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The presence of angiotensin IV (ANG IV)-specific receptors in pulmonary arterial endothelial cells has been studied. The activation of nitric oxide synthase (ecNOS) by ANG IV and the increase in cGMP production along with pulmonary arterial vasodilation due to ecNOS activation have also been investigated. The endothelial cells have been obtained from the main pulmonary artery of six to seven-month old pigs and propagated in monolayers. Results show that ANG IV activates ecNOS in lung endothelial cells through a novel AT4 receptor in the cells.

Experiments were performed to study the regions of the octapeptide angiotensin II (ANG II) type I (AT1) receptor involved in the initiation of intracellular changes that lead to changes in intracellular K (IK) and intracellular Ca (ICa). Results indicated the importance of the third cytoplasmic loop of the AT1a receptor in the activation of G protein and the regulation of IK and ICa. A mechanism that was postulated for the significance of the third loop in the processes was its ability to bind to ANG II and change the conformation of the G protein.

An investigation of voltage-dependent potassium and calcium conductance elicited by angiotensin II (ANG II) in neurons isolated from Sprague-Dawley rats showed that protein kinase C (PKC) and inositol 1,4,5-triphosphate (IP3) play an important role in the modulation of these ion currents after ANG II type 1 receptor activation. Addition of PKC antagonists further enhanced the ANG II-induced decrease in K+ current and abolished the ANG II-induced increase in Ca2+ current, whereas addition of IP3 reduced K+ current.

Changes in single rabbit aortic Ca2+-activated K+ (BK) channels due to pinacidil and cromakalim and its effect on the vasorelaxant properties of these drugs were investigated. An amplitude and slope information-using algorithm was used for the characterization of unitary single-channel currents. The correlation of the qualities of BK channels for both smooth and non-smooth muscle preparations and its function in the modulation of vasorelaxant actions were established.

Apical membrane of native [OMCD.sub.i] cells has nonselective cation channels. Am J Physiol Renal Physiol 280: F48-F55, 2001.--The purpose of this study was to examine cation channel activity in the apical membrane of the outer medullary collecting duct of the inner stripe ([OMCD.sub.i]) using the patch-clamp technique. In freshly isolated and lumen-opened rabbit [OMCD.sub.i], we have observed a single channel conductance of 23.3 [+ or -] 0.6 pS (n = 17) in cell-attached (c/a) patches with high KCl in the bath and in the pipette at room temperature. Channel open probability varied among patches from 0.06 [+ or -] 0.01 at -60 mV (n = 5) to 0.31 [+ or -] 0.04 at 60 mV (n = 6) and consistently increased upon membrane depolarization. In inside-out (i/o) patches with symmetrical KCl solutions, the channel conductance (22.8 [+ or -] 0.8 pS; n = 10) was similar as in the c/a configuration. Substitution of the majority of [Cl.sup.-] with gluconate from KCl solution in the pipette and bath did not significantly alter reversal potential ([E.sub.rev]) or the channel conductance (19.7 [+ or -] 1.1 pS in asymmetrical potassium gluconate, n = 4; 21.4 [+ or -] 0.5 pS in symmetrical potassium gluconate, n = 3). Experiments with 10-fold lower KCl concentration in bath solution in i/o patches shifted [E.sub.rev] to near the [E.sub.rev] of [K.sup.+]. The estimated permeability of [K.sup.+] vs. [Cl.sup.-] was over 10, and the conductance was 13.4 [+ or -] 0.1 pS (n = 3). The channel did not discriminate between [K.sup.+] and [Na.sup.+], as evidenced by a lack of a shift in the [E.sub.rev] with different [K.sup.+] and [Na.sup.+] concentration solutions in i/o patches (n = 3). The current studies demonstrate the presence of cation channels in the apical membrane of native [OMCD.sub.i] cells that could participate in [K.sup.+] secretion or [Na.sup.+] absorption. collecting duct; hydrogen-potassium-adenosinetriphosphatase; potassium channel; sodium channel; patch-clamp recording; inner stripe of the outer medullary collecting duct

ANG II-mediated inhibition of neuronal delayed rectifier [K.sup.+] current: role of protein kinase C-[Alpha]. Am J Physiol Cell Physiol 281: C17-C23, 2001.--It was previously determined that ANG II and phorbol esters inhibit Kv current in neurons cultured from new-born rat hypothalamus and brain stem in a protein kinase C (PKC)- and [Ca.sup.2+]-dependent manner. Here, we have further defined this signaling pathway by investigating the roles of 'physiological' activators of PKC and different PKC isozymes. The cell-permeable PKC activators, diacylglycerol (DAG) analogs 1,2-dioctanoyl-sn-glycerol (1 [micro]mol/l, n = 7) and 1-oleoyl-2-acetyl-sn-glycerol (1 [micro]mol/l, n = 6), mimicked the effect of ANG II and inhibited Kv current. These effects were abolished by the PKC inhibitor chelerythrine (1 [micro]mol/l, n = 5) or by chelation of internal [Ca.sup.2+] (n = 8). PKC antisense (AS) oligodeoxynucleotides (2 [micro]mol/l) against [Ca.sup.2+]-dependent PKC isoforms were applied to the neurons to manipulate the endogenous levels of PKC. PKC-[Alpha]-AS (n = 4) treatment abolished the inhibitory effects of ANG II and 1-oleoyl-2-acetyl-sn-glycerol on Kv current, whereas PKC-[Beta]-AS (n = 4) and PKC-[Gamma]-AS (n = 4) did not. These results suggest that the angiotensin type 1 receptor-mediated effects of ANG II on neuronal Kv current involve activation of PKC-[Alpha]. antisense; calcium; angiotensin type 1 receptor

Patel, Jawaharlal M., Yong D. Li, Jianliang Zhang, Craig H. Gelband, Mohan K. Raizada, and Edward R. Block. Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L794-L801, 1999.-- This study demonstrates that ANG IV-induced activation of lung endothelial cell nitric oxide synthase (ecNOS) is mediated through mobilization of [Ca.sup.2+] concentration and by increased expression and release of the [Ca.sup.2+] binding protein calreticulin in pulmonary artery endothelial cells (PAEC). In [Ca.sup.2+] -free medium and in the presence of the ANG II [AT.sub.1] and [AT.sub.2] receptor antagonists losartan and PD-123319 (1 [micro]M each), respectively, ANG IV (5, 50, and 500 nM) significantly increased intracellular [Ca.sup.2+] release in PAEC (P [is less than] 0.05 for all concentrations). In contrast, ANG IV-mediated activation of ecNOS was abolished by the intracellular [Ca.sup.2+] chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid AM. ANG IV stimulation resulted in significantly increased expression of calreticulin in cells as well as release of calreticulin into the medium of cells as early as 2 h after ANG IV stimulation (P < 0.05). Catalytic activity of purified ecNOS in the absence of calmodulin was increased in a concentration-dependent fashion by calreticulin. Immunocoprecipitation studies revealed that ecNOS and calreticulin were coprecipitated in ANG IV-stimulated PAEC. These results demonstrate that ANG IV-mediated activation of ecNOS is regulated by intracellular [Ca.sup.2+] mobilization and by increased expression of calreticulin, which appears to involve interaction of ecNOS and calreticulin proteins in PAEC. endothelial cell nitric oxide synthase; calcium; angiotensin IV; protein interaction; protein synthesis; protein-protein interaction

Katovich, Michael J., Craig H. Gelband, Phyllis Reaves, Hong-Wei Wang, and Mohan K. Raizada. Reversal of hypertension by angiotensin II type 1 receptor antisense gene therapy in the adult SHR. Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H1260-H1264, 1999.--Pharmacological blockade of the renin-angiotensin system in both hypertensive patients and animal models such as the spontaneously hypertensive rat (SHR) effectively reduces blood pressure (BP). Recent studies have established that virally mediated delivery (vector LNSV) of antisense to the angiotensin II type I receptor (LNSV-[AT.sub.1]R-AS) will attenuate or abolish the development of hypertension in the SHR. However, the effectiveness of this gene therapy approach to reduce high BP once it is established in the adult has not been ascertained. In this study, we investigated the hypothesis that viral delivery of [AT.sub.1]R-AS into the adult SHR will reduce BP and reverse the vascular reactivity associated with the hypertension. Intracardiac injection of virus particles containing LNSV-[AT.sub.1]R-AS into adult SHR resulted in a 30- to 60-mmHg reduction in BP that was maintained for up to 36 days compared with SHR treated with virus alone (LNSV without antisense). Measurement of renal resistance arteriolar reactivity demonstrated a leftward shift in the KCl and phenylephrine concentration-response relationships and an impaired endothelium-dependent relaxation to ACh in LNSV-treated SHR compared with control Wistar-Kyoto rats. These vascular alterations were reversed in the LNSV-[AT.sub.1]R-AS-treated SHR. Collectively, these data demonstrate that virally mediated gene delivery of [AT.sub.1]R-AS can effectively reduce BP and reverse renovascular pathophysiology associated with the hypertensive state when administered to the adult SHR. renovascular responsiveness; blood pressure

Gene therapy has been explored as a useful and improved method for treating hypertension. This has been brought about by advances in the field of gene delivery/gene transfer coupled with a better understanding of renin-angiotensin system (RAS) pharmacology. Based on the inhibition of RAS at a genetic level, all existing data indicate that gene therapy is an effective therapeutic approach for because of its advantages over traditional drug therapy and its potential of controlling hypertension on a permanent basis.