Evidence that renal arginine transport is impaired in spontaneously hypertensive rats.

Low renal nitric oxide (NO) bioavailability contributes to the development and maintenance of chronic hypertension. We investigated whether impaired l-arginine transport contributes to low renal NO bioavailability in hypertension. Responses of renal medullary perfusion and NO concentration to renal arterial infusions of the l-arginine transport inhibitor l-lysine (10 µmol·kg(-1)·min(-1); 30 min) and subsequent superimposition of l-arginine (100 µmol·kg(-1)·min(-1); 30 min), the NO synthase inhibitor N(G)-nitro-l-arginine (2.4 mg/kg; iv bolus), and the NO donor sodium nitroprusside (0.24 µg·kg(-1)·min(-1)) were examined in Sprague-Dawley rats (SD) and spontaneously hypertensive rats (SHR). Renal medullary perfusion and NO concentration were measured by laser-Doppler flowmetry and polarographically, respectively, 5.5 mm below the kidney surface. Renal medullary NO concentration was less in SHR (53 ± 3 nM) compared with SD rats (108 ± 12 nM; P = 0.004). l-Lysine tended to reduce medullary perfusion (-15 ± 7%; P = 0.07) and reduced medullary NO concentration (-9 ± 3%; P = 0.03) while subsequent superimposition of l-arginine reversed these effects of l-lysine in SD rats. In SHR, l-lysine and subsequent superimposition of l-arginine did not significantly alter medullary perfusion or NO concentration. Collectively, these data suggest that renal l-arginine transport is impaired in SHR. Renal l-[(3)H]arginine transport was less in SHR compared with SD rats (P = 0.01). Accordingly, we conclude that impaired arginine transport contributes to low renal NO bioavailability observed in the SHR kidney.

Role of L-arginine uptake mechanisms in renal blood flow responses to angiotensin II in rats.

AIM: To examine whether reduced renal arginine transport increases the responsiveness of the renal circulation to angiotensin II in salt sensitivity, renal perfusion responses to angiotensin II were examined in the presence of L-arginine transport inhibitor, L-lysine and subsequent L-arginine in Sprague Dawley (SD) and Dahl salt-sensitive (Dahl S) rats. METHODS: Laser Doppler probes and a transonic flow probe were used to measure regional renal perfusion and total renal perfusion respectively. Renal perfusion responses to intravenous (i.v.) angiotensin II were sequentially examined under control conditions and during i.v. infusion of L-lysine, L-arginine or nitric oxide synthase inhibitor, N(G)-nitro-L-arginine. RESULTS: Angiotensin II (10 and 100 ng kg(-1) min(-1) , i.v.) reduced total renal (-10 ± 3 and -36 ± 5%) and cortical (-10 ± 2 and -28 ± 4%) but not medullary perfusion in SD rats. In these rats L-lysine enhanced the renal perfusion response (P = 0.003), whereas subsequent L-arginine reversed this effect (P = 0.04). Angiotensin II reduced total renal, cortical and medullary perfusion in Dahl S rats. In Dahl S rats fed high salt, L-lysine did not affect renal perfusion responses to angiotensin II, but subsequent L-arginine blunted the renal blood flow response (P = 0.01) and increased the medullary perfusion during angiotensin II infusion (P = 0.006). CONCLUSION: Intact renal L-arginine transport attenuates the vasoconstrictor effects of circulating angiotensin II in the renal cortex in SD rats. L-arginine also plays an important role in protecting the renal medullary circulation from the ischemic effects of angiotensin II in Dahl S rats.

Analysis of the genome sequence of an alpaca coronavirus.

Coronaviral infection of New World camelids was first identified in 1998 in llamas and alpacas with severe diarrhea. In order to understand this infection, one of the coronavirus isolates was sequenced and analyzed. It has a genome of 31,076 nt including the poly A tail at the 3' end. This virus designated as ACoV-00-1381 (ACoV) encodes all 10 open reading frames (ORFs) characteristic of Group 2 bovine coronavirus (BCoV). Phylogenetic analysis showed that the ACoV genome is clustered closely (>99.5% identity) with two BCoV strains, ENT and LUN, and was also closely related to other BCoV strains (Mebus, Quebec, DB2), a human corona virus (strain 043) (>96%), and porcine hemagglutinating encephalomyelitis virus (>93% identity). A total of 145 point mutations and one nucleotide deletion were found relative to the BCoV ENT. Most of the ORFs were highly conserved; however, the predicted spike protein (S) has 9 and 12 amino acid differences from BCoV LUN and ENT, respectively, and shows a higher relative number of changes than the other proteins. Phylogenetic analysis suggests that ACoV shares the same ancestor as BCoV ENT and LUN.

Importance of the renin-angiotensin system in the regulation of arterial blood pressure in conscious mice and rats.

AIM: The present experiments were designed to determine the mechanism(s) for increased sensitivity to blockade of the renin-angiotensin system in mice in comparison with rats. METHODS: Mice and rats, with indwelling femoral arterial and venous catheters, were chronically administered angiotensin II or pharmacological inhibitors of the renin-angiotensin system as sodium intake was altered. RESULTS: Increasing sodium intake led to suppression of circulating renin, angiotensin II, and aldosterone in rats and mice in the absence of alterations in arterial blood pressure. Additional experiments demonstrated that continuous intravenous infusion of angiotensin II (20 ng kg(-1) min(-1)) significantly increased arterial blood pressure by approximately 35 mmHg in conscious rats at all levels of sodium intake (n = 6). In contrast, arterial pressure was unaffected by angiotensin II infusion in conscious mice under conditions of low sodium intake, although arterial pressure was increased by 16 mmHg when mice were administered a high sodium intake while infused with angiotensin II (n = 6). In comparison, blockade of the endogenous renin-angiotensin system led to significantly greater effects on arterial pressure in mice than rats. Continuous infusion of captopril (30 microg kg(-1) min(-1)) or losartan (100 microg kg(-1) min(-1)) resulted in a 55-90% greater fall in blood pressure in conscious mice in comparison with conscious rats. CONCLUSION: The present studies indicate that arterial pressure in mice is more dependent upon the endogenous renin-angiotensin system than it is in rats, but mice are more resistant to the hypertensive effects of exogenous angiotensin II.

Thermal stress and the disruption of redox-sensitive signalling and transcription factor activation: possible role in radiosensitization.

In spite of ongoing research efforts, the specific mechanism(s) of heat-induced alterations in the cellular response to ionizing radiation (IR) remain ambiguous, in part because they likely involve multiple mechanisms and potential targets. One such group of potential targets includes a class of cytoplasmic signalling and/or nuclear transcription factors known as immediate early response genes, which have been suggested to perform cytotoxic as well as cytoprotective roles during cancer therapy. One established mechanism regulating the activity of these early response elements involves changes in cellular oxidation/reduction (redox) status. After establishing common alterations in early response genes by oxidative stress and heat exposure, one could infer that heat shock may have similarities to other forms of environmental antagonists that induce oxidative stress. In this review, recent evidence supporting a mechanistic link between heat shock and oxidative stress will be summarized. In addition, the hypothesis that one mechanism whereby heat shock alters cellular responses to anticancer agents (including hyperthermic radiosensitization) is through heat-induced disruption of redox-sensitive signalling factors will be discussed.

Heat shock and the activation of AP-1 and inhibition of NF-kappa B DNA-binding activity: possible role of intracellular redox status.

The early response genes comprising the AP-1 and NF-kappa B transcription factors are induced by environmental stress and thought to modulate responses to injury processes through the induction of target genes. Exposure to heat and ionizing radiation (IR) has been shown to affect signalling machinery involved in AP-1 and NF-kappa B activation. Furthermore, regulation of the signalling pathways leading to the activation of these transcription factors has been linked to changes in intracellular oxidation/reduction (redox) reactions. The hypothesis is proposed that exposure to thermal stress and/or IR might alter metabolic processes impacting upon cellular redox state and thereby modify the activity of redox-sensitive transcription factors such as AP-1 and NF-kappa B. Gel electromobility shift assays (EMSA) demonstrated that heat shock-induced AP-1 DNA-binding activity but inhibited IR-induced activation of NF-kappa B. A time course showed that activation of the AP-1 complex occurs between 4 and 5 h following thermal stress, and inhibition of IR-induced NF-kappa B activation also occurs during this time interval. Using a redox-sensitive fluorescent probe [5-(and -6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate], a shift to 40% less intracellular dye oxidation was observed in HeLa cells 0-4 h post-heat shock (45 degrees C, 15 min) relative to cells held at 37 degrees C. This was followed by a shift to greater dye oxidation between 4 and 12 h after treatment (about 1.8-fold) that returned to control levels by 24 h post-heating. These results show changes in DNA-binding activity closely paralleled apparent heat-induced changes in the intracellular redox state. Taken together, these results provide correlative evidence for disruption of redox-sensitive IR-induced signalling pathways by heat shock and support the hypothesis that this mechanism might play a role in heat-induced alterations in radiation response.

Polyamines as olfactory stimuli in the goldfish Carassius auratus.

Electrophysiological responses of goldfish olfactory receptor neurons (ORNs) and goldfish behavioral responses to polyamines were investigated in vivo. Electro-olfactogram (EOG) recordings indicated that polyamines (putrescine, cadaverine and spermine) are potent olfactory stimuli for goldfish with estimated electrophysiological thresholds of 10(-8)-10(-7) mol l(-1), similar to that for L-arginine, the most stimulatory amino acid. Although thresholds were similar, the magnitude of the EOG responses to intermediate (10(-5)-10(-4) mol l(-1)) and high (10(-3) mol l(-1)) concentrations of polyamines dwarfed the responses to amino acids and related single amine containing compounds (amylamine and butylamine). The EOG responses to 0.1 mmol l(-1) putrescine, cadaverine and spermine were, respectively, 4.2x, 4.3x and 10.3x the response of the standard, 0.1 mmol l(-1) L-arginine. Electrophysiological cross-adaptation experiments indicated that polyamine receptor sites are independent from those to L-amino acids (alanine, arginine, glutamate, lysine, methionine and ornithine), bile salts (sodium taurocholate and taurolithocholate), the single amine containing compounds (amylamine and butylamine) and ATP. Further, the cross-adaptation experiments revealed the existence of independent receptor sites for the different polyamines tested. Pharmacological experiments suggested that polyamine odorant transduction does not primarily involve the cyclic AMP and IP(3) second messenger pathways. Behavioral assays indicated that polyamines are attractants that elicit feeding behavior similar to that elicited by L-amino acids.

Role of the renin-angiotensin system during alterations of sodium intake in conscious mice.

The present studies were performed to quantify circulating components of the renin-angiotensin-aldosterone axis and to determine the functional importance of this system during alterations in sodium intake in conscious mice. Increasing sodium intake from approximately 200 to 1,000 microeq/day significantly decreased plasma renin concentration from 472 +/- 96 to 304 +/- 83 ng ANG I. ml(-1). h(-1) (n = 5) but did not alter plasma renin activity from the low-sodium level of 7.7 +/- 1.1 ng ANG I. ml(-1). h(-1). Despite the elevated plasma renin concentration, plasma ANG II in mice on low-sodium level averaged 14 +/- 3 pg/ml and was significantly suppressed to 6 +/- 1 pg/ml by high-sodium intake (n = 7). Consistent with the modulation of ANG II, plasma aldosterone significantly decreased from 41 +/- 8 to 8 +/- 3 ng/dl when sodium intake was elevated (n = 6). In a final set of experiments, the continuous infusion of ANG II (20 ng. kg(-1). min(-1)) led to a mild salt-sensitive increase in mean arterial pressure from 108 +/- 2 to 131 +/- 2 mmHg as sodium intake was varied from low to high (n = 7). In vehicle-infused mice, mean arterial pressure was unaltered from 109 +/- 2 mmHg when sodium intake was increased (n = 6). These studies indicate that the physiological suppression of circulating ANG II may be required to maintain a constancy of arterial pressure during alterations in sodium intake in normal mice.

The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney.

The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.

Comparison of arterial blood pressure in different strains of mice.

The present study was performed to compare the resting level of arterial blood pressure when monitored for 24 h/day in outbred Swiss Webster (SW) and inbred C57BL/ 6J, A/J, C3HeB/FeJ, and SWR/J mice. Mean arterial pressure (MAP) and heart rate (HR) varied throughout the day, with maximal values observed in the hours of darkness. Systolic (SAP), MAP, and diastolic (DAP) arterial blood pressure averaged 122 +/- 2, 112 +/- 2, and 102 +/- 2 mm Hg, respectively, in conscious SW mice (N = 6). No differences were detected in the 24-h averages of MAP between SW, C57BL/6J (N = 7), A/J (N = 5), C3HeB/FeJ (N = 5), or SWR/J (N = 7) mice maintained on a normal sodium diet. Average daily heart rate (HR) was highest in the C3HeB/FeJ (665 +/- 15 beats/min) and lowest in the C57BL/6J (594 +/- 9 beats/min). The MAP was significantly increased in SW mice administered L-NAME (133 +/- 2 mm Hg, N = 5) and significantly decreased in SW mice administered captopril (99 +/- 2 mm Hg, N = 5). These studies demonstrate similar levels of resting arterial pressure in different mouse strains under baseline conditions.

Corynebacterium jeikeium bacteremia in bone marrow transplant patients with Hickman catheters.

Prior studies suggest that Corynebacterium jeikeium bacteremia in immunocompromised patients results in frequent morbidity that may be decreased by prompt removal of the indwelling catheter. To summarize recent experience, charts of 53 bone marrow transplant recipients with Hickman catheters and C. jeikeium bacteremia were reviewed. Forty-one patients were treated with vancomycin without catheter removal and 10 patients underwent catheter removal with subsequent vancomycin therapy. No patient in either group died with C. jeikeium bacteremia as the proximate cause. Salvage of the intravascular catheter was successful in 38 of 41 (93%) attempts. Three patients (7%) in the catheter-salvage group and one patient (10%) in the catheter-removal group experienced recurrent bacteremia. In both catheter-salvage and catheter-removal groups, median time to negative blood culture was 2 days. Thus, time to clearance of bacteremia and patient clinical outcome did not differ between treatment groups. In many patients with Hickman catheters, C. jeikeium bacteremia may be treated successfully with vancomycin and without removal of the catheter.

Characterization of the Oregon isolate of Neospora hughesi from a horse.

Neospora hughesi was isolated in cell cultures inoculated with homogenate of spinal cord from a horse in Oregon. Tachyzoites of this Oregon isolate of N. hughesi were maintained continuously by cell culture passage and tachyzoites were infective to immunosuppressed mice. Gamma interferon gene knockout (KO) mice injected with tachyzoites developed fatal myocarditis and numerous tachyzoites were seen in lesions. Gerbils (Meriones unguiculatus) inoculated with tachyzoites developed antibodies (> or = 1:500) as indicated by the Neospora caninum agglutination test but did not develop clinical signs, and Neospora organisms were not demonstrable in their tissues. Tissue cysts were not found in gerbils, nude mice, KO mice, immunosuppressed outbred Swiss Webster mice, or BALB/c mice injected with the Oregon isolate of N. hughesi. Ultrastructurally, tachyzoites of the Oregon isolate from the myocardium of infected KO mice and from cell culture were similar to N. caninum tachyzoites. Western blot analysis using NcSAG1 and NcSRS2 polyclonal and monoclonal antibodies and characterization of the internal transcribed spacer 1 sequences from the equine isolates and different isolates of N. caninum from dogs and cattle indicated that the Oregon isolate of N. hughesi is distinct from N. caninum isolates from cattle and dogs.

Characteristics of a recent isolate of Sarcocystis neurona (SN7) from a horse and loss of pathogenicity of isolates SN6 and SN7 by passages in cell culture.

An isolate of Sarcocystis neurona (SN7) was obtained from the spinal cord of a horse with neurologic signs. The parasite was isolated in cultures of bovine monocytes and equine spleen cells. The organism divided by endopolygeny and completed at least one asexual cycle in cell cultures in 3 days. The parasite was maintained by subpassages in bovine monocytes for 10 months when it was found to be non-pathogenic to gamma interferon knockout (KO) mice. Revival of a low passage (10th passage) of the initial isolate stored in liquid nitrogen for 18 months retained its pathogenicity for KO mice. Merozoites (10(6)) of the late passage (22nd passage) were infective to only one of four KO mice inoculated. Similar results were obtained with SN6 isolate of S. neurona. No differences were found in Western blot patterns using antigens from the low and high passage merozoites of the SN7 and SN6 isolates. These results suggest that prolonged passage in cell culture may affect the pathogenicity of some isolates of S. neurona.

Measurement of regional blood flow in the kidney using laser-Doppler flowmetry.

The study of regional blood flow in the kidney has been fraught with difficulties. Quantitation of perfusion of the inner cortex and medulla of the kidney has been especially challenging to measure because there is no way to directly visualize the large or small blood vessels in these areas. Furthermore, there has been a lack of reliable techniques to measure tissue blood flow in the kidney. In order to appreciate fully the technical demands of measuring changes in regional blood flow within the kidney, it is necessary to first understand the complex structure of the renal microcirculation.

Characterization of L-arginine transporters in rat renal inner medullary collecting duct.

Previous work from our laboratory has demonstrated that the inner medullary collecting duct (IMCD) expresses a large amount of nitric oxide synthase (NOS) activity. The present study was designed to characterize the transport of NOS substrate, L-arginine, in a suspension of bulk-isolated IMCD cells from the Sprague-Dawley rat kidney. Biochemical transport studies demonstrated an L-arginine transport system in IMCD cells that was saturable and Na(+) independent (n = 6). L-Arginine uptake by IMCD cells was inhibited by the cationic amino acids L-lysine, L-homoarginine, and L-ornithine (10 mmol/l each) and unaffected by the neutral amino acids L-leucine, L-serine, and L-glutamine. Both L-ornithine (n = 6) and L-lysine (n = 6) inhibited NOS enzymatic activity in a dose-dependent manner in IMCD cells, supporting the important role of L-arginine transport for NO production by this tubular segment. Furthermore, RT-PCR of microdissected IMCD confirmed the presence of cationic amino acid transporter CAT1 mRNA, whereas CAT2A, CAT2B, and CAT3 were not detected. These results indicate that L-arginine uptake by IMCD cells occurs via system y(+), is encoded by CAT1, and may participate in the regulation of NO production in this renal segment.

Comparative development and merozoite production of two isolates of Sarcocystis neurona and Sarcocystis falcatula in cultured cells.

The development and merozoite production of Sarcocystis falcatula and 2 isolates (SN6 and SN2) of Sarcocystis neurona were studied in various cultured cell lines inoculated with culture-derived merozoites. All 3 parasites underwent multiple cycles of schizogony in VERO cells, bovine monocytes (M617 cells), and bovine pulmonary artery endothelial cells (CPA). Sarcocystis neurona strains SN6 and SN2 formed schizonts in rat myoblasts (L6) but not in quail myoblasts (QM7); S. falcatula formed schizonts in QM7 cells but not in L6 cells. Merozoites did not develop to sarcocysts in the myoblast cells lines. During a 47-day culture period in VERO cells, SN6 produced substantially more merozoites than did SN2 or S. falcatula. M617 cells produced substantially more merozoites of SN6 than did VERO or CPA cells. During a 17-day culture period of SN6, M617 cells produced mean totals of 4.7 x 10(8) merozoites, VERO cells produced 1.9 x 10(8) merozoites, and CPA cells produced 5.9 x 10(7) merozoites. At 4-12 days after inoculation of cultured cells with SN6, M617 cells cultured in the presence of 10% fetal bovine serum (FBS) produced a mean merozoite total of 5.1 x 10(8) compared to 3.6 x 10(8) for culture medium containing 1% FBS.

Control of arterial blood pressure and renal sodium excretion by nitric oxide synthase in the renal medulla.

Work in our laboratory has focused on the role of nitric oxide synthase (NOS) in the regulation of renal medullary function. Biochemical studies demonstrated that the renal medulla is enriched in immunoreactive NOS protein and NOS enzymatic activity when compared with the renal cortex. Further experiments showed large amounts of NOS activity in the inner medullary collecting ducts, while moderate NOS activity was found in glomeruli and vasa recta and minimal NOS activity was detected in other nephron segments examined. In subsequent functional studies, selective renal medullary infusion of NOS stimulators (bradykinin or acetylcholine) or inhibitors (L-NAME) preferentially altered medullary blood flow. The alterations in medullary flow were associated with parallel changes in sodium and water excretion. Similar to the effects observed in anaesthetized rats, chronic infusion of L-NAME directly into the renal medullary interstitial space of conscious, uninephrectomized SD rats selectively decreased renal medullary blood flow throughout a 5-day L-NAME infusion period. The decrease in medullary blood flow was associated with retention of sodium and the development of hypertension, and the effects were reversible. In contrast to the effects of chronic NOS inhibition, renal medullary infusion of NOS substrate L-arginine prevented the development of sodium-sensitive hypertension in the Dahl salt-sensitive rat placed on a high sodium diet. The data reviewed in this paper indicate that NOS isoforms expressed in the renal medulla have a potent influence on renal medullary tubular and vascular function with consequential effects on fluid and electrolyte homeostasis and arterial blood pressure.

Nitric oxide synthase activity and isoforms in rat renal vasculature.

Experiments were performed to quantify nitric oxide synthase (NOS) activity and identify the NOS isoforms present in the Sprague-Dawley rat renal vasculature. NOS enzymatic activity was measured by adding [(3)H]arginine to microdissected renal blood vessels and quantifying the conversion to [(3)H]citrulline by reverse-phase high-performance liquid chromatography. Total NOS activity was greatest in microdissected vasa recta (123+/-41 pmol. mg(-1). h(-1), n=5) and significantly less in glomeruli (46+/-9 pmol. mg(-1). h(-1), n=6) and afferent arterioles (42+/-10 pmol. mg(-1). h(-1), n=6) and averaged <5 pmol. mg(-1). h(-1) in arcuate (n=8) and interlobular (n=9) arteries. Addition of 1.0 mmol/L EDTA to the reaction decreased NOS activity to <5 pmol. mg(-1). h(-1) in afferent arterioles, glomeruli, and vasa recta (n=5 each), indicating that the NOS enzymatic activity in these segments is primarily a result of constitutive NOS. Both neuronal and endothelial NOS mRNA were identified in each vascular segment by reverse transcription-polymerase chain reaction, but inducible NOS mRNA was detected only in microdissected arcuate arteries. The present experiments indicate that the vasa recta, glomeruli, and afferent arterioles contain large amounts of calcium-dependent NOS enzymatic activity and that neuronal NOS and endothelial NOS mRNA are present in these segments.