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PubMed2003

Generation of antibodies to the urokinase receptor (uPAR) by DNA immunization of uPAR knockout mice: membrane-bound uPAR is not required for an antibody response.

The urokinase receptor (uPAR) is a glycolipid-anchored cell surface glycoprotein that plays a central role in extracellular proteolysis during tissue remodeling processes including cancer invasion. Furthermore, uPAR is found on the surface of both dendritic cells (DCs) and T cells, and has been proposed to play a role in DC-induced T-cell activation and, therefore, in the induction of an immune response. In order to investigate the possibility of using DNA immunization for the generation of poly- and monoclonal antibodies to uPAR, we injected wild-type mice and mice deficient in uPAR (uPAR knockouts) intramuscularly with plasmid DNA encoding a carboxy-terminal truncated soluble form of the human uPAR. Multiple injections of 100 micro g of DNA resulted in a strong and specific antibody response in all mice irrespective of genotype. Antisera with a maximum titre of 32,000 were obtained, comparable with that obtained after immunization with recombinant uPAR. The subclass distribution of uPAR-specific antibodies in the sera demonstrated the induction of a mixed TH1/TH2 response, irrespective of the genotype of the mice. Our results demonstrate the possibility of generating high titre antibodies to uPAR by DNA immunization of wild-type as well as uPAR knockout mice, and that cell surface uPAR is not indispensable for the generation of a humoral immune response.

Animals↗

PubMed2002

Intravenous cocaine induced-activity and behavioural sensitization in norepinephrine-, but not dopamine-transporter knockout mice.

Previously, it was reported that both norepinephrine transporter (NET) and dopamine transporter (DAT) knockout (KO) mice were sensitive to the reinforcing effects of cocaine. However, assessing the locomotor-stimulant effects of cocaine in these subjects has proven difficult due to significant differences in their baseline activity compared to wild-type controls. The present studies were designed to clarify the role of NET and DAT in the stimulant effects of acute and repeated cocaine utilizing these knockout mice, and thereby assess the role of these substrates in the locomotor stimulant effects of cocaine. Mice were habituated to the test environment for sufficient time to ensure equal baselines at the time of cocaine administration. Mice then received cocaine (3-25 mg/kg) intravenously according to a within-session cumulative dose-response design. Cocaine dosing was repeated at 48-h intervals for four sessions to assess behavioural sensitization. NET-KO mice exhibited a reduced response to acute cocaine administration compared to wild-type (WT) controls. However, comparable sensitization developed in NET-KO and WT mice. The DAT-KO and DAT-heterozygote (HT) mice displayed no locomotor activation following either acute or repeated cocaine administration. These data suggest a role for the NET in the acute response to cocaine, but no involvement in sensitization to cocaine. In contrast, DAT appears to be necessary for both the acute locomotor response to cocaine and the subsequent development of sensitization. In addition to existing data concerning the reinforcing effects of cocaine in DAT-KO mice, these data suggest a dissociation between the reinforcing and locomotor stimulant effects of cocaine.

Animals↗

PubMed2003

5-HT1B receptor knockout mice show a compensatory reduction in 5-HT2C receptor function.

Although null mutant ('knockout') mice have provided valuable animal models to complement traditional approaches to psychopharmacology, such animals may also show complex adaptations to the induced mutation. Here we demonstrate that serotonin1B (5-HT1B) receptor knockout (KO) mice show adaptations in serotonin2C (5-HT2C) receptor-mediated functions. They show smaller reductions in food intake and locomotor activity in response to administration of 5-HT2C receptor agonists that are not accounted for by altered drug disposition. These effects are not mimicked by pretreatment of wildtype (WT) mice with a 5-HT1B receptor antagonist showing that they result from a longer term adaptation to the loss of 5-HT1B receptor function and not from a short-term interaction between 5-HT1B- and 5-HT2C-mediated functions. In addition, we show that 5-HT1B receptor KO mice have a lowered hypothalamic c-fos response to the administration of 5-HT2C receptor agonists. These results demonstrate that compensatory adaptations to the constitutive loss of 5-HT1B receptors may be an important determinant of the altered response of 5-HT1B KO mice to a variety of pharmacological challenges.

Animals↗

PubMed2001

Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G-protein activation.

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G-protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [(35)S]guanosine-5'-(gamma-O-thio) triphosphate ([(35)S]GTPgammaS) binding by the high efficacy agonist WIN 55,212-2 was reduced by only 20-25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and (9)-tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212-2 were generally unchanged. Saturation analysis of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding showed that decreased stimulation by WIN 55,212-2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net-stimulated [(35)S]GTPgammaS binding. The apparent maximal number of binding sites (B(max)) values of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net-stimulated [(35)S]GTPgammaS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region-dependent increases in the apparent efficiency of CB1 receptor-mediated G-protein activation in heterozygous CB1 knockout mice.

Animals↗

PubMed2004

In vivo efflux of serotonin in the dorsal raphe nucleus of 5-HT1A receptor knockout mice.

In the dorsal raphe nucleus (DR), extracellular serotonin (5-HT) regulates serotonergic transmission through 5-HT1A autoreceptors. In this work we used in vivo microdialysis to examine the effects of stressful and pharmacological challenges on DR 5-HT efflux in 5-HT1A receptor knockout (5-HT1A-/-) mice and their wild-type counterparts (5-HT1A+/+). Baseline 5-HT concentrations did not differ between both lines of mice, which is consistent with a lack of tonic control of 5-HT1A autoreceptors on DR 5-HT release. (R)-(+)-8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT, 0.5 mg/kg) reduced 5-HT levels to 30% of basal values in 5-HT1A+/+ mice, but not in 5-HT1A-/- mice. The selective 5-HT1B receptor agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP 93129, 300 micro m) reduced dialysate 5-HT to the same extent (30-40% of baseline) in the two genotypes, which suggests a lack of compensatory changes in 5-HT1B receptors in the DR of such mutant mice. Both a saline injection and handling for 3 min increased DR dialysate 5-HT in mutants, but not in 5-HT1A+/+ mice. Fluoxetine (5 and 20 mg/kg) elevated 5-HT in a dose-dependent manner in both genotypes. However, this effect was markedly more pronounced in the 5-HT1A-/- mice. The increased responsiveness of the extracellular 5-HT in the DR of 5-HT1A receptor knockout mice reflects a lack of the autoinhibitory control exerted by 5-HT1A autoreceptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1998

Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor gene-knockout mice.

Angiotensin type-1a (AT1a) receptor gene-knockout (AT1a-/-) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a-/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a-/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a-/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a-/- mice (205.5 +/- 26.1 ng/ml/hr) than in wild-type mice (8.0 +/- 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin gene expression were 12-times and 2.6-times higher in AT1a-/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a-/- mice compared with angiotensinogen-knockout mice. When AT1a-/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a-/- mice.

Angiotensin I↗

PubMed2000

Conditional knockout of mouse insulin-like growth factor-1 gene using the Cre/loxP system.

Insulin-like growth factor-1 (IGF-1) is an essential growth factor for normal intrauterine development and postnatal growth. Mice with a complete deficiency of IGF-1 (IGF-1-null mice), created by homologous recombination, were found to exhibit postnatal lethality, growth retardation, infertility, and profound defects in the development of major organ systems. Furthermore, IGF-1-null mice were resistant to growth hormone (GH) treatment in peri-pubertal somatic growth. Using the Cre/loxP-induced conditional knockout system, we generated a mouse that lacks IGF-1 specifically in the liver, the primary site of IGF-1 production. Interestingly, although circulating and serum levels of IGF-1 were decreased by approximately 75% in these mice, they exhibited no defect in growth or development. When administered exogenously, GH stimulated IGF-1 production in several extra-hepatic tissues as well as body growth. The "Somatomedin hypothesis" originally proposed that circulating IGF-1 acting in various tissues mediate the effects of GH. These striking in vivo results, obtained using homologous recombination technology, call for a major modification of the Somatomedin hypothesis. These gene targeting studies confirm that IGF-1 is essential for GH-stimulated postnatal body growth. However, liver-derived (endocrine) IGF-1 is not essential for normal postnatal growth, though it does exert a negative feedback on GH secretion. Instead, local production of IGF-1, acting in a paracrine/autocrine fashion, appears to mediate GH-induced somatic growth. This review will discuss the effects of tissue-specific IGF-1 gene deficiency created by the Cre/loxP system versus the conventional IGF-1 knockout.

Animals↗

PubMed2001

Mouse gene knockout models for the CLN2 and CLN3 forms of ceroid lipofuscinosis.

The childhood neuronal ceroid-lipofuscinoses (NCLs) are autosomal-recessively inherited neurodegenerative disorders that result in severe cognitive decline and premature death. The genetic bases for a number of different forms of NCL, including those designated CLN2 and CLN3, have now been determined. However, the mechanisms by which the gene defects cause the disease pathology are not known and no effective treatments for these disorders have been developed. To provide tools for studying the mechanisms underlying the disease pathologies and for screening potential therapeutic interventions, work is under way to develop mouse models for the CLN2 and CLN3 disorders. Targeted gene replacement was used to generate mice in which the murine orthologue of the CLN3 gene has been knocked out. Mice that are homozygous for the Cln3 knockout allele develop a number of pathological features similar to those that occur in the human disorder. Among these are accumulation of autofluorescent lysosomal storage bodies, behavioural abnormalities, retinal degeneration, and premature death. On a mixed strain genetic background, the appearance of these symptoms was quite variable, suggesting that other genes can modify the effects of CLN3 mutations. Work to develop a similar mouse gene knockout model for the CLN2 disorder is well under way. Chimaeric mice have been developed with cells that carry an induced mutation in the mouse orthologue of the CLN2 gene that would prevent synthesis of a functional CLN2 protein in mice that are homozygous for the mutation. Mice will be developed that are homozygous for this mutation, and these animals will be evaluated for the development of pathologies similar to those that occur in the human disorder.

Aminopeptidases↗

PubMed2003

What do knockout models teach us about the enteric nervous system?

Since the first histological studies, enormous strides have been made in understanding the genetics and cell biology of enteric nervous system (ENS) formation. Several mitogenic and trophic factors have been implicated in the process of neural cell proliferation and differentiation. A number of natural (piebald-lethal mice [s l], lethal spotting mice [ls], spotting lethal rats [sl]) or target (Gfralpha1-deficient mice, ret.k - mice, and NT-4 knockout mice) mutations have been reported to produce developmental defects in neural crest cell migration, differentiation or survival. Study of these mutations continues to provide new insights into this complex system. In the present investigation, we showed that a lack of basic fibroblast growth factor (FGF) or growth hormone (GH) leads to morphological abnormalities of the enteric nervous system. Because knockouts, neither of FGF nor of GH, produce enteric nervous system defects substantial enough to compromise the ability of the gut to support life, we postulate that FGF and GH affect only a relatively small subset of neurons and/or that compensatory effects of other growth factors might occur.

Animals↗

PubMed2001

Malignant transformation of the esophageal mucosa is enhanced in p27 knockout mice.

OBJECTIVE: In a previous study, we showed that experimentally induced gastroduodenal-esophageal reflux in mice treated with a carcinogen can result in Barrett esophagus and Barrett-associated adenocarcinoma. Since we have shown that most Barrett-associated adenocarcinomas in human beings have lost the tumor suppressor gene p27, we sought to determine whether cancer would be more likely to develop in p27 knockout mice than in p27 heterozygous or p27 wild type mice. METHODS: Three groups of mice were treated by esophagojejunostomy resulting in gastroduodenal-esophageal reflux and by a carcinogen (N -methyl-N -benzylnitrosamine): group I (50 wild type), group II (45 p27 heterozygous), and group III (50 p27 knockout). The mice were killed 18 to 20 weeks after operation and studied macroscopically and histopathologically. RESULTS: Barrett esophagus developed in 7 (14%) mice in group I, 4 (8.9%) mice in group II, and 13 (26%) mice in group III. Cancers developed in 30 (60%) mice in group I, 31 (68%) mice in group II, and 43 (86%) mice in group III. Ten percent of the cancers in group I were adenocarcinomas, as were 16.1% in group II, and 23.3% in group III. The difference between rates of Barrett esophagus in groups I and II compared with group III was statistically significant (P =.035), as was true of the cancer rates (P =.006). The percentage of cancers that were adenocarcinomas was highest in group III, but not significantly different from groups I and II. CONCLUSIONS: This experimental mouse model of Barrett esophagus and Barrett- associated adenocarcinoma is similar to what occurs in human beings and may be useful in developing methods to inhibit malignant transformation of Barrett esophagus.

Animals↗

PubMed2005

Accelerated chemokine receptor 7-mediated dendritic cell migration in Runx3 knockout mice and the spontaneous development of asthma-like disease.

The Runx3 transcription factor is a key regulator of lineage-specific gene expression in several developmental pathways and could also be involved in autoimmunity. We report that, in dendritic cells (DC), Runx3 regulates TGFbeta-mediated transcriptional attenuation of the chemokine receptor CCR7. When Runx3 is lost, i.e., in Runx3 knockout mice, expression of CCR7 is enhanced, resulting in increased migration of alveolar DC to the lung-draining lymph nodes. This increased DC migration and the consequent accumulation of activated DC in draining lymph nodes is associated with the development of asthma-like features, including increased serum IgE, hypersensitivity to inhaled bacterial lipopolysaccharide, and methacholine-induced airway hyperresponsiveness. The enhanced migration of DC in the knockout mice could be blocked in vivo by anti-CCR7 antibodies and by the drug Ciglitazone, known to inhibit CCR7 expression. The data indicate that Runx3 transcriptionally regulates CCR7 and that, when absent, the dysregulated expression of CCR7 in DC plays a role in the etiology of asthmatic conditions that recapitulate clinical symptoms of the human disease. Interestingly, human RUNX3 resides in a region of chromosome 1p36 that contains susceptibility genes for asthma and hypersensitivity against environmental antigens. Thus, mutations in RUNX3 may be associated with increased sensitivity to asthma development.

Animals↗

PubMed2006

The metabolic syndrome resulting from a knockout of the NEIL1 DNA glycosylase.

Endogenously formed reactive oxygen species continuously damage cellular constituents including DNA. These challenges, coupled with exogenous exposure to agents that generate reactive oxygen species, are both associated with normal aging processes and linked to cardiovascular disease, cancer, cataract formation, and fatty liver disease. Although not all of these diseases have been definitively shown to originate from mutations in nuclear DNA or mitochondrial DNA, repair of oxidized, saturated, and ring-fragmented bases via the base excision repair pathway is known to be critical for maintaining genomic stability. One enzyme that initiates base excision repair of ring-fragmented purines and some saturated pyrimidines is NEIL1, a mammalian homolog to Escherichia coli endonuclease VIII. To investigate the organismal consequences of a deficiency in NEIL1, a knockout mouse model was created. In the absence of exogenous oxidative stress, neil1 knockout (neil1-/-) and heterozygotic (neil1+/-) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia. In humans, this combination of clinical manifestations, including hypertension, is known as the metabolic syndrome and is estimated to affect >40 million people in the United States. Additionally, mitochondrial DNA from neil1-/- mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. These data suggest an important role for NEIL1 in the prevention of the diseases associated with the metabolic syndrome.

Animals↗

PubMed2003

Inducible protein knockout reveals temporal requirement of CaMKII reactivation for memory consolidation in the brain.

By integrating convergent protein engineering and rational inhibitor design, we have developed an in vivo conditional protein knockout andor manipulation technology. This method is based on the creation of a specific interaction interface between a modified protein domain and sensitized inhibitors. By introducing this system into genetically modified mice, we can readily manipulate the activity of a targeted protein, such as alpha-Ca(2+)calmodulin-dependent protein kinase II (alphaCAMKII), on the time scale of minutes in specific brain subregions of freely behaving mice. With this inducible and region-specific protein knockout technique, we analyzed the temporal stages of memory consolidation process and revealed the first postlearning week as the critical time window during which a precise level of CaMKII reactivation is essential for the consolidation of long-term memories in the brain.

Amino Acid Sequence↗

PubMed2003

Retention of NMDA receptor NR2 subunits in the lumen of endoplasmic reticulum in targeted NR1 knockout mice.

Glutamate is a major excitatory neurotransmitter in the mammalian central nervous system, and the N-methyl-D-aspartate-selective glutamate receptor (NR) consisting of the NR1 subunit and an NR2 or NR3 subunit plays crucial roles in synaptic transmission, plasticity, and learning and memory. By using a knockout mouse strain, in which the NR1 gene deletion is primarily targeted to the CA1 pyramidal cells of the hippocampus, we investigated the in vivo effect of the loss of the NR1 subunit on the cellular expression and intracellular distribution of the NR2 subunits. The NR1 gene deletion had no apparent effect on the levels of NR2A or NR2B mRNA but led to severe reductions of NR2A and NR2B protein in dendrites of CA1 pyramidal cells. This reduced dendritic distribution of the NR2 subunits accompanied their robust accumulation in perikarya, where they were condensed in the lumen of the endoplasmic reticulum as electron-dense granules. These granules were also observed in CA1 pyramidal cells of the control mice but they were much fewer and contained no detectable levels of the NR2 subunit. The effect of the NR1 knockout on intracellular localization of the NR2 subunits was specific in that no such effect was observed for the GluR1 and PSD-95, two other major postsynaptic proteins. These results suggest that the NR1 subunit plays a crucial role in the release of the NR2 subunit from the endoplasmic reticulum in hippocampal pyramidal cells in vivo, and when the NR1 subunit is unavailable, the NR2 subunits are retained and aggregate into intracisternal granules.

Aging↗

PubMed2003

Protein kinase involved in lung injury susceptibility: evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment.

Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal in vivo role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.

Amino Acid Sequence↗

PubMed2003

Probucol prevents early coronary heart disease and death in the high-density lipoprotein receptor SR-BI/apolipoprotein E double knockout mouse.

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment with the lipid-lowering, antiatherosclerosis, and antioxidation drug probucol extended life to as long as 60 weeks (mean 36 weeks), and at 5-6 weeks of age, virtually completely reversed the cardiac and most RBC pathologies and corrected the unesterified to total cholesterol ratio (0.3) and associated distinctive abnormal lipoprotein morphologies. Manipulation of the timing of administration and withdrawal of probucol could control the onset of death and suggested that critical pathological changes usually occurred in untreated double knockout mice between approximately 3 (weaning) and 5 weeks of age and that probucol delayed heart failure even after development of substantial CHD. The ability of probucol treatment to modulate pathophysiology in the double knockout mice enhances the potential of this murine system for analysis of the pathophysiology of CHD and preclinical testing of new approaches for the prevention and treatment of cardiovascular disease.

Age Factors↗

PubMed2002

Knockout of the alpha 1A/C-adrenergic receptor subtype: the alpha 1A/C is expressed in resistance arteries and is required to maintain arterial blood pressure.

alpha 1-adrenergic receptors (ARs) play a major role in blood pressure regulation. The three alpha 1-AR subtypes (A/C, B, and D) stimulate contraction of isolated arteries, but it is uncertain how different subtypes contribute to blood pressure regulation in the intact animal. We studied the role of the alpha 1A/C subtype by using gene knockout. alpha 1A/C knockout (KO) mice were viable and overtly normal. The LacZ reporter gene replaced alpha 1A/C coding sequence in the KO, and beta-galactosidase staining was present in resistance arteries and arterioles, but not in the thoracic aorta or its main branches. By tail cuff manometer and arterial catheter in conscious mice, alpha 1A/C KO mice were hypotensive at rest, with an 8-12% reduction of blood pressure dependent on alpha 1A/C gene copy number. A61603, an alpha 1A/C-selective agonist, caused a pressor response that was lost in the KO and reduced but significant in heterozygous mice with a single copy of the alpha 1A/C. A subtype-nonselective agonist [phenylephrine (PE)] caused a pressor response in KO mice, but the final arterial pressure was only 85% of wild type. The baroreflex was reset in the KO, and heart rate variability was decreased. After baroreflex blockade with atropine, PE increased blood pressure but did not change heart rate. Cardiac and vascular responses to the beta-AR agonist isoproterenol were unchanged, and the arterial lumen area was not altered. We conclude that the alpha 1A/C-AR subtype is a vasopressor expressed in resistance arteries and is required for normal arterial blood pressure regulation. alpha 1A/C-selective antagonists might be desirable antihypertensive agents.

Animals↗

PubMed2001

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2(-/-) mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2(-/-) mice, we used suppressive subtractive hybridization between livers from Usf2(-/-) and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2(-/-) hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Animals↗