes, was not inducible in either cell type, and contributed <3% to total System y+ activity. Although astroglial iNOS Km approximately 10 microM L-arginine for intracellular substrate, hyperbolic kinetics of inducible iNOS activity measured as a function of extracellular L-arginine concentration gave Km approximately 50 microM L-arginine with intact cells. The same Km approximately 50 microM was obtained for induced membrane transport System y+ activity. iNOS activity was reduced to zero in the absence of extracellular L-arginine uptake via System y+. These findings expand the current understanding of NO biosynthesis modulation and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane L-arginine transport in brain.

Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway.

Angiotensin II (Ang II) stimulates norepinephrine transporter (NET) and tyrosine hydroxylase (TH) in the neurons, but the signal transduction mechanism of this neuromodulation is not understood. Treatment of neuronal cultures of hypothalamus-brainstem with Ang II resulted in a time- and dose-dependent activation of Ras, Raf-1, and mitogen-activated protein kinase. This activation was mediated by the interaction of Ang II with the AT1, receptor subtype and was associated with the redistribution of AT1 receptor with Ras and Raf-1 on the neuronal membrane. Treatment with antisense oligonucleotide (AON) to mitogen-activated protein kinase decreased mitogen-activated protein kinase immunoreactivity by 70% and attenuated Ang II stimulation of c-fos, NET, and TH mRNA levels. This demonstrates that induction of these genes requires mitogen-activated protein kinase activation by Ang II. In contrast, AON to mitogen-activated protein kinase failed to inhibit Ang II stimulation of plasminogen activator inhibitor-1 mRNA levels. These results suggest that AT1 receptors are coupled to a Ras-Raf-1 mitogen-activated protein kinase signal transduction pathway that is responsible for stimulation of NET and TH, two neuro-modulatory actions of Ang II in the brain.

Angiotensin II regulation of plasminogen activator inhibitor-1 gene expression in neurons of normotensive and spontaneously hypertensive rat brains.

Neuronal cells in primary culture from the hypothalamus-brain stem areas of normotensive [Wistar-Kyoto (WKY)] and spontaneously hypertensive (SH) rat brains have been used in the present study to investigate an interaction between the brain renin-angiotensin II system and the plasminogen activator system. This is an attempt to further our understanding of the role of brain Ang II in the control of neuronal development and differentiation through its regulation of the extracellular matrix. Ang II caused a 10-fold stimulation of plasminogen activator inhibitor-1 (PAI-1) messenger RNA (mRNA) in WKY rat brain neuronal cultures. The stimulation was mediated by the AT1 receptor subtype and was accompanied by an increase in PAI-1 gene transcription and the synthesis of cellular PAI-1 protein. The stimulation involved activation of protein kinase C, and alterations in the intracellular Ca2+ pool caused a significant inhibition of Ang II stimulation of PAI mRNA. Ang II stimulation of PAI-1 mRNA succeeded its action on c-fos mRNA and was attenuated by c-fos antisense oligonucleotide. Although PAI-1 gene expression was also stimulated by Ang II in neuronal cultures of SH rat brain, two differences between WKY and SH rat brain neurons were observed: 1) the level of Ang II stimulation in SH rat neurons was 50% of that in WKY rat neurons; and 2) Ang II stimulation of c-fos was 2.4-fold higher in SH neurons than in WKY neurons, but c-fos antisense oligonucleotide did not attenuate the stimulatory action of Ang II on PAI-1 mRNA in SH neurons. These observations suggest that the changes in the Ang II-mediated signaling pathways and/or the regulatory region(s) of the PAI-1 gene may contribute to the differential actions of Ang II in WKY and SH rat brain neurons.