ANP in regulation of arterial pressure and fluid-electrolyte balance: lessons from genetic mouse models.

The recent development of genetic mouse models presenting life-long alterations in expression of the genes for atrial natriuretic peptide (ANP) or its receptors (NPR-A, NPR-C) has uncovered a physiological role of this hormone in chronic blood pressure homeostasis. Transgenic mice overexpressing a transthyretin-ANP fusion gene are hypotensive relative to the nontransgenic littermates, whereas mice harboring functional disruptions of the ANP or NPR-A genes are hypertensive compared with their respective wild-type counterparts. The chronic hypotensive action of ANP is determined by vasodilation of the resistance vasculature, which is probably mediated by attenuation of vascular sympathetic tone at one or several prejunctional sites. Under conditions of normal dietary salt consumption, the hypotensive action of ANP is dissociated from the natriuretic activity of the hormone. However, during elevated dietary salt intake, ANP-mediated antagonism of the renin-angiotensin system is essential for maintenance of blood pressure constancy, inasmuch as the ANP gene "knockout" mice (ANP -/-) develop a salt-sensitive component of hypertension in association with failure to adequately downregulate plasma renin activity. These findings imply that genetic deficiencies in ANP or natriuretic receptor activity may be underlying causative factors in the etiology of salt-sensitive variants of hypertensive disease and other sodium-retaining disorders, such as congestive heart failure and cirrhosis.

Altered expression of natriuretic peptide receptors in proANP gene disrupted mice.

BACKGROUND: The atrial natriuretic peptide (ANP) family is a complex system consisting of at least three polypeptides and at least three types of receptor. Each peptide interacts with different types of receptor at varying degrees of affinity. To determine if natriuretic peptide levels influence natriuretic peptide receptor expression and regulation, we examined the expression of guanylyl cyclase linked GC-A, GC-B and C-receptor in the lungs of mice with a mutation that inactivates the ANP gene (Nppa). METHODS: The mRNA level of GC-A, GC-B and C-receptor in the lung were studied by ribonuclease protection assays (RPA). RESULTS: Results of RPA showed that although the mRNA level of GC-A and GC-B of heterozygous ANP+/- was not different from wild type ANP+/+ mice, they were significantly higher in the homozygous mutant ANP-/- mice. In addition, C-receptor mRNA level in ANP+/- and ANP-/- was significantly lower than ANP+/+ mice. The C-receptor results were confirmed by receptor binding assays and affinity cross-linking studies. CONCLUSIONS: Taken together these data suggest that permanent removal of ANP from the natriuretic peptide system results in an up-regulation of GC-A and GC-B, and a corresponding down-regulation of C-receptor in the lung of proANP gene disrupted mice. We postulated that changes in the natriuretic peptide receptor population may result in chronic hypertension and cardiac hypertrophy in the ANP-/- mice.

Chronic regulation of arterial blood pressure in ANP transgenic and knockout mice: role of cardiovascular sympathetic tone.

OBJECTIVE: Atrial natriuretic peptide (ANP) lowers arterial blood pressure (ABP) chronically, in association with vasodilation of the resistance vasculature. The mechanism mediating the chronic relaxant effect of ANP is likely indirectly mediated by interactions with tonic vasoeffector mechanisms, inasmuch as the resistance vasculature is relatively insensitive to direct cGMP-mediated relaxation by ANP. On the basis of evidence that ANP has widespread sympatholytic activity, the current study investigated whether the chronic hypotensive effect of ANP is mediated by attenuation of tonic cardiovascular sympathetic tone. METHODS: Total plasma catecholamine concentration and changes in basal ABP and heart rate (HR) following autonomic ganglionic blockade were measured as indices of underlying sympathetic nerve activity in hypotensive ANP-overexpressing transgenic mice (TTR-ANP), hypertensive ANP knockout mice (-/-) and the genetically-matched wild type (NT and +/+, respectively) control mice. Pressor and chronotropic responses to norepinephrine infusion were measured in ganglion-blocked mice of all genotypes, and norepinephrine receptor binding was assessed in representative tissues of -/- and +/+ mice, in order to determine whether peripheral adrenergic receptor responsiveness is altered by ANP-genotype. RESULTS: Basal ABP was significantly lower in TTR-ANP and higher in -/- compared to their wild-type controls. Basal HR did not differ significantly between mutant and control mice. Autonomic ganglionic blockade reduced ABP and HR in all genotypes, however, the relative decrease in ABP was significantly smaller in TTR-ANP and greater in -/- mice than in their respective controls. Total plasma catecholamine was significantly higher in -/- than in +/+ mice but did not differ significantly between TTR-ANP and NT mice. Norepinephrine infusion during ganglionic blockade elicited quantitatively similar pressor and chronotropic responses in mutant and control mice. Tissue norepinephrine binding did not differ significantly between -/- and +/+ mice. CONCLUSIONS: The present study shows that differences in endogenous ANP activity in mice, resulting in chronic alterations in ABP are accompanied by directional changes in underlying cardiovascular sympathetic tone, and suggests that the chronic vasodilator effect of ANP is, at least partially, dependent on attenuation of vascular sympathetic tone, possibly at a prejunctional site(s).

An A/T-rich cis-element is essential for rat angiotensin II type 1A receptor transcription in vascular smooth muscle cells.

Transcriptional mechanisms regulating the expression of the rat angiotensin II type 1A receptor (rAT1AR) gene were investigated in cultured rat vascular smooth muscle cells (VSMC). Transcriptional analyses of various 5'-deletion mutants of the rAT1AR promoter region, fused upstream from the firefly luciferase gene, demonstrated that a 71 base pair (bp) region (-557 to -486 bp, with respect to transcription initiation) was necessary for expression of this gene in VSMC. Electrophoretic mobility shift assays demonstrated that specific protein-DNA complexes were formed with the -516 to -486 bp region of the rAT1AR promoter when incubated with VSMC extract. Computer analysis of this region indicated the presence of an A/T-rich sequence (i.e., TTTAAAAATAAA) which is similar to a myocyte enhancer binding factor 2 (MEF2) cis-regulatory element (i.e., CTTAAAAATAAC). Site-directed mutagenesis of this A/T-rich sequence inhibited rAT1AR promoter activity in VSMC, suggesting that this region was necessary for expression of this gene in these cells. Immuno-gel shift experiments suggest that MEF2 heterodimers may interact with the A/T-rich sequence in the rAT1AR promoter. Additionally, it was demonstrated that a transcription factor non-homologous to MEF2 can also interact with this A/T-rich site in the rAT1AR promoter. Taken together, our results suggest that MEF2 heterodimers, and/or transcription factors non-homologous to MEF2, are required to regulate the expression of the rAT1AR gene in VSMC.

Estradiol induces C-type natriuretic peptide gene expression in mouse uterus.

Previous experiments have demonstrated that C-type natriuretic peptide (CNP) expression in the uterus varies during the estrous cycle with maximal expression at proestrus. The present study was designed to determine whether exogenous steroid hormones regulate uterine CNP expression in ovariectomized mice. Estradiol increased significantly (3-fold) uterine immunoreactive CNP (irCNP) rapidly and dose dependently in ovariectomized mice as measured by radioimmunoassay. Other steroids produced either no significant change (testosterone, 1 mg; 2-methoxyestradiol, 1 microgram) or weak induction (estriol, 1 microgram) from vehicle controls. Progesterone (1 mg) significantly attenuated the estrogen-stimulated irCNP response by 50% when injected 30 min before estrogen (1 microgram) in estrogen-primed ovariectomized mice. Estrogen-stimulated increases in uterine CNP transcripts detected by ribonuclease protection analyses were blocked by actinomycin D (160 micrograms) or ICI-164,384 (20 micrograms), a specific nuclear estrogen receptor antagonist. These results indicate that a nuclear estrogen receptor is required for estrogen to stimulate uterine CNP transcription and that progesterone negatively regulates estrogen-stimulated CNP expression.

Coronary vascular and endothelial reactivity changes in transgenic mice overexpressing atrial natriuretic factor.

Recent advances in genetic methods permit the introduction of random and defined mutations into the mouse germ line, producing novel mouse strains, some of which affect the heart and vasculature. A TTR-ANF transgenic strain of mice, which constitutively expresses a fusion gene consisting of the transthyretin promoter and the ANF structural gene, has been shown to result in a lifelong elevation of plasma atrial natriuretic factor (ANF) and a chronically lowered arterial blood pressure. However, no established method for efficient functional analysis of possible alterations in coronary vascular function in mice has been reported. In the present study, we describe an isolated mouse coronary artery preparation that permits an effective and reproducible evaluation of coronary endothelial and vascular function. Both left main and right coronary arteries (resting luminal diam 70-90 microns) were isolated and pressurized, and changes in luminal diameter were determined by videomicroscopy. The coronary pressure-luminal diameter relationship was not significantly different between TTR-ANF transgenic and nontransgenic littermates. Relaxation of coronaries to 0.1-100 nM ANF was significantly reduced in transgenic [maximum effect (Emax) = 43 +/- 10% (mean +/- SE) of 11 vessels] compared with nontransgenic (Emax = 73 +/- 7%, n = 15) mice. Similarly, the relaxant response to an endothelium-dependent dilator, acetylcholine, but not to endothelium-independent dilators sodium nitroprusside and isoproterenol, was significantly decreased in transgenic (Emax = 46 +/- 10%, n = 12) compared with nontransgenic (Emax = 85 +/- 5%, n = 14) mice. In contrast, the dose-dependent vasoconstriction to endothelin-1, KCl and the thromboxane mimetic U-46619 was not significantly different between the two groups. These results indicate that lifelong ANF elevation in mice is associated with a decreased responsiveness of coronary vasorelaxation to ANF, possibly resulting from receptor downregulation and/or desensitization. Endothelium-dependent relaxation was also significantly depressed in transgenic mouse coronary arteries, but smooth muscle-specific dilation and constriction were not affected. The present findings are consistent with previous studies of TTR-ANF transgenic mice showing that ANF regulates arterial blood pressure and vascular function.

Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide.

Genomic sequences encoding mouse C-type natriuretic peptide (CNP) were isolated from bacteriophage libraries and characterized by restriction enzyme and sequence analysis. The mouse CNP gene (Nppc) comprised at least two exons and one intron and included several cis-regulatory elements in the 5'-flanking sequence. The deduced amino acid sequence of mouse CNP-22 was identical to other mammalian CNPs. Analysis of allele distributions in interspecific back-cross and recombinant inbred strains assigned Nppc to chromosome 1. CNP transcripts were detected by ribonuclease protection analysis in brain, ovary, and uterus, with lower levels in testes and epididymus. Uterine CNP transcripts and protein were low in sexually immature mice and adults at estrus and increased at proestrus, but similar variations in ovarian CNP expression were not statistically significant. Atrial natriuretic peptide and B-type natriuretic peptide transcripts were not detected in mouse ovary or uterus. Thus CNP gene expression is regulated by tissue-specific and inducible mechanisms in female reproductive organs. Correlations between CNP expression and uterine fluid content suggest that CNP may regulate uterine fluid balance in mice and other mammals.

Organization of the mouse cardiac natriuretic peptide locus encoding BNP and ANP.

The genes encoding the mouse atrial natriuretic peptide and B-type natriuretic peptide were previously shown to be physically linked on mouse chromosome 4 (Steinhelper ME, 1993, Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene. Circ Res 72: 984-992). In the present study the spatial relationship and orientation of the mouse atrial natriuretic peptide and B-type natriuretic peptide transcription units were identified and a physical map of the mouse cardiac natriuretic peptide locus was obtained. To this end, genomic clones encoding atrial natriuretic peptide and B-type natriuretic peptide were isolated from a mouse genomic library in bacteriophage P1. Three independent clones encoding atrial natriuretic peptide were isolated and two of these also encode B-type natriuretic peptide. Both transcripts were shown to arise from the same DNA strand, with B-type natriuretic peptide encoded approximately 15 kb 5'-of atrial natriuretic peptide based on field inversion gel electrophoresis of fragments amplified with specific oligonucleotides. This finding was confirmed by isolation of subclones comprising the entire locus and by blot hybridization analysis of mouse genomic DNA. The results show that the genes encoding the two natriuretic peptides expressed predominantly in mammalian cardiac myocytes are organized in tandem on mouse chromosome 4. This information provides a physical framework for investigating mechanisms that regulate transcription of the cardiac natriuretic peptide locus.

Overexpression of NGF within the heart of transgenic mice causes hyperinnervation, cardiac enlargement, and hyperplasia of ectopic cells.

Nerve growth factor (NGF) supports the survival of developing sympathetic and a subpopulation of sensory neurons. In the adult it participates in maintenance of the neurotransmitter phenotype of responsive neurons. The amount of NGF synthesized by a given target tissue determines its final innervation density; those developing neurons that fail to receive sufficient NGF undergo apoptosis. In order to examine the ramifications of this principle in the context of a specific target organ, a transgenic mouse model was developed in which NGF expression was increased in developing and adult cardiac tissue by placing a NGF minigene under the transcriptional control of the cardiac-specific alpha-myosin heavy chain promoter. Transgenic mice developed cardiac enlargement secondary to both an increase in myocardial mass and the presence of an abundant ectopic cell population. Immunohistochemical analyses with the neural marker S-100 revealed staining of a subpopulation of ectopic cells, suggesting their derivation from the neural crest. Whereas immunostaining for the neuronal-specific protein neuron-specific enolase demonstrated labeling of another subpopulation of ectopic cells within the heart. Measurements of cardiac tissue catecholamine levels revealed a marked elevation in transgenic mice, consistent with sympathetic hyperinnervation. Analysis of mediastinal sympathetic ganglia revealed increases in both the size and the number of neurons. In this model, increased expression of NGF produced hyperinnervation of the heart, pathological cardiac growth, and the recruitment and/or expansion of an ectopic, neural crest-derived cell type.

Spontaneous activity in transgenic mouse heart: comparison of primary atrial tumor with cultured AT-1 atrial myocytes.

INTRODUCTION: We have generated transgenic animals that heritably develop atrial tumors composed of differentiated proliferating cardiomyocytes. Experiments were initiated to characterize the electrical properties of these cells. METHODS AND RESULTS: We show that the primary atrial tumors are composed of discrete foci that exhibit spontaneous automatic activity. A direct correlation was observed between tumor size and firing rate of these foci. In addition to the primary atrial tumors, we examined the properties of cultured cardiomyocytes isolated from a transplantable transgenic tumor lineage (designated AT-1 cells). Cultured AT-1 cells are also spontaneously automatic. The action potential configuration from these preparations is similar to that observed in nontransgenic atrial cardiomyocytes, albeit somewhat more depolarized and of longer duration. As would be expected for cardiomyocytes of atrial origin, the transgenic cardiomyocyte preparations hyperpolarize during muscarinic stimulation due to increased K+ conductance mediated by a pertussis toxin sensitive G-protein. Assessment of pharmacologic blockage of the "if" pacemaker current suggests that the automaticity of both transgenic cardiomyocyte preparations may be of novel origin. In this context, the cultured AT-1 cells showed spontaneous behavior that was clearly of cellular origin; this activity was manifest as transient bursts of electrical activity followed by periods of electrical quiescence. This bursting pattern is unusual for normal adult cardiomyocytes, but has been observed in several other cell types. In the primary tumors, automatic behavior may arise from a similar cellular origin or alternatively from a microreentrant phenomena. CONCLUSION: Primary tumors and AT-1 cells show essential atrial electrophysiology with important novel features.

Cardiopulmonary responses to chronic hypoxia in transgenic mice that overexpress ANP.

Elevated plasma atrial natriuretic peptide (ANP) levels have been shown to blunt pulmonary hemodynamic responses to chronic hypoxia, but whether elevated circulating ANP levels negatively feedback on cardiac expression of the ANP gene is unknown. Using a recently developed strain of transgenic mouse (TTR-ANF) that expresses a transthyretin promoter-ANP fusion gene in the liver, we studied the effect of chronically elevated plasma ANP levels on cardiac hypertrophic and pulmonary hemodynamic responses and expression of the endogenous cardiac ANP gene during chronic hypoxia. Plasma ANP levels were 10-fold higher in TTR-ANF mice than in their non-transgenic littermates. After 3 wk of hypobaric hypoxia (0.5 atm), right ventricular hypertrophy and pulmonary hypertension had developed in both groups of mice, but TTR-ANF mice had lower right ventricle-to-left ventricle plus septum weight ratios (0.39 +/- 0.01 vs. 0.45 +/- 0.02), right ventricular systolic pressures (25 +/- 2 vs. 29 +/- 2 mmHg), and lung dry weight-to-body weight ratios (0.48 +/- 0.03 vs. 0.57 +/- 0.01 mg/g) and less muscularization of peripheral pulmonary vessels (8.3 +/- 1.4 vs. 17.4 +/- 2.5%) than nontransgenic controls. Right atrial and ventricular steady-state ANP mRNA levels were the same in both groups of mice under normoxic and hypoxic conditions despite much higher plasma ANP levels and less pulmonary hypertension in TTR-ANF mice. We conclude that chronically elevated plasma ANP levels attenuate the development of hypoxic pulmonary hypertension in mice but do not suppress cardiac expression of the endogenous ANP gene under normoxic conditions nor blunt the upregulation of right ventricular ANP expression during chronic hypoxia.

Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene.

The structure of the mouse natriuretic peptide type-B (BNP) gene was determined by isolating and sequencing genomic clones. The mouse BNP gene was structurally similar to other natriuretic peptide genes and comprised three exons and two introns. Expression of the mouse BNP gene was found only in cardiac tissue as determined by ribonuclease protection analyses. Initiation of transcription was 31 bp downstream from a consensus TATA box as determined by primer extension analysis of cardiac RNA. Comparative DNA sequence analysis identified several DNA elements with potential transcriptional regulatory function. Comparative amino acid sequence analysis showed that the N-terminal portion of the mouse and rat BNP precursors was more conserved than the C-terminal 45-amino-acid sequence that constitute the bioactive BNP-45 peptide. The proteolytic processing site (RXXR-S) generating bioactive BNPs was highly conserved among all BNP precursors and was identical to the consensus site of furin, a calcium-dependent serine endoprotease. Finally, the BNP gene was mapped using recombinant inbred DNA and a polymerase chain reaction-based restriction fragment-length polymorphism assay to mouse chromosome 4 near the atrial natriuretic factor (Anf) locus. No recombination event between Bnp and Anf was evident in the 39 recombinant inbred and inbred strains examined. This physical linkage between the two natriuretic peptide genes expressed in cardiac tissue may be important for their transcriptional regulation.

Cardiomyocyte proliferation in mice expressing alpha-cardiac myosin heavy chain-SV40 T-antigen transgenes.

To determine the proliferative potential of adult ventricular cardiomyocytes, we have generated transgenic mice that express the SV40 large T-antigen oncogene in the heart. A fusion gene comprised of the rat alpha-cardiac myosin heavy chain promoter and the SV40 early region was used to target oncogene expression to the myocardium. Expression of SV40 large T-antigen was observed in both atrial and ventricular cardiomyocytes in adult transgenic animals. T-antigen expression was associated with hyperplasia in the targeted cells. Immunohistological analysis indicated that the proliferating cells continued to express sarcomeric myosin. Electron microscopic examination demonstrated that cardiomyocytes in various stages of the cell cycle retained ultrastructural characteristics typical of mitotic cardiac muscle cells in vivo. Cardiomyocytes isolated from transgenic tumors were able to proliferate in culture and retained a differentiated phenotype, as evidenced by spontaneous contractile activity. Preliminary studies indicate that these cells can undergo a limited number of passages while retaining this differentiated phenotype. These studies demonstrate that both ventricular and atrial cardiomyocytes from transgenic mice proliferate in response to targeted T-antigen expression.

Gene expression and atrial natriuretic factor processing and secretion in cultured AT-1 cardiac myocytes.

BACKGROUND: Studies were carried out to characterize several biochemical features of cultured AT-1 cells. METHODS AND RESULTS: These cells were obtained from a transplantable atrial cardiomyocyte tumor lineage. Reverse transcriptase-polymerase chain reaction-based analyses demonstrated that the pattern of gene expression of cultured AT-1 cells was similar to that of adult atrial myocytes. AT-1 cells expressed atrial natriuretic factor (ANF), alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. Radioimmunoassays verified that the cells synthesized, stored, and secreted ANF. Through size-exclusion, reversed-phase, and carboxymethyl-ion-exchange high-performance liquid chromatography, it was shown that cultured AT-1 cells stored ANF as pro-ANF (ANF-[1-126]), which was cosecretionally processed quantitatively to ANF-(1-98) and the bioactive 28-amino-acid ANF-(99-126). In addition, cultured AT-1 cells secreted ANF at almost a sixfold greater rate in response to endothelin-1, a potent secretagogue of ANF. KCl, metenkephalinamide, isoproterenol, phenylephrine, and 12-O-tetradecanoyl-phorbol-13-acetate also stimulated ANF release. CONCLUSIONS: These studies, in combination with previous findings, demonstrated that cultured AT-1 cells, while maintaining the ability to proliferate, have retained functional, biochemical, and ultrastructural features that are characteristic of adult atrial myocytes.

Engineering the cardiovascular system. Blood pressure regulation.

We have generated several lineages of transgenic mice that exhibit chronic elevations in the steady-state concentration of atrial natriuretic factor (ANF) in the peripheral circulation. ANF, a peptide hormone synthesized primarily by atrial cardiomyocytes, is a potent natriuretic and diuretic. ANF also reduces blood pressure transiently when acutely administered. To address the potential role of ANF in chronic cardiovascular regulation, we generated transgenic mice that express the ANF gene in the liver. The fusion genes comprised either the mouse transthyretin (TTR) or rat phosphoenolpyruvate carboxykinase (PEPCK) promoters fused to the mouse ANF structural gene and were designed to target to the liver constitutive and inducible expression of pre-pro-ANF, respectively. Transgenic animals harboring the TTR-ANF fusion gene expressed chimeric ANF transcripts exclusively in the liver. In contrast, mice harboring the PEPCK-ANF fusion gene did not express detectable amounts of ANF mRNA in liver even after induction (24-hour fasting). In the TTR-ANF mice, hepatic and plasma immunoreactive ANF concentrations were proportional to the concentration of hepatic ANF transcripts. Moreover, mean arterial blood pressure recorded in conscious transgenic mice was inversely proportional to hepatic ANF expression. These transgenic models demonstrate that chronically elevated ANF concentration can induce sustained hypotension.

Segregation of atrial-specific and inducible expression of an atrial natriuretic factor transgene in an in vivo murine model of cardiac hypertrophy.

To study the mechanisms that activate expression of the atrial natriuretic factor (ANF) gene during pressure-induced hypertrophy, we have developed and characterized an in vivo murine model of myocardial cell hypertrophy. We employed microsurgical techniques to produce a stable 35- to 45-mmHg pressure gradient across the thoracic aorta of the mouse that is associated with rapid and transient expression of an immediate-early gene program (c-fos/c-jun/junB/Egr-1/nur-77), an increase in heart weight/body weight ratio, and up-regulation of the endogenous ANF gene. These responses that are identical to those in cultured cell and other in vivo models of hypertrophy. To determine whether tissue-specific and inducible expression of the ANF gene can be segregated, we used a transgenic mouse line in which 500 base pairs of the human ANF promoter region directs atrial-specific expression of the simian virus 40 large tumor antigen (T antigen), with no detectable expression in the ventricles. Thoracic aortic banding of these mice led to a 20-fold increase in the endogenous ANF mRNA in the ventricle but no detectable expression of the T-antigen marker gene. This result provides evidence that atrial-specific and inducible expression of the ANF gene can be segregated, suggesting that a distinct set of regulatory cis sequences may mediate the up-regulation of the ANF gene during in vivo pressure overload hypertrophy. This murine model demonstrates the utility of microsurgical techniques to study in vivo cardiac physiology in transgenic mice and should allow the application of genetic approaches to identify the mechanisms that activate ventricular expression of the ANF gene during in vivo hypertrophy.

Acrylyl-PAF, a synthetic platelet-activating factor analogue.

Acrylyl-PAF, a novel platelet-activating factor analogue with substantial agonist properties, was synthesized from lyso-PAF and acrylyl chloride. Incubation of acrylyl-PAF with partially purified human serum acetylhydrolase inhibited the action of acetylhydrolase on PAF. Kinetic studies using several concentrations of PAF as well as acrylyl-PAF suggested that acrylyl-PAF was a competitive inhibitor of the acetylhydrolase. Reciprocal Lineweaver-Burk plots and Dixon plots showed that acrylyl-PAF has an apparent Ki of 2.6 microM. Radiolabeled 18: 0-acrylyl-PAF was also synthesized. Studies comparing the effectiveness of acrylyl-PAF as a substrate for the acetylhydrolase versus PAF show that approximately 60% more PAF is metabolized by acetylhydrolase than acrylyl-PAF under identical conditions. Acrylyl-PAF was shown to aggregate washed rabbit platelets and to stimulate the release of glucose in the perfused rat liver. Dose-response curves illustrate that acrylyl-PAF was about half an order of magnitude less potent than PAF as an agonist. Tissue extracts of freeze-clamped livers that were perfused with radiolabeled acrylyl-APF indicated that approximately 26% of the label associated with acrylyl-PAF washed through the liver into the perfusate, while 48% of the acrylyl-PAF remained intact in the liver. Only 9% of the acrylyl-PAF was converted to k lyso-PAF with the ramainder metabolized to other lipids. This observations stands in contrast with livers perfused with radiolabeled PAF where less than 1% of the activity associated with PAF washed through the liver while about 70% remained as intact PAF and 30% was converted to lyso-PAF. Repeated bolus infusions of acrylyl-PAF exhibited desensitization of the glycogenolytic response of the perfused rat liver. The present study describes teh synthesis and characterization of acrylyl-PAF, which was shown to be: (a) poorly hydrolyzed by the human serum acetylhydrolase; (b) a competitive inhibitor of the acetylhydrolase; and (c) an agonist with potent activity in both platelet aggregation and hepatic glycogenolytic responses.

Kidney function in ANF-transgenic mice: effect of blood volume expansion.

Transgenic mice, created from inbred C3HeB/FeJ embryos, were used to overexpress selectively in the liver a fusion gene comprising mouse transthyretin (TTR) regulatory and atrial natriuretic factor (ANF) structural sequences. Animals were anesthetized, and kidney function was studied before and after blood volume expansion. Baseline urine volumes and electrolyte excretions were not significantly different from those of non-transgenic littermates, despite a markedly lower arterial blood pressure in the experimental group. A slightly lower glomerular filtration rate (GFR) in transgenics was not different statistically. Plasma ANF levels measured by radioimmunoassay were approximately 10-fold higher in the transgenic animals, compared with their nontransgenic siblings. After acute blood volume expansion, the diuretic, natriuretic, kaliuretic, and chloruretic responses were markedly enhanced in the transgenic group. Arterial pressure was increased as a result of hypervolemia, although it remained relatively depressed relative to the controls. GFR again was not different. We conclude that transgenic mice overexpressing ANF can maintain normal excretion of salt and water, possibly via ANF-induced reduction of renal perfusion pressure. After acute blood volume expansion, an increase in pressure may allow full renal expression of the chronically elevated ANF levels.

Proliferation in vivo and in culture of differentiated adult atrial cardiomyocytes from transgenic mice.

Transgenic mice expressing atrial natriuretic factor-SV40 T-antigen fusion genes (ANF-TAG) developed unilateral right atrial tumors composed of differentiated dividing cardiomyocytes. The atrial tumors could be propagated as transplantable tumor lineages in syngeneic animals. Cardiomyocytes derived from ANF-TAG atrial tumors did not proliferate in tissue culture. However, cardiomyocytes derived from the transplantable tumor lines proliferated in culture, and these proliferating cardiomyocytes could be passaged in culture and recovered from frozen stocks. Cardiomyocytes from either tumor source were highly differentiated as determined by diverse functional and structural criteria. The cells continued to express numerous cardiac-specific proteins and retained ultrastructural features characteristic of cardiomyocytes including well-formed myofibrils, transverse tubules, and intercalated disks. In addition, the cultured cells displayed spontaneous electrical and contractile activities. These atrial tumor cardiomyocytes are a novel experimental resource for the identification of genes regulating the cardiomyocyte cell cycle.