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Mohan K Raizada

Publications and source records attributed to Mohan K Raizada.

11 recordsLinked to original sources
PubMed2003

PI3-kinase inhibitors abolish the enhanced chronotropic effects of angiotensin II in spontaneously hypertensive rat brain neurons.

Angiotensin II (Ang II), acting at Ang II type 1 receptors (AT1Rs), increases the firing rate of neurons from Wistar-Kyoto (WKY) rat brain via protein kinase C (PKC)- and calcium-calmodulin kinase II (CaMKII)-dependent mechanisms. The objectives of this study were twofold; first, to compare the Ang-II-stimulated increase in firing of neurons from WKY and spontaneous hypertensive rats (SHR) and second, to elucidate the signaling mechanisms involved. Action potentials were measured in neurons cultured from SHR and WKY rat brains using the whole cell configuration of the patch-clamp technique in the current-clamp mode. Ang II (100 nM) caused three- and sixfold increases in neuronal firing rate in WKY rat and SHR neurons, respectively; effects that were abolished by the AT1R antagonist Losartan (1 microM). Co-administration of calphostin C (10 microM, a PKC inhibitor) and KN-93 (10 microM, a CaMKII inhibitor) completely blocked this Ang II action in WKY rat neurons, while they caused only a approximately 50% attenuation in SHR neurons. The residual increase in firing rate produced by Ang II in SHR neurons was blocked by inhibitors of phosphatidylinositol 3 kinase (PI3-kinase), either LY 294002 (10 microM) or wortmannin (100 nM). These observations suggest that a PI3-kinase signaling pathway may be responsible for the enhanced chronotropic effect produced by Ang II in SHR neurons.

Action Potentials↗
PubMed2003

Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo.

The aim of this study was to develop an efficient method for packaging and concentrating lentiviral vectors that consistently yields high-titer virus on a scale suitable for in vivo applications. Transient cotransfection of 293T packaging cells with DNA plasmids encoding lentiviral vector components was optimized using SuperFect, an activated dendrimer-based transfection reagent. The use of SuperFect allowed reproducible and efficient production of high-titer lentiviral vector at concentrations greater than 1 x 10(7) transducing units per ml (TU/ml) and required less than one-third of the total amount of DNA used in traditional calcium phosphate transfection methods. Viral titers were further increased using a novel concentration protocol that yielded an average final titer of 1.4 x 10(10) TU/ml. Lentiviruses produced using these methods exhibited efficient transduction of central nervous system and peripheral tissues in vivo. The method is reproducible and can be scaled up to facilitate the use of these vectors in animal studies.

Alkaline Phosphatase↗
PubMed2003

Brain renin-angiotensin system dysfunction in hypertension: recent advances and perspectives.

This review focuses on the dysfunction of the intrinsic brain renin-angiotensin system (RAS) in the pathogenesis of hypertension. Hyperactivity of the brain RAS plays a critical role in mediating hypertension in both humans and animal models of hypertension, including the spontaneously hypertensive rat (SHR). The specific mechanisms by which increased brain RAS activity results in hypertension are not well understood but include increases in sympathetic vasomotor tone and impaired arterial baroreflex function. We discuss the contribution of endogenous angiotensin (Ang) II actions on presympathetic vasomotor rostral ventrolateral medulla neurons to enhance sympathetic activity and maintain hypertension. In addition, we discuss Ang II-induced attenuation of afferent baroreceptor feedback within the nucleus tractus solitarius and its relevance to the development of hypertension. We also outline the cellular and molecular mechanisms of Ang II signal transduction that may be critical for the initiation and establishment of hypertension. In particular, we present evidence for a phosphoinositide-3-kinase-dependent signaling pathway that appears to contribute to hypertension in the SHR, possibly via augmented Ang II-induced increases in neuronal firing rate and enhanced transcriptional noradrenaline neuromodulation. Finally, we outline future directions in utilizing our understanding of the brain RAS dysfunction in hypertension for the development of improved therapeutic intervention in hypertension.

Angiotensin II↗
PubMed2003

Endothelial-independent prevention of high blood pressure in L-NAME-treated rats by angiotensin II type I receptor antisense gene therapy.

It has previously been established that a single systemic administration of retroviral vector containing angiotensin II type I receptor antisense (AT(1)R-AS) in the neonatal spontaneously hypertensive rat (SHR) prevents development of hypertension, and in addition cardiac hypertrophy and endothelial dysfunction. However, these studies could not determine whether the effects of AT(1)R-AS on high blood pressure (BP) and endothelial function were independent. Angiotensin receptor blockers have been shown to reduce BP in the L-NAME (N (omega)-nitro-L-arginine methyl ester hydrochloride)-induced rat model of hypertension. Our objective in the present study was to use the L-NAME model of hypertension to determine whether AT(1)R-AS treatment would lower high BP and attenuate cardiac hypertrophy under conditions of permanent endothelial damage. A single bolus of LNSV-AT(1)R-AS viral particles in neonatal Wistar-Kyoto (WKY) rats was without affect on basal BP. Efficacy of the transgene incorporation was assessed by observing a significant reduction in angiotensin-induced dipsogenic response in the AT(1)R-AS-treated animals. Introduction of L-NAME in the drinking water for 10 weeks resulted in the establishment of hypertension only in the WKY rats treated with vector alone. These hypertensive (BP, 179 +/- 4 mmHg) animals showed a 17 % increase in heart weight/body weight ratio and a 60 % reduction in ACh-induced vasorelaxation in phenylephrine-preconstricted arteries. The L-NAME-induced high BP and cardiac hypertrophy were attenuated in rats expressing AT(1)R-AS. However, endothelial dysfunction could not be prevented with the antisense therapy. These observations demonstrate that attenuation of endothelial dysfunction is not a prerequisite for the antihypertensive effects of AT(1)R-AS treatment.

Animals↗
PubMed2002

Transduction of a functional domain of the AT1 receptor in neurons by HIV-Tat PTD.

Despite advances in transgenic and gene transfer technologies, in vivo structure-function studies of the angiotensin II type I receptor (AT1R) have revealed limited information on the diverse actions of angiotensin II. Our objective in the present study was to determine if protein transduction technology with the use of the HIV-Tat protein transduction domain could fill this gap. Recombinant HIV-Tat protein transduction domain fused to EGFP and to the third intracellular loop of the AT1R was expressed. Incubation of hypothalamus and brainstem neurons with this peptide indicated an efficient transport of the protein to most of the cells. This transduction was accompanied by an increase in neuronal firing rate, an effect similar to that observed with angiotensin II stimulation of the neuronal AT1R. The characteristics of the chronotropic effects of recombinant third intracellular loop and its synthetic counterpart were similar and comparable to the effects of angiotensin II on these neurons. In addition, in the presence of the protein kinase C inhibitor calphostin C, the peptide failed to increase firing rate. These observations demonstrated that transduction of neurons with the third intracellular loop of the AT1R produces chronotropic effects similar to those induced by angiotensin II. The data suggests that protein transduction technology could be useful for in vivo AT1R domain transduction.

Action Potentials↗
PubMed2002

Hypertension-linked decrease in the expression of brain gamma-adducin.

Gene profiling data coupled with adducin polymorphism studies led us to hypothesize that decreased expression of this cytosolic protein in the brain could be a key event in the central control of hypertension. Thus, our objectives in the present study were to (1) determine which adducin subunit gene demonstrates altered expression in the hypothalamus and brainstem (two cardioregulatory-relevant brain areas) in two genetic strains of hypertensive rats and (2) analyze the role of adducins in neurotransmission at the cellular level. All three adducin subunits (alpha, beta, and gamma) were present in the hypothalamus and brainstem of Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats. However, only the gamma-adducin subunit expression was 40% to 60% lower in the SH rat compared with WKY rat. A similar decrease in gamma-adducin expression was observed in the hypothalamus and brainstem of the renin transgenic rat compared with its normotensive control. Losartan treatment of the SH rat failed to normalize gamma-adducin gene expression. A hypertension-linked decrease of gamma-adducin was confirmed by demonstrating a decrease in gamma-adducin expression in hypothalamic/brainstem neuronal cultures from prehypertensive SH rats. Neuronal firing rate was evaluated to analyze the role of this protein in neurotransmission. Perfusion of a gamma-adducin-specific antibody caused a 2-fold increase in the neuronal firing rate, an effect similar to that observed with angiotensin II. Finally, we observed that preincubation of neuronal cultures for 8 hours with 100 nmol/L angiotensin II caused a 60% decrease in endogenous gamma-adducin and was associated with a 2-fold increase in basal firing rate. These observations support our hypothesis that a decrease in gamma-adducin expression in cardioregulatory-relevant brain areas is linked to hypertension possibly by regulating the release of neurotransmitters.

Action Potentials↗
PubMed2002

ANG II stimulation of neuritogenesis involves protein kinase B in brain neurons.

Stimulation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) signal transduction pathway has been linked to the neuromodulatory action of ANG II in the brain neurons of the spontaneously hypertensive rat (Yang H and Raizada MK. J Neurosci 19: 2413-2423, 1999). The cellular consequences of this signaling pathway, however, remain unknown in the brain neurons from the normotensive rat. The present study was designed to test the hypothesis that the PI3K-PKB signaling cascade activates an ANG II-mediated neuritogenic action by stimulating cellular growth-associated protein-43 (GAP-43) and neurite extension in Wistar-Kyoto rat brain neurons. ANG II activation of the ANG II type 1 receptor caused increases in PKB activity, cellular GAP-43 levels, and neurite extension in a time- and dose-dependent manner. Depletion of PKB by specific antisense oligonucleotides attenuated ANG II stimulation of both GAP-43 and neurite extension. PKB involvement in neuritogenic action is further supported by the observation that neurons that overexpress PKB develop extensive neuronal processes in the absence of ANG II. These observations demonstrate that PKB is directly involved in ANG II-mediated effects and may recruit both nuclear and cytoplasmic signaling systems for this action.

Angiotensin II↗
PubMed2002

Blood pressure-independent attenuation of cardiac hypertrophy by AT(1)R-AS gene therapy.

Our studies have established that a single intracardiac administration of the retroviral vector containing angiotensin II type I receptor antisense gene causes prolonged antihypertensive actions in the spontaneously hypertensive rat. These results suggest that antisense gene therapy is a conceptually valid strategy for the control of hypertension at the genetic level. To evaluate whether attenuation of the pathophysiological aspects of hypertension are dependent on the blood pressure lowering actions of antisense gene therapy, we chose the renin transgenic rat as a hypertensive animal model and cardiac hypertrophy as the hypertension-associated pathophysiology. A single intracardiac administration of the retroviral vector containing angiotensin II type I receptor antisense in the neonatal rat resulted in long-term expression of the antisense transgene in various cardiovascular-relevant tissues, including the heart. This expression was associated with a significant attenuation of cardiac hypertrophy despite its failure to normalize high blood pressure. Developmental studies indicated that cardiac hypertrophy was evident as early as 16 days of age in viral vector-treated control transgenic rats, despite these animals exhibiting normal blood pressure. These observations demonstrate that, in the renin-transgenic rat, the onset of cardiac hypertrophy occurs during development and is prevented without normalization of high blood pressure. Collectively, these results provide further proof of the concept and indicate that antisense gene therapy could successfully target the local tissues' renin-angiotensin system to produce beneficial cardiovascular outcomes.

Actins↗
PubMed2002

Obligatory role of protein kinase Cbeta and MARCKS in vesicular trafficking in living neurons.

Neurotransmitter release from neurons involves both vesicular trafficking and subsequent fusion of synaptic vesicles with the plasma membrane. The mechanisms involving the formation and fusion of vesicles that allow the exocytotic release of transmitters are understood well. Little is known, however, about the signaling mechanism involved in the trafficking of vesicles along the neurites. In this study, we used real-time confocal microscopy to search for evidence that vesicular trafficking in neurons requires the activation of protein kinase Cbeta (PKCbeta) and the myristoylated alanine-rich C kinase substrate (MARCKS) signaling pathway. Dopamine-beta-hydroxylase fused to green fluorescent protein has been used to trace vesicular movement. Angiotensin II, an established neuromodulatory hormone, stimulates translocation of green fluorescent protein-dopamine-beta-hydroxylase vesicles from the cell body to neurites. This translocation was blocked by an antisense oligonucleotide to PKCbeta and MARCKS. Stimulation of PKC by other means, such as phorbol-12-myristate-13-acetate or carbachol, also resulted in the redistribution of fluorescence in a manner similar to that observed for angiotensin II. These observations demonstrate that PKCbeta-MARCKS signaling may be a general mechanism for the stimulation of vesicular trafficking in brain neurons.

Angiotensin II↗
PubMed2002

Chronotropic action of angiotensin II in neurons via protein kinase C and CaMKII.

Angiotensin II (Ang II) plays an important role in the central control of blood pressure and baroreflexes. These effects are initiated by stimulation of Ang II type 1 (AT(1)) receptors on neurons within the hypothalamus and brain stem, and involve increasing the activity of noradrenergic, substance P, and glutamatergic pathways. The goal of this study is to investigate the intracellular signaling molecules, which are involved in mediating the Ang II-induced increases in neuronal activity. Using neurons in primary culture from newborn rat hypothalamus and brain stem, we have previously determined that Ang II elicits an AT(1) receptor-mediated inhibition of delayed rectifier K(+) current, a stimulation of Ca(2+) current, and a consequent increase in firing rate. In the present study we have demonstrated that this chronotropic action of Ang II in neuronal cultures involves activation of Ca(2+)-dependent signaling molecules. The Ang II-induced increase in firing rate was abolished by inhibition of phospholipase C with U73122 (10 micromol/L), and was attenuated by the protein kinase C inhibitor calphostin C (10 micromol/L) or by the calcium/calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 (10 micromol/L). A combination of calphostin C and KN-93 completely inhibited this Ang II action. These results indicate that the AT(1) receptor-mediated increase in neuronal firing rate involves activation of both PKC and CaMKII, and suggest that these enzymes are potential targets for manipulating the central actions of Ang II.

Angiotensin II↗