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PubMed2003

Dopamine modulation of membrane excitability in striatal spiny neurons is altered in DARPP-32 knockout mice.

The phosphoprotein DARPP-32 (dopamine and cAMP-regulated phosphoprotein 32 kDa) plays a central role in mediating the actions of a variety of neurotransmitters in medium spiny neurons of the striatum (Greengard, 1990; Fienberg et al., 1998). This study examines D1 and D2 dopamine (DA) agonist effects on the membrane properties of identified striatal neurons recorded in slices obtained from wild-type and DARPP-32-knockout mice. In wild-type spiny cells, DA D1 receptor activation decreased cell excitability, causing a 58.8 +/- 13.5% increase in rheobase current required to evoke spike discharge. In contrast, D1 agonist administration did not alter cell excitability when applied to spiny cells in slices prepared from the DARPP-32 knockout mice. D2 agonist administration decreased cell excitability in both wild-type and knockout mice. The response produced by combined D1 and D2 agonist stimulation was dependent on the sequence of agonist administration. Thus, the D1 agonist-induced decrease in excitability was reversed to a facilitation of spiking upon subsequent D2 agonist administration. In contrast, D2 agonist applied simultaneously with the D1 agonist only produced a reduction in excitability. This type of D1-dependent modulation was not present in slices from the DARPP-32 knockout mice.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2004

Impaired alveologenesis and maintenance of secretory mammary epithelial cells in Jak2 conditional knockout mice.

Jak2 is a hormone-receptor-coupled kinase that mediates the tyrosine phosphorylation and activation of signal transducers and activators of transcription (Stat). The biological relevance of Jak2-Stat signaling in hormone-responsive adult tissues is difficult to investigate since Jak2 deficiency leads to embryonic lethality. We generated Jak2 conditional knockout mice to study essential functions of Jak2 during mammary gland development. The mouse mammary tumor virus-Cre-mediated excision of the first coding exon resulted in a Jak2 null mutation that uncouples signaling from the prolactin receptor (PRL-R) to its downstream mediator Stat5 in the presence of normal and supraphysiological levels of PRL. Jak2-deficient females were unable to lactate as a result of impaired alveologenesis. Unlike Stat5a knockouts, multiple gestation cycles could not reverse the Jak2-deficient phenotype, suggesting that neither other components of the PRL-R signaling cascade nor other growth factors and their signal transducers were able to compensate for the loss of Jak2 function to activate Stat5 in vivo. A comparative analysis of Jak2-deficient mammary glands with transplants from Stat5a/b knockouts revealed that Jak2 deficiency also impairs the pregnancy-induced branching morphogenesis. Jak2 conditional mutants therefore resemble PRL-R knockouts more closely, which suggested that Jak2 deficiency might affect additional PRL-R downstream mediators other than Stat5a and Stat5b. To address whether Jak2 is required for the maintenance of PRL-responsive, differentiating alveolar cells, we utilized a transgenic strain that expresses Cre recombinase under regulatory elements of the whey acidic protein gene (Wap). The Wap-Cre-mediated excision of Jak2 resulted in a negative selection of differentiated alveolar cells, suggesting that Jak2 is required not only for the proliferation and differentiation of alveolar cells but also for their maintenance during lactation.

Animals↗

PubMed2004

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells.

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N(omega)-monomethyl-l-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation.

Animals↗

PubMed2002

Modulation by NO of acetylcholine release in the ileum of wild-type and NOS gene knockout mice.

Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.

Acetylcholine↗

PubMed1999

Nerve-mediated motility of ileal segments isolated from NK(1) receptor knockout mice.

Tachykinins such as substance P (SP) and neurokinin A (NKA) acting on neurokinin (NK) receptors modulate the nonadrenergic noncholinergic (NANC) neurotransmission in the gastrointestinal tract of several species, but the information about the mouse small intestine is scanty. Both SP and NKA induced concentration-dependent contractions of ileal segments isolated from wild-type mice that were blocked by NK(1) and NK(2) antagonists, respectively. In contrast, segments isolated from NK(1) receptor (NK(1)-R) knockout mice responded only to elevated concentrations of SP. To reveal the inhibitory NANC (iNANC) responses, tissues were pretreated with atropine and guanethidine. Under these conditions, a tetrodotoxin-sensitive relaxation in response to electrical field stimulation (EFS) was observed. NK(1)-R knockout mice presented a trend toward an increase in iNANC responses, whereas the NK(1)-R antagonist significantly potentiated iNANC relaxation in tissues isolated from wild-type mice. N(G)-nitro-L-arginine methyl ester (100 microM) transformed the relaxant response to EFS into a tetrodotoxin-sensitive, frequency-dependent contraction characteristic of an excitatory NANC (eNANC) system. A NK(1)-R antagonist abolished the contractile responses of the mouse ileum to EFS, whereas a NK(2) receptor antagonist had a trend toward reducing EFS-induced contraction. The eNANC component was absent in NK(1)-R knockout mice. Measurement of SP-like immunoreactivity indicated similar amounts of SP per gram of tissue isolated from wild-type and NK(1)-R knockout mice, indicating that the observed differences in response to EFS were not due to a differential peptide content. It is concluded that, in the mouse ileum, both NK(1) and NK(2) receptors modulated the responses to exogenous tachykinins, whereas NK(1) is the primary tachykinin receptor involved in both iNANC and eNANC transmission.

Animals↗

PubMed2001

ICAM-1 is involved in the mechanism of alcohol-induced liver injury: studies with knockout mice.

To test the hypothesis that leukocyte infiltration mediated by intercellular adhesion molecule (ICAM)-1 is involved in early alcohol-induced liver injury, male wild-type or ICAM-1 knockout mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin for 4 wk. There were no differences in mean urine alcohol concentrations between the groups fed ethanol. Alcohol administration significantly increased liver size and serum alanine aminotransferase levels in wild-type mice over high-fat controls, effects that were blunted significantly in ICAM-1 knockout mice. Dietary ethanol caused severe steatosis, mild inflammation, and focal necrosis in livers from wild-type mice. Furthermore, livers from wild-type mice fed ethanol showed significant increases in the number of infiltrating leukocytes, which were predominantly lymphocytes. These pathological changes were blunted significantly in ICAM-1 knockout mice. Tumor necrosis factor (TNF)-alpha mRNA expression was increased in wild-type mice fed ethanol but not in ICAM-1 knockout mice. These data demonstrate that ICAM-1 and infiltrating leukocytes play important roles in early alcohol-induced liver injury, most likely by mechanisms involving TNF-alpha.

Alanine Transaminase↗

PubMed2002

Adenylate kinase AK1 knockout heart: energetics and functional performance under ischemia-reperfusion.

Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-P(i) and creatine phosphate-to-P(i) ratios. Postischemic AK1 knockout hearts maintained approximately 40% of beta-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.

Adenosine Triphosphate↗

PubMed1997

beta-Adrenergic regulation of cAMP and protein phosphorylation in phospholamban-knockout mouse hearts.

The stimulatory effects of beta-adrenergic agonists reflect increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels and phosphorylation of key regulatory proteins in the heart. One of these phosphoproteins is phospholamban (PLB) in sarcoplasmic reticulum, and ablation of PLB is associated with attenuation of the contractile responses to beta-adrenergic stimulation in the mouse heart. To determine whether this attenuation of beta-stimulation is due to altered phosphorylation characteristics of the other key cardiac phosphoproteins and/or to compensatory responses occurring in the absence of PLB, PLB-knockout and wild-type hearts were perfused and their protein phosphorylation patterns examined. The beta-adrenergic receptor density, adenylyl cyclase activity, tissue cAMP levels, and the basal phosphoprotein pattern were similar between PLB-knockout and wild-type hearts. Isoproterenol perfusion resulted in similar increases in the tissue cAMP levels and the degree of phosphorylation of troponin I, C protein, and the 21-kDa microsomal protein in wild-type and PLB-knockout hearts. These findings indicate that the attenuation of isoproterenol-mediated increases in contractility of the PLB-knockout hearts is not due to alterations in the beta-adrenergic signal transduction pathway or the degree of phosphorylation of the key cardiac regulatory phosphoproteins in myofibrils and sarcolemma.

Animals↗

PubMed1998

Alterations in dynamic heart rate control in the beta 1-adrenergic receptor knockout mouse.

beta 1-Adrenergic receptors (beta 1-ARs) are key targets of sympathetic nervous system activity and play a major role in the beat-to-beat regulation of cardiac chronotropy and inotropy. We employed a beta 1-AR gene knockout model to test the hypothesis that beta 1-AR function is critical for maintenance of resting heart rate and baroreflex responsiveness and, on the basis of its important role in regulating chronotropy and inotropy, is also required for maximal exercise capacity. Using an awake unrestrained mouse model, we demonstrate that resting heart rate and blood pressure are normal in beta 1-AR knockouts and that the qualitative responses to baroreflex stimulation are intact. Chronotropic reserve in beta 1-AR knockouts is markedly limited, with peak heart rates approximately 200 beats/min less than wild types. During graded treadmill exercise, heart rate is significantly depressed in beta 1-AR knockouts at all work loads, but despite this limitation, there are no reductions in maximal exercise capacity or metabolic indexes. Thus, in mice, the beta 1-AR is not essential for either maintenance of resting heart rate or for maximally stressed cardiovascular performance.

Adrenergic beta-Agonists↗

PubMed2002

The multifaceted phenotype of the knockout mouse for the KCNE1 potassium channel gene.

Mutations of the KCNE1 gene (IsK, minK) are related to hereditary forms of cardiac arrhythmias, so-called long QT syndromes (LQT). Here we review the phenotype of a mouse model for the recessive form of LQT known as Jervell and Lange-Nielsen syndrome. KCNE1 knockout mice exhibit an enhanced QT-RR adaptability, which is probably part of the pathophysiological mechanism leading to life-threatening tachyarrhythmia in patients. Like patients, knockout mice are deaf and show vestibular symptoms due to an impaired endolymph production. Knockout mice show urinary and fecal salt wasting and volume depletion. The renal phenotype is due to diminished reabsorption of Na(+) and glucose. The mice are hypokalemic and have increased aldosterone levels. Besides volume depletion, aldosterone is elevated via a set-point shift for sensing of extracellular K(+) in aldosterone-secreting glomerulosa cells, which physiologically express KCNE1. In conclusion, KCNE1 knockout leads to a complex phenotype resulting from direct loss of KCNE1 and compensatory mechanisms. Murine KCNE1 physiology could be helpful for the pathophysiological understanding and perhaps gene-specific treatment of long QT patients.

Aldosterone↗

PubMed1997

Exacerbated febrile responses to LPS, but not turpentine, in TNF double receptor-knockout mice.

We examined the effects of injections of systemic [lipopolysaccharide (LPS), 2.5 mg/kg or 50 pg/kg ip] or local (turpentine, 100 microl sc) inflammatory stimuli on fever, motor activity, body weight, and food intake in tumor necrosis factor (TNF) double receptor (TNFR)-knockout mice. A high dose of LPS resulted in exacerbated fevers in TNFR-knockout mice compared with wild-type mice for the early phase of fever (3-15 h); the late phase of fever (16-24 h) and fevers to a low dose of LPS were similar in both groups. Motor activity, body weight, and food intake were similarly reduced in both groups of mice after LPS administration. In response to turpentine, TNFR-knockout and wild-type mice developed virtually identical responses to all variables monitored. These results suggest that 1) TNF modulates fevers to LPS dose dependently, 2) TNF does not modulate fevers to a subcutaneous injection of turpentine, and 3) knockout mice may develop cytokine redundancy in the regulation of the acute phase response to intraperitoneally injected LPS or subcutaneously injected turpentine.

Animals↗

PubMed2000

Defective fluid and HCO(3)(-) absorption in proximal tubule of neuronal nitric oxide synthase-knockout mice.

Using renal clearance techniques and in situ microperfusion of proximal tubules, we examined the effects of N(G)-monomethyl-L-arginine methyl ester (L-NAME) on fluid and HCO(3)(-) transport in wild-type mice and also investigated proximal tubule transport in neuronal nitric oxide synthase (nNOS)-knockout mice. In wild-type mice, administration of L-NAME (3 mg/kg bolus iv) significantly increased mean blood pressure, urine volume, and urinary Na(+) excretion. L-NAME, given by intravenous bolus and added to the luminal perfusion solution, decreased absorption of fluid (60%) and HCO(3)(-) (49%) in the proximal tubule. In nNOS-knockout mice, the urinary excretion of HCO(3)(-) was significantly higher than in the wild-type mice (3.12 +/- 0.52 vs. 1. 40 +/- 0.33 mM) and the rates of HCO(3)(-) and fluid absorption were 62 and 72% lower, respectively. Both arterial blood HCO(3)(-) concentration (20.7 vs. 25.7 mM) and blood pH (7.27 vs. 7.34) were lower, indicating a significant metabolic acidosis in nNOS-knockout mice. Blood pressure was lower in nNOS-knockout mice (76.2 +/- 4.6 mmHg) than in wild-type control animals (102.9 +/- 8.4 mmHg); however, it increased in response to L-NAME (125.5 +/- 5.07 mmHg). Plasma Na(+) and K(+) were not significantly different from control values. Our data show that a large component of HCO(3)(-) and fluid absorption in the proximal tubule is controlled by nNOS. Mice without this isozyme are defective in absorption of fluid and HCO(3)(-) in the proximal tubule and develop metabolic acidosis, suggesting that nNOS plays an important role in the regulation of acid-base balance.

Acid-Base Equilibrium↗

PubMed2003

Tissue angiotensin-converting-enzyme (ACE) deficiency leads to a reduction in oxidative stress and in atherosclerosis: studies in ACE-knockout mice type 2.

UNLABELLED: Background- Angiotensin II, produced by angiotensin-converting-enzyme (ACE), enhances oxidative stress and atherogenesis. In this study, we analyzed whether tissue ACE deficiency in ACE-knockout mice type-2 would affect their oxidative status. Moreover, by crossbreeding the ACE-knockout mice with atherosclerotic apolipoprotein E (apo E)-deficient (E0) mice, we questioned whether tissue ACE deficiency affects atherogenesis. METHODS AND RESULTS: ACE-deficient mice type-2 (ACE+/-) exhibited reduced serum lipid peroxidation compared with ACE+/+ mice. Peritoneal macrophages from ACE+/- mice demonstrated lower oxidative status, as exhibited by decreases of 47%, 33% 56%, and 51%, in their lipid peroxides, superoxide release, dichlorofluorescein fluorescence, and LDL oxidation, respectively, compared with ACE+/+ mice. ACE+/- mice crossbred with E0 mice, resulting in atherosclerotic mice heterozygous for ACE (ACE+/-/E0 mice), exhibited reduced lipid peroxidation, increased paraoxonase activity, and lower macrophage LDL oxidation compared with E0 and ACE+/+/E0 mice. ACE+/-/E0 mice also exhibited reduced NADPH-induced aortic superoxide ion production by 52% and a reduction of 43% in their atherosclerotic lesion size compared with E0 mice. Finally, 2 animals genotyped as homozygous-knockout for both ACE and APOE genes (ACE-/-/E0), exhibited a striking reduction of 86% in their atherosclerotic lesion area compared with E0 mice. CONCLUSIONS: Reduction of tissue ACE with the ACE-knockout mouse type-2 model inhibited oxidative stress and atherogenesis.

Animals↗

PubMed2005

Role of C-reactive protein in atherogenesis: can the apolipoprotein E knockout mouse provide the answer?

OBJECTIVE: Human C-reactive protein (CRP) was reported to accelerate atherosclerotic lesion development in male but not in female apolipoprotein E (apoE) knockout mice. Here, mice expressing rabbit CRP (rbCRP) were crossbred onto apoE knockout animals, and the effect on atherogenesis was studied. METHODS AND RESULTS: Hemolytic complement activity could not be detected in apoE knockout mice. Furthermore, in contrast to human complement, neither rabbit nor human CRP complexed to modified low-density lipoprotein-activated murine complement. At 52 weeks, rbCRP levels were similar in male and female transgenic animals. Serum cholesterol levels were equivalent in female animals irrespective of rbCRP expression, whereas rbCRP-positive males had significantly higher serum cholesterol levels than the rbCRP-negative counterparts. All mice exhibited extensive atherosclerotic lesions, as studied en face, and no differences were noted between rbCRP-negative and rbCRP-positive animals. Atherosclerotic luminal obstruction of aortic arch and first-order neck branches did not differ significantly between rbCRP-positive and rbCRP-negative mice. There was no correlation between rbCRP levels and atherosclerotic lesion formation. CONCLUSIONS: No marked effect of rbCRP on the formation of moderately advanced atherosclerotic lesions could be discerned in the apoE knockout mouse. Because of the oddities of the mouse complement system, however, this may not be a good model to investigate the role of CRP in human atherosclerosis.

Animals↗

PubMed2005

Plaque rupture after short periods of fat feeding in the apolipoprotein E-knockout mouse: model characterization and effects of pravastatin treatment.

BACKGROUND: These studies examined the early time course of plaque development and destabilization in the brachiocephalic artery of the apolipoprotein E-knockout mouse, the effects of pravastatin thereon, and the effects of pravastatin on established unstable plaques. METHODS AND RESULTS: Male apolipoprotein E-knockout mice were fed a high-fat, cholesterol-enriched diet from the age of 8 weeks. Animals were euthanized at 1-week intervals between 4 and 9 weeks of fat feeding. Acutely ruptured plaques were observed in the brachiocephalic arteries of 3% of animals up to and including 7 weeks of fat feeding but in 62% of animals after 8 weeks, which suggests that there is a sharp increase in the number of plaque ruptures at 8 weeks. These acute plaque ruptures then appear to heal and form buried fibrous caps; after 9 weeks of fat feeding, mice had 1.05+/-0.15 buried fibrous caps at a single site in the brachiocephalic artery. Pravastatin (40 mg/kg of body weight per day for 9 weeks; resultant plasma concentration 16+/-4 nmol/L) had no effect on plasma cholesterol concentration in fat-fed apolipoprotein E-knockout mice but reduced the number of buried fibrous caps by 43% (P<0.0001). In longer-term experiments, the delay of pravastatin treatment until unstable plaques had developed reduced the incidence of acute plaque rupture by 36% (P<0.0001). CONCLUSIONS: Plaque rupture occurs at high frequency in the brachiocephalic arteries of male apolipoprotein E-knockout mice after 8 weeks of fat feeding. Pravastatin treatment inhibits early plaque rupture and is also effective when begun after unstable plaques have developed.

Animals↗

PubMed2002

Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice.

Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (+/-) and homozygous (-/-) for the knockout allele. Wild-type (+/+), cMyBP-C(+/-), and cMyBP-C(-/-) mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C(+/-) mice were indistinguishable from wild-type littermates, cMyBP-C(-/-) mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C(-/-) mice. Ca2+ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C(-/-) but not cMyBP-C(+/-) mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function.

Actin Cytoskeleton↗

PubMed2004

Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1.

The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.

Action Potentials↗

PubMed2001

Contribution of the 5-HT(1B) receptor to hypoxia-induced pulmonary hypertension: converging evidence using 5-HT(1B)-receptor knockout mice and the 5-HT(1B/1D)-receptor antagonist GR127935.

5-Hydroxytryptamine (5-HT)(1B) receptors mediate contraction in human pulmonary arteries, and 5-HT(1B) receptor-mediated contraction is enhanced in pulmonary arteries from hypoxic rats. Here we further examine the role of this receptor in the development of pulmonary hypertension (PHT) by examining (1) the effects of a 5-HT(1B/1D)-receptor antagonist (GR127935) on hypoxia-induced PHT (CHPHT) in rats and (2) CHPHT in 5-HT(1B)-receptor knockout mice. In rats, hypoxia increased right ventricular pressure and right ventricular hypertrophy and induced pulmonary vascular remodeling associated with an increase in pulmonary arterial wall thickness. GR127935 (3 mg. kg(-1). d(-1)) reduced all of these indices. 5-HT(1)-mediated contraction was enhanced in pulmonary arteries of the CHPHT rats. The effects of GR127935 on PHT indices were associated with an attenuation of the enhanced contractile responses to 5-HT and the 5-HT(1)-receptor agonist, 5-carboxamidotryptamine (5-CT), in isolated pulmonary arteries. In wild-type mice, hypoxia increased right ventricular hypertrophy, which was absent in 5-HT(1B)-receptor knockout mice. Hypoxia increased pulmonary vascular remodeling in wild-type mice, and this was reduced in the 5-HT(1B)-receptor knockout mice. Hypoxia increased 5-HT(1)-mediated contraction in pulmonary arteries from the wild-type mice and this was attenuated in the 5-HT(1B)-receptor knockout mice. In conclusion, the 5-HT(1B) receptor plays a role in the development of CHPHT. One possible mechanism may be via enhanced 5-HT(1) receptor-mediated contraction of the pulmonary arterial circulation.

Animals↗