Nerve high-resolution ultrasonography in tangier disease.

INTRODUCTION: Tangier disease (TD) is an autosomal recessive disorder characterized by severe reduction in high-density lipoprotein and accumulation of cholesterol esters in peripheral nerves and other tissues. The aim of this study was to evaluate whether nerve high-resolution ultrasonography (HRUS) can detect morphological nerve changes in TD. METHODS: Three related patients of a previously reported Italian family with Tangier disease, carrying the Y1698X mutation in ABCA1, underwent clinical, neurophysiological, and quantitative nerve HRUS evaluation. Nerve HRUS data were compared with normal controls. RESULTS: Despite neurophysiological abnormalities, no quantitative HRUS abnormality was detected in peripheral nerves. DISCUSSION: Normalcy of HRUS in neurophysiologically abnormal nerves suggests possible subtle abnormalities that escape quantitative HRUS detection. Systematic studies in larger TD cohorts with different mutations are needed to confirm our findings. Muscle Nerve 59:587-587, 2019.

Peripheral neuropathy in Tangier disease: A literature review and assessment.

Tangier disease (TD) (OMIM#205400) is a rare cause of inherited metabolic neuropathies characterized by marked deficiency of high-density lipoproteins and accumulation of cholesterol esters in various tissue resulting from reverse cholesterol transport deficiency. We report a case of a patient with TD with multifocal demyelinating neuropathy with conduction block who presents with winging scapula, tongue, and asymmetric extremity weakness. We also present a review of all studies published from 1960 to 2017 regarding peripheral neuropathy in TD. Our search identified 54 patients with TD with peripheral neuropathy. Syringomyelia-like neuropathy subtype (52.4%) was more frequent than multifocal sensorial and motor neuropathy subtype (26.2%), focal neuropathy subtype (19.1%), and distal symmetric polyneuropathy subtype (2.4%). Splenomegaly was the most common (40.7%) clinical manifestation in these patients. The pattern of electrodiagnostic abnormalities are: (1) demyelinating abnormalities were more predominant in the upper extremities than in the lower extremities and (2) slowing of motor nerve conduction was more prominent in the intermediate segment than in distal nerve segments. The sural-sparing pattern was present in 34.6% and conduction block was present in 11.5% of the patients. Our literature review and our case showed the clinical spectrum of TD neuropathy is quite wide and that it should be considered in the differential diagnosis of non-uniform demyelinating neuropathies.

Can Tangier disease cause male infertility? A case report and an overview on genetic causes of male infertility and hormonal axis involved.

Tangier disease is an autosomal recessive disorder caused by mutations in the ABCA1 gene and characterized by the accumulation of cholesteryl ester in various tissues and a near absence of high-density lipoprotein. The subject in this investigation was a 36-year-old Italian man with Tangier disease. He and his wife had come to the In Vitro Fertilization Unit, Pesaro Hospital (Azienda Ospedaliera Ospedali Riuniti Marche Nord) seeking help regarding fertility issues. The man was diagnosed with severe oligoasthenoteratozoospermia. Testosterone is the sex hormone necessary for spermatogenesis and cholesterol is its precursor; hence, we hypothesized that the characteristic cholesterol deficiency in Tangier disease patients could compromise their fertility. The aim of the study was to therefore to determine if there is an association between Tangier disease and male infertility. After excluding viral, infectious, genetic and anatomical causes of the subject's oligoasthenoteratozoospermia, we performed a hormonal analysis to verify our hypothesis. The patient was found to be negative for frequent bacteria and viruses. The subject showed a normal male karyotype and tested negative for Yq microdeletions and Cystic Fibrosis Transmembrane Conductance Regulator gene mutations. A complete urological examination was performed, and primary hypogonadism was also excluded. Conversely, hormonal analyses showed that the subject had a high level of follicle stimulating hormone and luteinizing hormone, low total testosterone and a significant decline in inhibin B. We believe that the abnormally low cholesterol levels typically found in subjects with Tangier disease may result in a reduced testosterone production which in turn could affect the hormonal axis responsible for spermatogenesis leading to a defective maturation of spermatozoa.

Clinical, Electrodiagnostic, and Genetic Features of Tangier Disease in an Adolescent Girl with Presentation of Peripheral Neuropathy.

Tangier disease (TD) is a rare, autosomal recessive inherited disorder caused by a mutation in the adenosine triphosphate-binding cassette transporter 1 (ABCA1) gene, which results in a decrease in plasma high-density lipoprotein (HDL) levels. Peripheral neuropathy can be seen in approximately 50% of patients with TD, which usually occurs after the age of 15 years, and is characterized by relapsing-remitting mono- or polyneuropathy or syringomyelia-like neuropathy. Herein, we report a 16-year-old female patient who was initially diagnosed with peripheral neuropathy at the age of 13 years. Whole exome sequencing was performed, and a nonsense mutation (p.Arg1817X) in ABCA1 was identified. The patient was investigated for systemic findings of TD after the genetic diagnosis was made, and low (< 5 mg/dL) levels of HDL cholesterol were detected by lipid electrophoresis. Other family members were reexamined after the diagnosis of the proband, and asymptomatic sister of the proband was diagnosed with TD. We would like to emphasize that TD should be considered in the differential diagnosis of pediatric patients presenting with peripheral neuropathy; furthermore detection of HDL levels by lipid electrophoresis is a simple but indicative diagnostic test.

ABCA1 mediates unfolding of apolipoprotein AI N terminus on the cell surface before lipidation and release of nascent high-density lipoprotein.

OBJECTIVE: To gain insight into the mechanism by which ABCA1 generates nascent high-density lipoprotein. APPROACH AND RESULTS: HEK293 cells were stably transfected with ABCA1 vectors, encoding wild type, and the W590S and C1477R Tangier disease mutation isoforms, along with the K939M ATP-binding domain mutant. Apolipoprotein AI (ApoAI) binding, plasma membrane remodeling, cholesterol efflux, apoAI cell surface unfolding, and apoAI cell surface lipidation were determined, the latter 2 measured using novel fluorescent apoAI indicators. The W590S isoform had decreased plasma membrane remodeling and lipid efflux activities, and the C1477R isoform had decreased apoAI binding, and lipid efflux activities, whereas the K939M isoform did not bind apoAI, remodel the membrane, or efflux cholesterol. However, all ABCA1 isoforms led to apoAI unfolding at the cell surface, which was higher for the isoforms that increased apoAI binding. ApoAI lipidation was not detected on ABCA1-expressing cells, only in the conditioned medium, consistent with rapid release of nascent high-density lipoprotein from ABCA1-expressing cells. CONCLUSIONS: We identified a third activity of ABCA1, the ability to unfold the N terminus of apoAI on the cell surface. Our results support a model in which unfolded apoAI on the cell surface is an intermediate in its lipidation and that, once apoAI is lipidated, it forms an unstable structure that is rapidly released from the cells to generate high-density lipoprotein.

Tangier disease: epidemiology, pathophysiology, and management.

Tangier disease is one of the most severe forms of familial high-density lipoprotein (HDL) deficiency. Since its discovery it has been diagnosed in about 100 patients and is characterized by severe plasma deficiency or absence of HDL, apolipoprotein A-I (apoA-I, the major HDL apolipoprotein) and by accumulation of cholesteryl esters in many tissues throughout the body. The biochemical signs of this condition are plasma HDL concentrations less than 5 mg/dL, low total plasma cholesterol (below 150 mg/dL), and normal or high plasma triglycerides. Tangier disease is caused by mutations in the 'ATP-Binding Cassette transporter A1' (ABCA1) gene, which encodes the membrane transporter ABCA1. This transporter plays a key role in the first step of reverse cholesterol transport, through which the efflux of free cholesterol from peripheral cells is transferred to lipid-poor apoA-I. The Tangier disease clinical phenotype is inherited as an autosomal recessive trait, the biochemical phenotype is inherited as an autosomal co-dominant trait. Nearly all the children affected by Tangier disease were identified on the basis of large, yellow-orange tonsils, while half of the adult patients affected by Tangier disease came to medical attention because of symptoms of neuropathy. Diagnosis in the remaining subjects was related to the clinical features of hepatomegaly, splenomegaly, premature myocardial infarction (about 30% of Tangier disease cases) or stroke, thrombocytopenia, anemia, gastrointestinal disorders, corneal opacities, hypocholesterolemia, low HDL cholesterol, or following a familial screening of Tangier patients. To date there is no specific treatment for Tangier disease. Old and recently designed drugs, known to increase HDL levels, have been shown to be ineffective in Tangier patients. The possible and more realistic therapeutic strategy should be designed to obtain a selective increase of mature HDL concentration to restore cholesterol efflux. Recently designed drugs like the cholesteryl ester transfer protein (CETP) inhibitors dalcetrapib and anacetrapib and reconstituted forms of HDL could be considered until the development of gene therapy.

Clinical and electrophysiological characteristics of neuropathy associated with Tangier disease.

Tangier disease (TD) (OMIM#205400) is a rare autosomal recessive disorder resulting from mutations in the ABCA1 gene, leading to decreased levels of plasma high-density lipoproteins (HDL). Peripheral neuropathy is a common finding in this disease, and may present as relapsing/remitting mono/polyneuropathies or as syringomyelia-like neuropathy. We retrospectively analyzed four patients, and report here their clinical, biological, electrophysiological, imaging, and genetic findings. Three patients had a typical pseudosyringomyelic neuropathy including facial diplegia, but asymmetrical onset was observed in one patient who had first been misdiagnosed with Lewis-Sumner syndrome. Electrophysiological pattern was heterogeneous, showing both signs of demyelination and axonal degeneration. Truncating mutations of the ABCA1 gene, including two previously undescribed mutations, were constantly found. Atypical symptom onset and demyelinating features on electrophysiological examination can be misleading in case of pseudosyringomyelic neuropathy. These reports illustrate two different neurological phenotypes in TD, namely the pseudosyringomyelic type and the Lewis-Sumner-like type, and advocate for a systematic assessment of lipid profile including HDL cholesterol in demyelinating neuropathies.

A novel role for ABCA1-generated large pre-beta migrating nascent HDL in the regulation of hepatic VLDL triglyceride secretion.

In Tangier disease, absence of ATP binding cassette transporter A1 (ABCA1) results in reduced plasma HDL and elevated triglyceride (TG) levels. We hypothesized that hepatocyte ABCA1 regulates VLDL TG secretion through nascent HDL production. Silencing of ABCA1 expression in oleate-stimulated rat hepatoma cells resulted in: 1) decreased large nascent HDL (>10 nm diameter) and increased small nascent HDL (<10 nm) formation, 2) increased large buoyant VLDL1 particle secretion, and 3) decreased phosphatidylinositol-3 (PI3) kinase activation. Nascent HDL-containing conditioned medium from rat hepatoma cells or HEK293 cells transfected with ABCA1 was effective in increasing PI3 kinase activation and reducing VLDL TG secretion in ABCA1-silenced hepatoma cells. Addition of isolated large nascent HDL particles to ABCA1-silenced hepatoma cells inhibited VLDL TG secretion to a greater extent than small nascent HDL. Similarly, addition of recombinant HDL, but not human plasma HDL, was effective in attenuating TG secretion and increasing PI3 kinase activation in ABCA1-silenced cells. Collectively, these data suggest that large nascent HDL particles, assembled by hepatic ABCA1, generate a PI3 kinase-mediated autocrine signal that attenuates VLDL maturation and TG secretion. This pathway may explain the elevated plasma TG concentration that occurs in most Tangier subjects and may also account, in part, for the inverse relationship between plasma HDL and TG concentrations in individuals with compromised ABCA1 function.

Cholesterol-binding toxins and anti-cholesterol antibodies as structural probes for cholesterol localization.

Cholesterol is one of the major constituents of mammalian cell membranes. It plays an indispensable role in regulating the structure and function of cell membranes and affects the pathology of various diseases. In recent decades much attention has been paid to the existence of membrane microdomains, generally termed lipid "rafts", and cholesterol, along with sphingolipids, is thought to play a critical role in raft structural organization and function. Cholesterol-binding probes are likely to provide useful tools for analyzing the distribution and dynamics of membrane cholesterol, as a structural element of raft microdomains, and elsewhere within the cell. Among the probes, non-toxic derivatives of perfringolysin O, a cholesterol-binding cytolysin, bind cholesterol in a concentration-dependent fashion with a strict threshold. They selectively recognize cholesterol in cholesterol-enriched membranes, and have been used in many studies to detect microdomains in plasma and intracellular membranes. Anti-cholesterol antibodies that recognize cholesterol in domain structures have been developed in recent years. In this chapter, we describe the characteristics of these cholesterol-binding proteins and their applications to studies on membrane cholesterol localization.

Lewis-Sumner syndrome and Tangier disease.

OBJECTIVE: To report unusual electrophysiologic data in a patient with Tangier disease in an effort to better understand the pathophysiologic features of the peripheral nerve lesions in this disease. DESIGN: Case report. PATIENT: A 15-year-old girl had subacute onset of asymmetric neuropathy with persistent conduction block, resembling Lewis-Sumner syndrome. MAIN OUTCOME MEASURES: Electrophysiologic data in Tangier disease. RESULTS: After initially unsuccessful treatment with intravenously administered immunoglobulins, the finding of an abnormal lipid profile led to the diagnosis of Tangier disease due to the R587W mutation in the adenotriphosphate-binding cassette transporter-1 gene (ABCA1) (OMIM 9q22-q31). CONCLUSIONS: Conduction block, which is the electrophysiologic hallmark of focal demyelination, can be present in Tangier disease. It could be induced by focal nerve ischemia or by preferential lipid deposition in the paranodal regions of myelinated Schwann cells. The presence of a conduction block in Tangier disease may lead to a misdiagnosis of dysimmune neuropathy.

Contribution of Cdc42 to cholesterol efflux in fibroblasts from Tangier disease and Werner syndrome.

Atherosclerotic cardiovascular disease is a life-threatening disorder. Cholesterol efflux from the cells is the rate-limiting step in regulating the intracellular cholesterol content as well as raft structure in the plasma membrane. The defect of cholesterol efflux leads to the development of atherosclerosis. Tangier disease (TD), a hereditary high-density lipoprotein deficiency, is characterized by the presence of defective cellular cholesterol efflux. Using the cDNA subtraction technique, we found that expression of Cdc42 was decreased markedly in fibroblasts and macrophages from patients with TD. Madin-Darby canine kidney cells expressing the dominant-negative form of Cdc42 had a reduced lipid efflux; inversely, cells expressing the active form had increased efflux. Furthermore, we found that cellular lipid efflux was defective and Cdc42 was reduced in fibroblasts from a premature aging disorder, Werner syndrome. Complementation experiments using an adenovirus carrying wild-type Cdc42 successfully corrected impaired lipid efflux in Werner syndrome cells. We concluded that Cdc42 may play important roles in cellular cholesterol efflux and that dysregulation of this type of RhoGTPase might lead to the development of atherosclerotic cardiovascular disease.

Identification of an ABCA1-dependent phospholipid-rich plasma membrane apolipoprotein A-I binding site for nascent HDL formation: implications for current models of HDL biogenesis.

It is well accepted that both apolipoprotein A-I (apoA-I) and ABCA1 play crucial roles in HDL biogenesis and in the human atheroprotective system. However, the nature and specifics of apoA-I/ABCA1 interactions remain poorly understood. Here, we present evidence for a new cellular apoA-I binding site having a 9-fold higher capacity to bind apoA-I compared with the ABCA1 site in fibroblasts stimulated with 22-(R)-hydroxycholesterol/9-cis-retinoic acid. This new cellular apoA-I binding site was designated "high-capacity binding site" (HCBS). Glyburide drastically reduced (125)I-apoA-I binding to the HCBS, whereas (125)I-apoA-I showed no significant binding to the HCBS in ABCA1 mutant (Q597R) fibroblasts. Furthermore, reconstituted HDL exhibited reduced affinity for the HCBS. Deletion of the C-terminal region of apoA-I (Delta187-243) drastically reduced the binding of apoA-I to the HCBS. Interestingly, overexpressing various levels of ABCA1 in BHK cells promoted the formation of the HCBS. The majority of the HCBS was localized to the plasma membrane (PM) and was not associated with membrane raft domains. Importantly, treatment of cells with phosphatidylcholine-specific phospholipase C, but not sphingomyelinase, concomitantly reduced the binding of (125)I-apoA-I to the HCBS, apoA-I-mediated cholesterol efflux, and the formation of nascent apoA-I-containing particles. Together, these data suggest that a functional ABCA1 leads to the formation of a major lipid-containing site for the binding and the lipidation of apoA-I at the PM. Our results provide a biochemical basis for the HDL biogenesis pathway that involves both ABCA1 and the HCBS, supporting a two binding site model for ABCA1-mediated nascent HDL genesis.

Senescent phenotypes of skin fibroblasts from patients with Tangier disease.

Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) approximately 10 compared with control cells. TD cells practically ceased proliferation at PDL approximately 30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated beta-galactosidase (SA-beta-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients.

Pseudomonas aeruginosa and sPLA2 IB stimulate ABCA1-mediated phospholipid efflux via ERK-activation of PPARalpha-RXR.

Bacterial infection triggers an acute inflammatory response that might alter phospholipid metabolism. We have investigated the acute-phase response of murine lung epithelia to Pseudomonas aeruginosa infection. Ps. aeruginosa triggered secretion of the pro-inflammatory lipase, sPLA2 IB (phospholipase A2 IB), from lung epithelium. Ps. aeruginosa and sPLA2 IB each stimulated basolateral PtdCho (phosphatidylcholine) efflux in lung epithelial cells. Pre-treatment of cells with glyburide, an inhibitor of the lipid-export pump, ABCA1 (ATP-binding cassette transporter A1), attenuated Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux. Effects of Ps. aeruginosa and sPLA2 IB were completely abolished in human Tangier disease fibroblasts, cells that harbour an ABCA1 genetic defect. Ps. aeruginosa and sPLA2 IB induced the heterodimeric receptors, PPARa (peroxisome-proliferator-activated receptor-a) and RXR (retinoid X receptor), factors known to modulate ABCA1 gene expression. Ps. aeruginosa and sPLA2 IB stimulation of PtdCho efflux was blocked with PD98059, a p44/42 kinase inhibitor. Transfection with MEK1 (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase 1), a kinase upstream of p44/42, increased PPARa and RXR expression co-ordinately with increased ABCA1 protein. These results suggest that pro-inflammatory effects of Ps. aeruginosa involve release of an sPLA2 of epithelial origin that, in part, via distinct signalling molecules, transactivates the ABCA1 gene, leading to export of phospholipid.

ABCA1, from pathology to membrane function.

The ABCA1 transporter is the prototype of the A class of mammalian adenosine triphosphate binding cassette transporters and one of the largest members of this family. ABCA1 has been originally identified as an engulfment receptor on macrophages and, more recently, it has been shown to play an essential role in the handling of cellular lipids. Indeed by promoting the effluxes of membrane phospholipids and cholesterol to lipid-poor apoprotein acceptors, ABCA1 controls the formation of high-density lipoproteins and thus the whole process of reverse cholesterol transport. A number of additional phenotypes have been found in the mouse model of invalidation of the ABCA1 gene. In spite of their clinical diversity, they all are extremely sensitive to variations in the physicochemical properties of the cell membrane, which ABCA1 controls as a lipid translocator.

Tangier disease four decades of research: a reflection of the importance of HDL.

Reduced circulating levels of high density lipoprotein cholesterol (HDL-C) are a frequent lipoprotein disorder in coronary heart disease patients and can be caused by either genetic and/or environmental factors (sedentary lifestyle, diabetes mellitus, smoking, obesity or a diet enriched in carbohydrates). Extremely low serum HDL-C levels occur in patients with Tangier disease (TD), which is caused by mutations in the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1). Clinical manifestations are related to the storage of cholesteryl esters in reticuloendothelial tissues and to peripheral neuropathy. This review focuses on the genetic and lipid abnormalities of TD, the consequence of these on clinical outcome and the possible treatment options. These abnormalities reflect the importance of HDL in the pathogenesis of vascular disease.

ABCA1-dependent but apoA-I-independent cholesterol efflux mediated by fatty acid-bile acid conjugates (FABACs).

FABACs (fatty acid-bile acid conjugates) are synthetic molecules that are designed to treat a range of lipid disorders. The compounds prevent cholesterol gallstone formation and diet-induced fatty liver, and increase reverse cholesterol transport in rodents. The aim of the present study was to investigate the effect of FABACs on cholesterol efflux in human cells. Aramchol (3beta-arachidylamido-7alpha,12alpha,5beta-cholan-24-oic acid) increased cholesterol efflux from human skin fibroblasts in a dose-dependent manner in the absence of known efflux mediators such as apoA-I (apolipoprotein A-I), but had little effect on phospholipid efflux. An LXR (liver X receptor) agonist strongly increased Aramchol-induced cholesterol efflux; however, in ABCA1 (ATP-binding cassette transporter A1)-deficient cells from Tangier disease patients, the Aramchol effect was absent, indicating that activity of ABCA1 was required. Aramchol did not affect ABCA1 expression, but plasma membrane levels of the transporter increased 2-fold. Aramchol is the first small molecule that induces ABCA1-dependent cholesterol efflux without affecting transcriptional control. These findings may explain the beneficial effect of the compound on atherosclerosis.

Confocal microscopy and corneal sensitivity in a patient with corneal manifestations of Tangier disease.

PURPOSE: Tangier disease is an autosomal recessive disorder in which cholesterol-rich lipids are deposited at various tissues of the body including the cornea. In this case report, the corneal changes in a patient with Tangier disease are described. METHODS: A 72-year-old patient who was diagnosed with Tangier disease 25 years before received a complete eye examination including confocal microscopy and Cochet-Bonnet esthesiometry. RESULTS: Slit-lamp biomicroscopy and confocal microscopy showed bilateral corneal opacifications caused by lipid accumulation. Confocal microscopy showed that pathologic changes in the cornea in Tangier disease are limited to the stroma. Neither a reduced corneal sensation nor lid abnormalities as previously described in Tangier disease were found. CONCLUSION: Confocal microscopy helps to identify corneal changes in the stroma caused by Tangier disease easily missed in a slit-lamp examination. The ocular manifestations of Tangier disease do not necessarily include a reduced corneal sensitivity and lid abnormalities.

Restoration of endothelial function by increasing high-density lipoprotein in subjects with isolated low high-density lipoprotein.

BACKGROUND: Loss-of-function mutations in the ATP-binding cassette (ABCA)-1 gene locus are the underlying cause for familial hypoalphalipoproteinemia, providing a human isolated low-HDL model. In these familial hypoalphalipoproteinemia subjects, we evaluated the impact of isolated low HDL on endothelial function and the vascular effects of an acute increase in HDL. METHODS AND RESULTS: In 9 ABCA1 heterozygotes and 9 control subjects, vascular function was assessed by venous occlusion plethysmography. Forearm blood flow responses to the endothelium-dependent and -independent vasodilators serotonin (5HT) and sodium nitroprusside, respectively, and the inhibitor of nitric oxide synthase NG-monomethyl-l-arginine (L-NMMA) were measured. Dose-response curves were repeated after systemic infusion of apolipoprotein A-I/phosphatidylcholine (apoA-I/PC) disks. At baseline, ABCA1 heterozygotes had decreased HDL levels (0.4+/-0.2 mmol/L; P<0.05), and their forearm blood flow responses to both 5HT (maximum, 49.0+/-10.4%) and L-NMMA (maximum, -22.8+/-22.9%) were blunted compared with control subjects (both P< or =0.005). Infusion of apoA-I/PC disks increased plasma HDL to 1.3+/-0.4 mmol/L in ABCA1 heterozygotes, which resulted in complete restoration of vasomotor responses to both 5HT and L-NMMA (both P