Differential neural control of intrarenal blood flow

To test whether renal sympathetic nerve activity (RSNA) can differentially regulate blood flow in the renal medulla (MBF) and cortex (CBF) of pentobarbital sodium-anesthetized rabbits, we electrically stimulated the renal nerves while recording total renal blood flow (RBF), CBF, and MBF. Three stimulation sequences were applied 1) varying amplitude (0.5-8 V), 2) varying frequency (0.5-8 Hz), and 3) a modulated sinusoidal pattern of varying frequency (0. 04-0.72 Hz). Increasing amplitude or frequency of stimulation progressively decreased all flow variables. RBF and CBF responded similarly, but MBF responded less. For example, 0.5-V stimulation decreased CBF by 20 +/- 9%, but MBF fell by only 4 +/- 6%. The amplitude of oscillations in all flow variables was progressively reduced as the frequency of sinusoidal stimulation was increased. An increased amplitude of oscillation was observed at 0.12 and 0.32 Hz in MBF and to a lesser extent RBF, but not CBF. MBF therefore appears to be less sensitive than CBF to the magnitude of RSNA, but it is more able to respond to these higher frequencies of neural stimulation.     

Angiotensin II and nitric oxide in neural control of intrarenal blood flow

We investigated the roles of the renin-angiotensin system and the significance of interactions between angiotensin II and nitric oxide, in responses of regional kidney perfusion to electrical renal nerve stimulation (RNS) in pentobarbital sodium-anesthetized rabbits. Under control conditions, RNS (0.5-8 Hz) reduced total renal blood flow (RBF; -89 +/- 3% at 8 Hz) and cortical perfusion (CBF; -90 +/- 2% at 8 Hz) more than medullary perfusion (MBF; -55 +/- 5% at 8 Hz). Angiotensin II type 1 (AT(1))-receptor antagonism (candesartan) blunted RNS-induced reductions in RBF (P = 0.03), CBF (P = 0.007), and MBF (P = 0.04), particularly at 4 and 8 Hz. Nitric oxide synthase inhibition with N(G)-nitro-L-arginine (L-NNA) enhanced RBF (P = 0.003), CBF (P = 0.001), and MBF (P = 0.03) responses to RNS, particularly at frequencies of 2 Hz and less. After candesartan pretreatment, L-NNA significantly enhanced RNS-induced reductions in RBF (P = 0.04) and CBF (P = 0.007) but not MBF (P = 0.66). Renal arterial infusion of angiotensin II (5 ng.kg(-1).min(-1)) selectively enhanced responses of MBF to RNS in L-NNA-pretreated but not in vehicle-pretreated rabbits. In contrast, greater doses of angiotensin II (5-15 ng.kg(-1).min(-1)) blunted responses of MBF to RNS in rabbits with intact nitric oxide synthase. These results suggest that endogenous angiotensin II enhances, whereas nitric oxide blunts, neurally mediated vasoconstriction in the renal cortical and medullary circulations. In the renal medulla, but not the cortex, angiotensin II also appears to be able to blunt neurally mediated vasoconstriction.     

Increased dietary sodium alters neural control of blood pressure during intravenous ANG II infusion

Increased dietary sodium enhances both excitatory and inhibitory blood pressure responses to stimulation of the central sympathetic nervous system (SNS) centers. In addition, long-term (hours to days) administration of ANG II increases blood pressure by activation of the SNS. These studies investigated the effects of increased dietary sodium on SNS control of blood pressure during 0- to 24-h infusion of ANG II in conscious, male rats consuming either tap water or isotonic saline (Iso) for 2 to 3 wk. The SNS component (evaluated by ganglionic blockade with trimetaphan) of both control blood pressure and the pressor response to intravenous ANG II was reduced in Iso animals. Furthermore, although the pressor response to intravenous ANG II infusion was similar between groups, the baroreflex-induced bradycardia during the initial 6 h of ANG II infusion was significantly greater, whereas the tachycardia accompanying longer infusion periods was significantly attenuated in Iso animals. These data suggest that in normal rats increased dietary sodium enhances sympathoinhibitory responses during intravenous ANG II.     

Translational regulation of ANG II type 1 receptors in proliferating vascular smooth muscle cells

The current study examined angiotensin receptor (ATR) regulation in proliferating rat aortic vascular smooth muscle cells (VSMCs) in culture. Radioligand competition analysis coupled with RNase protection assays (RPAs) revealed that angiotensin type 1a receptor (AT(1a)R) densities (B(max)) increased by 30% between 5 and 7 days in culture [B(max) (fmol/mg protein): day 5, 379 +/- 8.4 vs. day 7, 481 +/- 12, n = 3, P < 0.05] under conditions in which no significant changes in AT(1a)R mRNA expression occurred [in RPA arbitrary units (AU): day 5, 0.23 +/- 0.01 vs. day 7, 0.24 +/- 0.04, n = 4] or in mRNA synthesis determined by nuclear run-on assays [AU: day 5, 0.35 +/- 0.14 vs. day 7, 0.33 +/- 0.11, n = 5]. In contrast, polysome distribution analysis indicated that AT(1a)R mRNA was more efficiently translated in day 7 cells compared with day 5 [% of AT(1a)R mRNA in fraction 2 out of total AT1R mRNA recovered from the sucrose gradient: day 5, 20.9 +/- 9.9 vs. day 7, 56.8 +/- 5.6, n = 3, P < 0.001]. Accompanying the polysome shift was 50% less RNA-protein complex (RPC) formation between VSMC cytosolic RNA binding proteins in day 7 cells compared with 5-day cultures and the 5' leader sequence (5'LS) of the AT(1a)R [5'LS RPC (AU): day 5, 0.62 +/- 0.15 vs. day 7, 0.23 +/- 0.03; n = 4, P < 0.05] and also with exon 2 [Exon 2 RPC (AU): day 5, 35.0 +/- 5.7 vs. day 7, 17.2 +/- 3.6; n = 4, P < 0.05]. Taken together, these results suggest that AT(1a)R expression is regulated by translation during VSMC proliferation in part by RNA binding proteins that interact within exon 2 in the 5'LS of the AT(1a)R mRNA.     

Effects of ANG II type 1 and 2 receptors on oxidative stress,renal NADPH oxidase,and SOD expression

Oxidative stress accompanies angiotensin (ANG) II infusion, but the role of ANG type 1 vs. type 2 receptors (AT1-R and AT2-R, respectively) is unknown. We infused ANG II subcutaneously in rats for 1 wk. Excretion of 8-isoprostaglandin F2alpha (8-Iso) and malonyldialdehyde (MDA) were related to renal cortical mRNA abundance for subunits of NADPH oxidase and superoxide dismutases (SODs) using real-time PCR. Subsets of ANG II-infused rats were given the AT1-R antagonist candesartan cilexetil (Cand) or the AT2-R antagonist PD-123,319 (PD). Compared to vehicle (Veh), ANG II increased 8-Iso excretion by 41% (Veh, 5.4 +/- 0.8 vs. ANG II, 7.6 +/- 0.5 pg/24 h; P < 0.05). This was prevented by Cand (5.6 +/- 0.5 pg/24 h; P < 0.05) and increased by PD (15.8 +/- 2.0 pg/24 h; P < 0.005). There were similar changes in MDA excretion. Compared to Veh, ANG II significantly (P < 0.005) increased the renal cortical mRNA expression of p22phox (twofold), Nox-1 (2.6-fold), and Mn-SOD (1.5-fold) and decreased expression of Nox-4 (2.1-fold) and extracellular (EC)-SOD (2.1-fold). Cand prevented all of these changes except for the increase in Mn-SOD. PD accentuated changes in p22phox and Nox-1 and increased p67phox. We conclude that ANG II infusion stimulates oxidative stress via AT1-R, which increases the renal cortical mRNA expression of p22phox and Nox-1 and reduces abundance of Nox-4 and EC-SOD. This is offset by strong protective effects of AT2-R, which are accompanied by decreased expression of p22phox, Nox-1, and p67phox.     

Differential control of intrarenal blood flow during reflex increases in sympathetic nerve activity

The role of renal sympathetic nerve activity (RSNA) in the physiological regulation of medullary blood flow (MBF) remains ill defined, yet regulation of MBF may be crucial to long-term arterial pressure regulation. To investigate the effects of reflex increases in RSNA on intrarenal blood flow distribution, we exposed pentobarbital sodium-anesthetized, artificially ventilated rabbits (n = 7) to progressive hypoxia while recording RSNA, cortical blood flow (CBF), and MBF using laser-Doppler flowmetry. Another group of animals with denervated kidneys (n = 6) underwent the same protocol. Progressive hypoxia (from room air to 16, 14, 12, and 10% inspired O(2)) significantly reduced arterial oxygen partial pressure (from 99 +/- 3 to 65 +/- 2, 51 +/- 2, 41 +/- 1, and 39 +/- 2 mmHg, respectively) and significantly increased RSNA (by 8 +/- 3, 44 +/- 25, 62 +/- 21, and 76 +/- 37%, respectively, compared with room air) without affecting mean arterial pressure. There were significant reductions in CBF (by 2 +/- 1, 5 +/- 2, 11 +/- 3, and 14 +/- 2%, respectively) in intact but not denervated rabbits. MBF was unaffected by hypoxia in either group. Thus moderate reflex increases in RSNA cause renal cortical vasoconstriction, but not at vascular sites regulating MBF.     

Acute intrarenal infusion of ANG II does not stimulate immediate early gene expression in the kidney

ANG II is capable of stimulating expression of immediate early genes such as egr-1 and c-fos in a variety of cultured cells, including cells of renal origin. To investigate whether ANG II can stimulate early growth response gene expression in vivo, we studied the effects of acute renal artery infusion of low-dose ANG II (2.5 ng small middle dot kg(-1) small middle dot min(-1)) or vehicle on the renal expression of c-fos and egr-1 genes in rats. ANG II infusion for 30 or 240 min decreased renal vascular conductance by approximately 13 and 8%, respectively, compared with the vehicle group. Expression of the early growth response genes c-fos and egr-1 was analyzed using Northern blot hybridization. No significant upregulation of c-fos or egr-1 mRNA levels was detected in rats that received ANG II for either 30 or 240 min, compared with the vehicle groups. We conclude that ANG II, at doses that cause significant physiological effects, does not increase the renal expression of c-fos or egr-1 genes over periods of up to 4 h in vivo.     

Functional contribution of P2Y1 receptors to the control of coronary blood flow

Activation of ADP-sensitive P2Y(1) receptors has been proposed as an integral step in the putative "nucleotide axis" regulating coronary blood flow. However, the specific mechanism(s) and overall contribution of P2Y(1) receptors to the control of coronary blood flow have not been clearly defined. Using vertically integrative studies in isolated coronary arterioles and open-chest anesthetized dogs, we examined the hypothesis that P2Y(1) receptors induce coronary vasodilation via an endothelium-dependent mechanism and contribute to coronary pressure-flow autoregulation and/or ischemic coronary vasodilation. Immunohistochemistry revealed P2Y(1) receptor expression in coronary arteriolar endothelial and vascular smooth muscle cells. The ADP analog 2-methylthio-ADP induced arteriolar dilation in vitro and in vivo that was abolished by the selective P2Y(1) antagonist MRS-2179 and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. MRS-2179 did not alter baseline coronary flow in vivo but significantly attenuated coronary vasodilation to ATP in vitro and in vivo and the nonhydrolyzable ATP analog ATPγS in vitro. Coronary blood flow responses to alterations in coronary perfusion pressure (40-100 mmHg) or to a brief 15-s coronary artery occlusion were unaffected by MRS-2179. Our data reveal that P2Y(1) receptors are functionally expressed in the coronary circulation and that activation produces coronary vasodilation via an endothelium/nitric oxide-dependent mechanism. Although these receptors represent a critical component of purinergic coronary vasodilation, our findings indicate that P2Y(1) receptor activation is not required for coronary pressure-flow autoregulation or reactive hyperemia.     

Bioinformatics and type II G-protein-coupled receptors

The best known family B, or Type II, G-protein-coupled receptors (GPCRs) recognize peptides as ligands. The receptors for corticotrophin-releasing factor, parathyroid hormone and secretin typify this group. However, there are only 15 such GPCRs. Many other receptors share sequence homology and have been assigned to this family. The ten 'Frizzled' and one 'Smoothened' receptors show the lowest sequence homology and are not necessarily G-protein coupled. Drosophila genetics have enabled our understanding of their biology. In contrast, relatively little is known about the largest group with family B, the 33 'large amino termini' or large N-terminal family B seven-transmembrane (LNB 7TM) receptors. This review highlights the similarities found between family B receptors and provides a classification of LNB 7TM receptors.     

Adenoviral-mediated neuron specific transduction of angiotensin II type 2 receptors

The angiotensin II (Ang II) type 2 receptor (AT2R) is localized at specific nuclei within adult rat brain. However, a lack of specific approaches for manipulating the activity of neuronal AT2R has meant that the physiological actions of these sites in the brain remain to be established. Therefore, in this study, our aim was to develop a method by which AT2R can be specifically overexpressed in neurons and in rat brain, with the ultimate goal of a producing a system where discrete increases in AT2R levels in brain nuclei could reveal (and be linked to) physiological actions. Here, we have constructed an AT2R recombinant adenoviral vector, Ad5-SYN-AT2R-IRES-EGFP, which contains the AT2R gene and an IRES-linked EGFP reporter gene, both driven by the neuron-specific synapsin I (SYN) gene promoter. This vector efficiently transduces the AT2R into neuronal cells in culture and results in the expression of high levels of AT2R. These expressed receptors are functional in terms of inhibition of Erk mitogen activated protein kinases (Erk MAPK) and stimulation of neuronal K+ current. Furthermore, microinjection of this vector into adult rat brain elicits a long lasting ( approximately 1 month) expression of AT2R within neurons. In summary, we have developed a viral vector that can be used for the efficient transduction of AT2R into neurons both in vitro and in vivo, the use of which may help to define the physiological functions of brain AT2R in adult rats.     

Gill blood flow control

The arrangement of the fish gill vasculature is quite complex, and varies between the different fish groups. The use of vascular casting techniques has greatly enhanced our knowledge of the anatomy of the branchial microcirculation, not least through the contributions of Pierre Laurent and co-workers at Strasbourg. At different physiological situations, the contact surface between water and blood (functional surface area) varies to balance oxygen uptake against osmotic water flow ("respiratory-osmoregulatory compromise"). This is controlled by nerves and by blood-borne or locally released substances that affect blood flow patterns in the gill. Histochemical techniques have been used to demonstrate neurotransmitter substances in the branchial innervation. In combination with physioly-osmoregulatory compromise" at different physiological situations.     

Local control of blood flow

Organ blood flow is determined by perfusion pressure and vasomotor tone in the resistance vessels of the organ. Local factors that regulate vasomotor tone include myogenic and metabolic autoregulation, flow-mediated and conducted responses, and vasoactive substances released from red blood cells. The relative importance of each of these factors varies over time, from tissue to tissue, and among vessel generations.     

ANG II increases 2-deoxyglucose uptake in mouse embryonic stem cells

It is now suggested that all components of the renin-angiotensin system are present in many tissues, including the embryo and may play a major role in embryo development and differentiation. However, little is known regarding whether ANG II regulates glucose transport in mouse embryonic stem (ES) cells. Thus, the effects of ANG II on [3H]-2-deoxyglucose (2-DG) uptake and its related signal pathways were examined in mouse ES cells. ANG II significantly increased cell proliferation and 2-DG uptake in concentration- and time-dependent manner (>18 h, >10(-8) M) and increased mRNA and protein level of GLUT1 by 31+/-7% and 22+/-5% compared to control, respectively. Actinomycin D and cycloheximide completely blocked the effect of ANG II on 2-DG uptake. ANG II-induced increase of 2-DG uptake was blocked by losartan, an ANG II type 1 (AT1) receptor blocker, but not by PD 123319, an ANG II type 2 (AT2) receptor blocker. In addition, ANG II-induced stimulation of 2-DG uptake was attenuated by phospholipase C (PLC) inhibitors, neomycin and U 73122 and ANG II increased inositol phosphates (IPs) formation by 37+/-8% of control. Protein kinase C (PKC) inhibitors, staurosporine, bisindolylmaleimide I, and H-7 also blocked ANG II-induced stimulation of 2-DG uptake. Indeed, ANG II activated a PKC translocation from the cytosolic to membrane fraction, suggesting a role of PKC. A 23187 (Ca2+ ionophore) increased 2-DG uptake and nifedifine (L-type Ca2+ channel blocker) blocked it. In conclusion, ANG II increased 2-DG uptake by PKC activation via AT1 receptor in mouse ES cells.     

Effect of ANG II type I receptor antagonist and ACE inhibitor on vitamin D receptor-null mice

We recently showed that vitamin D receptor (VDR) inactivation results in deregulated stimulation of the renin-angiotensin system (RAS). To address further the relation between RAS activation and the abnormalities in electrolyte and volume homeostasis, we studied the effect of the ANG II type I receptor antagonist losartan and the angiotensin-converting enzyme inhibitor captopril on VDR-null mice. Treatment with losartan or captopril normalized the water intake and urine excretion of VDR-null mice. However, the increase in salt excretion in VDR-null mice was not affected by either drug, suggesting that this abnormality is independent of the RAS. Northern blot and immunohistochemical analyses revealed that both drugs caused a drastic stimulation of renin expression in wild-type and VDR-null mice, but renin expression remained much higher in the treated VDR-null mice than in the treated wild-type mice, suggesting that the ANG II feedback mechanism remains intact in the mutant mice. These data firmly established a causative relation between RAS overstimulation and the abnormal volume homeostasis in VDR-null mice and demonstrated that vitamin D repression of renin expression is independent of the ANG II feedback regulation in vivo.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.