Zinc-Finger Nuclease Knockout of Dual-Specificity Protein Phosphatase-5 Enhances the Myogenic Response and Autoregulation of Cerebral Blood Flow in FHH.1BN Rats

We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1^BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1^BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1^BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1^BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1^BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.     

Temporal characterization of the development of renal injury in FHH rats and FHH.1BN congenic strains

The present study examined the effect of transfer of portions of chromosome 1 that includes (FHH.1(BN) AR(+) strain) or excludes (control FHH.1(BN) AR(-) strain) a 4.3-Mb region from the Brown Norway (BN) rat that restores the autoregulation (AR) of renal blood flow (RBF) on the development of hypertension and renal injury in congenic strains of Fawn Hooded Hypertensive (FHH) rats. FHH and control AR(-) rats exhibited poor autoregulation of RBF, and glomerular capillary pressure (Pgc) rose by 19 ± 2 mmHg in FHH rats when renal perfusion pressure (RPP) was increased from 100 to 150 mmHg. In contrast, RBF was well autoregulated in the AR(+) strain, and Pgc only increased by 3 ± 1 mmHg when RPP was increased over this range. Baseline mean arterial pressure (MAP) at 12 wk of age was similar in all strains and averaged 122 mmHg. MAP increased significantly in FHH rats and was significantly higher by 12 mmHg in 21-wk-old FHH rats than in the FHH.1(BN) congenic strains. Protein excretion rose from 5 ± 1 to 397 ± 29 mg/day in 6- vs. 21-wk-old FHH rats. In contrast, protein excretion only increased to 139 ± 21 mg/day in the control AR(-) strain, and it did not increase significantly in the AR(+) strain. Glomerular permeability to albumin was similar in all strains at 6 wk of age. It increased significantly in 9-wk-old FHH and control AR(-) rats, but not in the AR(+) strain. The levels of matrix metalloproteinase (MMP)-2 and transforming growth factor (TGF)-β2 protein were significantly higher in the renal cortex of 9-wk-old FHH rats compared with the levels seen in the AR(+) strain. These data indicate that transfer of a 4.3-Mb region of BN chromosome 1 into the FHH genetic background improves autoregulation of RBF, normalizes Pgc, and slows the progression of renal disease.     

Introgression of chromosome 13 in Dahl salt-sensitive genetic background restores cerebral vascular relaxation

To evaluate the potential role of impaired renin-angiotensin system (RAS) function in contributing to reduced vascular relaxation in Dahl salt-sensitive (S) rats, responses to ACh (10(-6) mol/l) and hypoxia (Po(2) reduction to 40-45 mmHg) were determined in isolated middle cerebral arteries of Dahl S rats, Brown Norway (BN) rats, and consomic rats having chromosome 13 (containing the renin gene) or chromosome 16 of the BN rat substituted into the Dahl S genetic background (SS-13(BN) and SS-16(BN), respectively). Arteries of BN rats on a low-salt (LS) diet (0.4% NaCl) dilated in response to ACh and hypoxia, whereas dilation in response to these stimuli was absent in Dahl S rats on LS diet. Vasodilation to ACh and hypoxia was restored in SS-13(BN) rats on an LS diet but not in SS-16(BN) rats. High-salt diet (4% NaCl), to suppress ANG II, eliminated vasodilation to hypoxia and ACh in BN and in SS-13(BN) rats. Treatment of SS-13(BN) rats with the AT(1) receptor antagonist losartan also eliminated the restored vasodilation in response to ACh and hypoxia. These studies suggest that restoration of normal RAS regulation in SS-13(BN) consomic rats restores vascular relaxation mechanisms that are impaired in Dahl S rats.     

Restoration of normal vascular relaxation mechanisms in cerebral arteries by chromosomal substitution in consomic SS.13BN rats

This study sought to identify the mechanisms of vascular relaxation that are rescued in middle cerebral arteries (MCA) of SS.13BN consomic rats by substituting chromosome 13 containing the renin gene from Brown Norway (BN) rats into the Dahl salt-sensitive (SS) genetic background. Isolated MCA from SS rats exhibited an indomethacin-sensitive constriction in response to acetylcholine (ACh) and hypoxia. ACh-induced dilation was NO dependent and hypoxic dilations were cyclooxygenase (COX) dependent in BN and SS.13BN rats. In SS rats, hypoxic dilation was restored by indomethacin and abolished by inhibiting cytochrome P-450 epoxygenases, suggesting a role for epoxyeicosatrienoic acids. MCA from SS and SS.13BN rats constricted and MCA from BN rats dilated in response to the stable prostacyclin analog iloprost. MCA from SS.13BN and BN rats (but not SS rats) dilated in response to the prostaglandin E2 receptor agonist butaprost. Hypoxia increased prostacyclin release in cerebral arteries from all the strains, whereas thromboxane A2 production was reduced in BN rat vessels only. These data suggest that SS rats may be less sensitive to vasodilator prostaglandins and that normalization of renin-angiotensin system regulation causes a switch from production of COX-derived vasoconstrictor metabolites (in SS rats) toward NO-dependent relaxation in response to ACh- and prostaglandin-dependent dilation in response to hypoxia in SS.13(BN) rats.     

Oxidant stress and constrictor reactivity impair cerebral artery dilation in obese Zucker rats

This study tested the hypothesis that evolution of the metabolic syndrome in obese Zucker rats (OZR) leads to impaired dilator reactivity of cerebral resistance arteries vs. responses determined in lean Zucker rats (LZR). Middle cerebral arteries (MCA) from 17-wk-old male LZR and OZR were isolated and cannulated with glass micropipettes. Vascular reactivity was assessed in response to challenge with ACh, sodium nitroprusside (SNP), reductions and elevations in Po2, 5-HT, and increased intralumenal pressure. Vessels were treated with the free radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol) to assess the role of superoxide production in altering reactivity, and passive vascular wall mechanics was assessed in each vessel. Vascular superoxide production was assessed in isolated arteries using fluorescence microscopy. Vessel dilation to ACh and hypoxia was impaired in OZR vs. LZR, although responses to SNP were normal. Vessel constriction to 5-HT, elevated Po2, and elevated intralumenal pressure was enhanced in OZR vs. LZR. Fluorescence microscopy demonstrated an increased superoxide production in arteries of OZR vs. LZR, correctable by incubation with tempol. Although treatment of vessels from OZR with tempol improved dilation to ACh and hypoxia, constrictor responses to 5-HT, elevated Po2, and pressure were not altered by tempol treatment. Indexes of vessel wall mechanics were comparable between groups. These results suggest that vasodilator reactivity of MCA of OZR in response to endothelium-dependent dilator stimuli is impaired vs. LZR and that this may represent a reduced bioavailability of signaling molecules due to oxidant scavenging. However, oxidative stress-independent increases in myogenic tone and constrictor reactivity may contribute to blunted dilator responses of cerebral microvessels.     

NAD(P)H oxidase-derived reactive oxygen species contribute to age-related impairments of endothelium-dependent dilation in rat soleus feed arteries

We tested the hypothesis that age-related endothelial dysfunction in rat soleus muscle feed arteries (SFA) is mediated in part by NAD(P)H oxidase-derived reactive oxygen species (ROS). SFA from young (4 mo) and old (24 mo) Fischer 344 rats were isolated and cannulated for examination of vasodilator responses to flow and acetylcholine (ACh) in the absence or presence of a superoxide anion (O(2)(-)) scavenger (Tempol; 100 μM) or an NAD(P)H oxidase inhibitor (apocynin; 100 μM). In the absence of inhibitors, flow- and ACh-induced dilations were attenuated in SFA from old rats compared with young rats. Tempol and apocynin improved flow- and ACh-induced dilation in SFA from old rats. In SFA from young rats, Tempol and apocynin had no effect on flow-induced dilation, and apocynin attenuated ACh-induced dilation. To determine the role of hydrogen peroxide (H(2)O(2)), dilator responses were assessed in the absence and presence of catalase (100 U/ml) or PEG-catalase (200 U/ml). Neither H(2)O(2) scavenger altered flow-induced dilation, whereas both H(2)O(2) scavengers blunted ACh-induced dilation in SFA from young rats. In old SFA, catalase improved flow-induced dilation whereas PEG-catalase improved ACh-induced dilation. Compared with young SFA, in response to exogenous H(2)O(2) and NADPH, old rats exhibited blunted dilation and constriction, respectively. Immunoblot analysis revealed that the NAD(P)H oxidase subunit gp91phox protein content was greater in old SFA compared with young. These results suggest that NAD(P)H oxidase-derived reactive oxygen species contribute to impaired endothelium-dependent dilation in old SFA.     

Impaired endothelium-mediated relaxation in isolated cerebral arteries from insulin-resistant rats

Insulin resistance (IR) impairs vascular responses in peripheral arteries. However, the effects of IR on cerebrovascular control mechanisms are completely unexplored. We examined the vascular function of isolated middle cerebral arteries (MCAs) from fructose-fed IR and control rats. Endothelium-dependent vasodilation elicited by bradykinin (BK) was reduced in IR compared with control MCAs. Maximal dilation to BK (10(-6) M) was 38 +/- 3% (n = 13) in control and 19 +/- 3% (n = 10) in IR arteries (P < 0.01). N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) decreased responses to BK in control arteries by approximately 65% and inhibited the already reduced responses completely in IR MCAs. Indomethacin (10 microM) reduced relaxation to BK in control MCAs by approximately 40% but was largely ineffective in IR arteries. Combined L-NAME and indomethacin treatments eliminated the BK-induced dilation in both groups. Similarly to BK, endothelium-mediated and mainly cyclooxygenase (COX)-dependent dilation to calcium ionophore A23187 was reduced in IR arteries compared with controls. In contrast, vascular relaxation to sodium nitroprusside was similar between the IR and control groups. These findings demonstrate that endothelium-dependent dilation in cerebral arteries is impaired in IR primarily because of a defect of the COX-mediated pathways. In contrast, nitric oxide-mediated dilation remains intact in IR arteries.     

Impact of genetic background and aging on mesenteric collateral growth capacity in Fischer 344, Brown Norway, and Fischer 344 x Brown Norway hybrid rats

Available studies indicate that both genetic background and aging influence collateral growth capacity, but it is not known how their combination affects collateral growth. We evaluated collateral growth induced by ileal artery ligation in Fischer 344 (F344), Brown Norway (BN), and the first generation hybrid of F344 x BN (F1) rats available for aging research from the National Institute on Aging. Collateral growth was determined by paired diameter measurements in anesthetized rats immediately and 7 days postligation. In 3-mo-old rats, significant collateral growth occurred only in BN (35% +/- 11%, P < 0.001). The endothelial cell number in arterial cross sections was also determined, since this precedes shear-mediated luminal expansion. When compared with the same animal controls, the intimal cell number was increased only in BN rats (92% +/- 21%, P < 0.001). The increase in intimal cell number and the degree of collateral luminal expansion in BN rats was not affected by age from 3 to 24 mo. Immunohistochemical studies demonstrated that intimal cell proliferation was much greater in the collaterals of BN than of F1 rats. The remarkable difference between these three strains of rats used in aging research and the lack of an age-related impairment in the BN rats are novel observations. These rat strains mimic clinical observations of interindividual variation in collateral growth capacity and the impact of age on arteriogenesis and should be useful models to investigate the molecular mechanisms responsible for such differences.     

Carbon monoxide mediates vasodilator effects of glutamate in isolated pressurized cerebral arterioles of newborn pigs

The excitatory neurotransmitter glutamate causes dilation of newborn pig cerebral arterioles in vivo that is blocked by inhibition of carbon monoxide (CO) production. CO, a potent dilator in cerebral circulation in vivo, is produced endogenously in cerebral microvessels via heme oxygenase (HO). In isolated pressurized cerebral arterioles (approximately 200 microm) from newborn pigs, we investigated the involvement of CO and the endothelium in response to glutamate. A CO-releasing molecule, dimanganese decacarbonyl (10(-8)-10(-6) M), dilated cerebral arterioles. Glutamate (10(-6)-10(-4) M) and 1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD; 10(-6)-10(-5) M), a N-methyl-D-aspartate (NMDA) receptor agonist, caused cerebral vascular dilation. Dilation of cerebral arterioles to glutamate and cis-ACPD was abolished by chromium mesoporphyrin (CrMP; 10(-6) M), a HO inhibitor. In contrast, CrMP did not alter dilation to isoproterenol, a -adrenergic receptor agonist. Endothelium-denuded cerebral arterioles did not dilate to glutamate or bradykinin (endothelium-dependent dilator), whereas responses to isoproterenol were preserved. These data indicate that cerebral arterioles from newborn pigs may directly respond to glutamate and the NMDA receptor agonists by endothelium-dependent dilation that involves stimulation of CO production via the HO pathway in the endothelium.     

Role of 20-HETE in the antihypertensive effect of transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats

This study examined whether substitution of chromosome 5 containing the CYP4A genes from Brown Norway rat onto the Dahl S salt-sensitive (SS) genetic background upregulates the renal production of 20-HETE and attenuates the development of hypertension. The expression of CYP4A protein and the production of 20-HETE were significantly higher in the renal cortex and outer medulla of SS.5(BN) (chromosome 5-substituted Brown Norway rat) consomic rats fed either a low-salt (LS) or high-salt (HS) diet than that seen in SS rats. The increase in the renal production of 20-HETE in SS.5(BN) rats was associated with elevated expression of CYP4A2 mRNA. MAP measured by telemetry rose from 117 ± 1 to 183 ± 5 mmHg in SS rats fed a HS diet for 21 days, but only increased to 151 ± 5 mmHg in SS.5(BN) rats. The pressure-natriuretic and diuretic responses were twofold higher in SS.5(BN) rats compared with SS rats. Protein excretion rose to 354 ± 17 mg/day in SS rats fed a HS diet for 21 days compared with 205 ± 13 mg/day in the SS.5(BN) rats, and the degree of glomerular injury was reduced. Baseline glomerular capillary pressure (Pgc) was similar in SS.5(BN) rats (43 ± 1 mmHg) and Dahl S (44 ± 2 mmHg) rats. However, Pgc increased to 59 ± 3 mmHg in SS rats fed a HS diet for 7 days, while it remained unaltered in SS.5(BN) rats (43 ± 2 mmHg). Chronic administration of an inhibitor of the synthesis of 20-HETE (HET0016, 10 mg·kg(-1)·day(-1) iv) reversed the antihypertensive phenotype seen in the SS.5(BN) rats. These findings indicate that the transfer of chromosome 5 from the BN rat onto the SS genetic background increases the renal expression of CYP4A protein and the production of 20-HETE and that 20-HETE contributes to the antihypertensive and renoprotective effects seen in the SS.5(BN) consomic strain.     

Ventilatory phenotypes among four strains of adult rats.

Our purpose in this study was to identify different ventilatory phenotypes among four different strains of rats. We examined 114 rats from three in-house, inbred strains and one outbred strain: Brown Norway (BN; n = 26), Dahl salt-sensitive (n = 24), Fawn-hooded Hypertensive (FHH: n = 27), and outbred Sprague-Dawley rats (SD; n = 37). We measured eupneic (room air) breathing and the ventilatory responses to hypoxia (12% O(2)-88% N(2)), hypercapnia (7% CO(2)), and two levels of submaximal exercise. Primary strain differences were between BN and the other strains. BN rats had a relatively attenuated ventilatory response to CO(2) (P < 0.001), an accentuated ventilatory response to exercise (P < 0.05), and an accentuated ventilatory roll-off during hypoxia (P < 0.05). Ventilation during hypoxia was lower than other strains, but hyperventilation during hypoxia was equal to the other strains (P > 0.05), indicating that the metabolic rate during hypoxia decreased more in BN rats than in other strains. Another strain difference was in the frequency and timing components of augmented breaths, where FHH rats frequently differed from the other strains, and the BN rats had the longest expiratory time of the augmented breaths (probably secondary to the blunted CO(2) sensitivity). These strain differences not only provide insight into physiological mechanisms but also indicate traits (such as CO(2) sensitivity) that are genetically regulated. Finally, the data establish a foundation for physiological genomic studies aimed at elucidating the genetics of these ventilatory control mechanisms.     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

Epileptic seizures cause extended postictal cerebral vascular dysfunction that is prevented by HO-1 overexpression

The extended postictal state is characterized by neurological problems in patients. Inadequate blood supply to the brain and impaired cerebral autoregulation may contribute to seizure-induced neuronal damage. Recent evidence in newborn pigs indicates that activation of the antioxidative enzyme heme oxygenase (HO) at the onset of seizures is necessary for increased cerebral blood flow during the ictal episode and for normal cerebral vascular functioning during the immediate postictal period. We hypothesized that seizures cause prolonged postictal cerebral vascular dysfunction that can be accentuated by HO inhibition and rescued by HO overexpression. Cerebral vascular responses to endothelium-dependent (hypercapnia, bradykinin) and -independent (isoproterenol, sodium nitroprusside) stimuli were assessed 48 h after bicuculline-induced seizures in: 1) saline-control newborn piglets, 2) HO-inhibited animals (HO was inhibited by tin protoporphyrin, SnPP, 3 mg/kg iv), and 3) HO-overexpressing piglets (HO-1 was upregulated by cobalt protoporphyrin, CoPP, 50 mg/kg ip). Extended alterations of HO expression in cerebral microvessels were confirmed by measuring CO production and inducible HO (HO-1) and constitutive HO (HO-2) proteins. Our data provide evidence that seizures cause a severe, sustained, postictal cerebral vascular dysfunction as reflected by impaired vascular reactivity to physiologically relevant dilators. During the delayed postictal state, vascular reactivity to all dilator stimuli was reduced in saline control and, to a greater extent, in HO-inhibited animals. In CoPP-treated piglets, no reduction in postictal cerebral vascular reactivity was observed. These findings may indicate that CoPP prevents postictal cerebral vascular dysfunction by upregulating HO-1, a finding that might have implications for preventing postictal neurological complications.     

Genetic control of the development of experimental allergic encephalomyelitis in rats. Separation of MHC and non-MHC gene effects

Experimental allergic encephalomyelitis (EAE)-susceptible Lew and EAE-resistant Brown Norway (BN) rats and the corresponding MHC congenic strains were examined for their ability to develop clinical and histologic EAE. The ability of T cells from these animals to proliferate in vitro in response to whole guinea pig (GP) myelin basic protein (MBP), rat MBP, and to the major encephalitogenic peptide of GP MBP 66-88 (GP 68-88) was also assessed. We found that Lewis (Lew) was highly susceptible and showed good T cell responses to GP, MBP, rat MBP, and GP 68-88. Lew.1N (BN MHC on Lew background) and BN were not susceptible and T cells from these strains showed significant responses to GP MBP, but not to rat MBP or GP 68-88. Although BN.B1 (Lew MHC on BN background) was not susceptible to actively induced EAE, MBP-specific Lew T cells could transfer severe disease to BN.B1. BN.B1 T cells showed responses to GP-MBP, rat MBP, and GP 68-88 and, when transferred to naive BN.B1 or Lew, induced only mild clinical EAE in both strains. Increasing the number of T cells from BN.B1 had no effect on the severity of clinical symptoms in either recipient, suggesting some deficiency in the T cell repertoire that is necessary for induction of severe EAE. These results suggest that 1) the T cell response to rat MBP and GP68-88 (but not to sites other than 68-88 in GP MBP) is necessary for susceptibility to EAE; 2) the ability to respond to both rat MBP and GP 68-88 is determined by the MHC gene products on APC; and 3) given a permissive MHC, the T cell response that results in EAE is influenced by non-MHC genes.     

Hindlimb unweighting alters endothelium-dependent vasodilation and ecNOS expression in soleus arterioles.

The purpose of this study was to test the hypothesis that endothelium-dependent dilation is impaired in soleus resistance arteries from hindlimb-unweighted (HLU) rats. Male Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 14) or weight-bearing control (Con, n = 14) conditions for 14 days. After the 14-day treatment period, soleus first-order (1A) arterioles were isolated and cannulated with micropipettes to assess vasodilator responses to an endothelium-dependent dilator, ACh (10(-9)-10(-4) M), and an endothelium-independent dilator, sodium nitroprusside (SNP, 10(-9)-10(-4) M). Arterioles from HLU rats were smaller than Con arterioles (maximal passive diameter = 140 +/- 4 and 121 +/- 4 microm in Con and HLU, respectively) but developed similar spontaneous myogenic tone (43 +/- 3 and 45 +/- 3% in Con and HLU, respectively). Arteries from Con and HLU rats dilated in response to increasing doses of ACh, but dilation was impaired in arterioles from HLU rats (P = 0.03), as was maximal dilation to ACh (85 +/- 4 and 65 +/- 4% possible dilation in Con and HLU, respectively). Inhibition of nitric oxide (NO) synthase (NOS) with N(omega)-nitro-L-arginine (300 microM) reduced ACh dilation by approximately 40% in arterioles from Con rats and eliminated dilation in arterioles from HLU rats. The cyclooxygenase inhibitor indomethacin (50 microM) did not significantly alter dilation to ACh in either group. Treatment with N(omega)-nitro-L-arginine + indomethacin eliminated all ACh dilation in Con and HLU rats. Dilation to sodium nitroprusside was not different between groups (P = 0.98). To determine whether HLU decreased expression of endothelial cell NOS (ecNOS), mRNA and protein levels were measured in single arterioles with RT-PCR and immunoblot analysis. The ecNOS mRNA and protein expression was significantly lower in arterioles from HLU rats than in Con arterioles (20 and 65%, respectively). Collectively, these data indicate that HLU impairs ACh dilation in soleus 1A arterioles, in part because of alterations in the NO pathway.     

Role of genetic modifiers in an orthologous rat model of ARPKD

Human data and animal models of autosomal recessive polycystic kidney disease (ARPKD) suggest that genetic factors modulate the onset and severity of the disease. We report here for the first time that ARPKD susceptibility is attenuated by introgressing the mutated Pkhd1 disease allele from the polycystic kidney (PCK) rat onto the FHH (Fawn-Hooded Hypertensive) genetic background. Compared with PCK, the FHH.Pkhd1 strain had significantly decreased renal cyst formation that coincided with a threefold reduction in mean kidney weights. Further analysis revealed that the FHH. Pkhd1 is protected from increased blood pressure as well as elevated plasma creatinine and blood urea nitrogen levels. On the other hand, liver weight and biliary cystogenesis revealed no differences between PCK and FHH.Pkdh1, indicating that genes within the FHH genetic background prevent the development of renal, but not hepatic, manifestations of ARPKD. Microarray expression analysis of kidneys from 30-day-old PCK rats revealed increased expression of genes previously identified in PKD renal expression profiles, such as inflammatory response, extracellular matrix synthesis, and cell proliferation genes among others, whereas the FHH.Pkhd1 did not show activation of these common markers of disease. This newly developed strain can serve as a tool to map modifier genes for renal disease in ARPKD and provides further insight into disease variability and pathophysiology.     

Sequential hydrolysis of proline-containing peptides with immobilized aminopeptidases

Proline-containing polypeptides are shown to be sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains, but does not cleave the N-terminal secondary peptide bonds involving a prolyl nitrogen. Aminopeptidase P (EC 3.4.11.9) cleaves exclusively such secondary bonds. The two enzymes were immobilized by coupling them covalently to porous amino glass beads. Highly stable preparations were obtained with unchanged pH optimum and thermal stability. The applicability of clostridial aminopeptidase to sequence determination was demonstrated by the time-dependent hydrolysis of enkephalin and Substance P octapeptide. Sequential hydrolysis with the two immobilized enzymes was demonstrated with the proline-containing (Pro-Gly-Pro)10, [Asn1, Val5]angiotensin II, bradykinin, Substance P and tuftsin. Absence of endopeptidase activities was demonstrated by resistance of cytochrome c to hydrolysis and by the ordered release of amino acids during the sequential degradation by immobilized clostridial aminopeptidase and aminopeptidase P.     

Aminopeptidase P from rat brain. Purification and action on bioactive peptides.

Aminopeptidase P (EC 3.4.11.9) was purified from rat brain cytosol. A subunit Mr of 71,000 was determined for the reduced, denaturated protein whereas an Mr of 143,000 was determined for the native enzyme. The purified aminopeptidase P selectively liberated all unblocked, preferentially basic or hydrophobic ultimate amino acids from di-, tri- and oligopeptides with N-terminal Xaa-Pro- sequences. Corresponding peptides with penultimate Ala instead of Pro were cleaved with much lower rates; oligopeptides with residues other than Pro or Ala in the penultimate position appeared not to be substrates for the enzyme. Several bioactive peptides with Xaa-Pro sequences, especially bradykinin, substance P, corticortropin-like intermediate lobe peptide, casomorphin and [Tyr]melanostatin were shortened by the N-terminal amino acid by aminopeptidase P action. Rat brain aminopeptidase P was optimally active at pH 7.6-8.0 in the presence of Mn2+. Chelating agents and SH-reacting reagents inhibited the enzyme, but common inhibitors of aminopeptidases, like amastatin or bestatin, of prolidase or of dipeptidyl peptidases II and IV, like N-benzoyloxycarbonyl-proline or epsilon-benzyl-oxycarbonyl-lysyl-proline, as well as antibiotics like beta-lactam ones, bacitracin or puromycin, had little or no effect.     

Rosuvastatin improves cerebrovascular function in Zucker obese rats by inhibiting NAD(P)H oxidase-dependent superoxide production

Insulin-resistance induces cerebrovascular dysfunction and increases the risk for stroke. We investigated whether rosuvastatin (RSV), a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, can reverse reduced cerebrovascular responsiveness in insulin-resistant rats. Dilator responses of the basilar artery (BA) were examined after 1-day or 4-wk RSV (2 mg.kg(-1).day(-1)) treatment in anesthetized 12-wk-old insulin-resistant Zucker obese (ZO) and lean (ZL) rats by using a cranial window preparation. Vehicle-treated ZO rats had significantly higher fasting insulin, total cholesterol (TC), and triglyceride (TG) levels compared with ZL rats. In addition, in the ZO rats, dilator responses of the BA to acetylcholine, iloprost, cromakalim, and potassium chloride were significantly reduced when compared with ZL rats. One-day RSV treatment improved dilator responses of the ZO BAs without altering lipid levels. Four-week RSV treatment lowered both TC and TG by 30% and also improved dilator responses of the ZO BAs, although without additional effects compared with the 1-day RSV treatment. NAD(P)H oxidase-dependent superoxide production was significantly higher in the cerebral arteries of vehicle-treated ZO rats compared with ZL rats, but both 1-day and 4-wk RSV treatments normalized elevated superoxide levels in the ZO arteries. These findings demonstrate that RSV improves cerebrovascular function in insulin-resistance independently from its lipid-lowering effect by the inhibition of NAD(P)H oxidase.     

An integrated genetic linkage map of the laboratory rat

The laboratory rat, Rattus novegicus, is a major model system for physiological and pathophysiological studies, and since 1966 more than 422,000 publications describe biological studies on the rat (NCBI/Medline). The rat is becoming an increasingly important genetic model for the study of specific diseases, as well as retaining its role as a major preclinical model system for pharmaceutical development. The initial genetic linkage map of the rat contained 432 genetic markers (Jacob et al. 1995) out of 1171 developed due to the relatively low polymorphism rate of the mapping cross used (SHR × BN) when compared to the interspecific crosses in the mouse. While the rat genome project continues to localize additional markers on the linkage map, and as of 11/97 more than 3,200 loci have been mapped. Current map construction is using two different crosses (SHRSP × BN and FHH × ACI) rather than the initial mapping cross. Consequently there is a need to provide integration among the different maps. We set out to develop an integrated map, as well as increase the number of markers on the rat genetic map. The crosses available for this analysis included the original mapping cross SHR × BN reciprocal F2 intercross (448 markers), a GH × BN intercross (205 markers), a SS/Mcw × BN intercross (235 markers), and a FHH/Eur × ACI/Hsd intercross (276 markers), which is also one of the new mapping crosses. Forty-six animals from each cross were genotyped with markers polymorphic for that cross. The maps appear to cover the vast majority of the rat genome. The availability of these additional markers should facilitate more complete whole genome scans in a greater number of strains and provide additional markers in specific genomic regions of interest. Received: 3 December 1997 / Accepted: 20 February 1998