Fetal growth of left-sided structures and postnatal surgical outcome in borderline left heart varies by cardiac phenotype.

OBJECTIVES: There are two borderline left-heart phenotypes in the fetus: (1) severe aortic stenosis (AS), which is associated with a 'short, fat', globular left ventricle (LV), LV systolic dysfunction and LV growth arrest; and (2) severe left-heart hypoplasia (LHH), which is associated with a 'long, skinny' LV, laminar flow across hypoplastic mitral and aortic valves and arch hypoplasia. Both phenotypes may be counseled for possible single-ventricle palliation. We aimed to compare the rates of left-sided cardiac structure growth and Z-score change over gestation and to describe the postnatal outcomes associated with these two phenotypes. We hypothesized that the left-sided structures would grow faster in fetuses with LHH compared to those with AS, and that those with LHH would be more likely to achieve biventricular circulation. METHODS: This was a retrospective cohort study using data collected in an institutional cardiology database between April 2001 and April 2018. We included fetuses with severe AS or severe LHH, and with at least two fetal echocardiograms. Inclusion criteria for the AS group included: aortic-annulus Z-score < -2.0, severe AS, depressed LV function, retrograde systolic flow in the aortic arch and endocardial fibroelastosis. Inclusion criteria for the LHH group included: aortic-annulus Z-score < -2.0, hypoplastic but apex-forming LV, normal LV function and retrograde systolic flow in the aortic arch. Exclusion criteria were: abnormal cardiac connections, other forms of structural congenital heart disease, cardiomyopathy, history of fetal aortic valvuloplasty and participation in a maternal hyperoxygenation study. Measurements and respective Z-scores for the aortic-valve annulus, mitral-valve annulus, LV long- and short-axis dimensions, along with aortic-arch measurements, were collected longitudinally for each fetus and plotted over time for both cohorts. Mean slopes of change in dimension and Z-scores over gestation were calculated and compared between the two groups using mixed generalized linear regression accounting for repeated measures. A subanalysis was performed, matching six fetuses from each group for initial aortic-annulus Z-score and gestational age, due to the significant differences in these two variables between the original cohorts. RESULTS: In total, 53 fetuses with 158 echocardiograms were included. In the AS cohort, there were 20 (38%) fetuses with 65 echocardiograms, and in the LHH cohort there were 33 (62%) fetuses with 93 echocardiograms. On the first echocardiogram, LHH fetuses had a later gestational age and a larger aortic-annulus diameter. The rate of aortic-annulus growth was greater in the LHH group compared with the AS group (mean ± SD, 0.15 ± 0.01 mm/week for LHH vs 0.07 ± 0.01 mm/week for AS (P < 0.001)). While the LHH group had a decrease in aortic-annulus Z-score over time, this was at a slower rate than the decrease in the AS group (mean ± SD, -0.04 ± 0.02/week for LHH vs -0.13 ± 0.02/week for AS (P < 0.001)). A similar pattern was seen for the mitral-valve and LV short-axis-dimension Z-scores. Subanalysis of six fetuses from each group matched for initial aortic-annulus Z-score and gestational age demonstrated similar findings, with the LHH group Z-scores decreasing at a slower rate than those in the AS group. Fifty-two of the 53 fetuses were liveborn, one LHH fetus dying in utero. Of the 20 liveborn in the AS cohort, 15 (75%) infants underwent single-ventricle palliation, two (10%) underwent biventricular repair and three (15%) died prior to intervention. Of the 32 liveborn in the LHH cohort, three (9.4%) underwent single-ventricle palliation, 28 (87.5%) achieved biventricular circulation, of which six required no surgery, and one (3.1%) died prior to intervention. CONCLUSIONS: The left-sided cardiac structures grow at a faster rate in fetuses with severe LHH than they do in fetuses with severe AS, and the Z-scores decrease at a slower rate in fetuses with severe LHH than they do in those with severe AS. The majority of infants in the LHH group did not undergo single-ventricle palliation. This information can be useful in counseling families on the expected growth potential of the fetus's aortic valve, mitral valve and LV, depending on the cardiac phenotype. © 2022 International Society of Ultrasound in Obstetrics and Gynecology.

Disrupted nitric oxide signaling due to GUCY1A3 mutations increases risk for moyamoya disease, achalasia and hypertension.

Moyamoya disease (MMD) is a progressive vasculopathy characterized by occlusion of the terminal portion of the internal carotid arteries and its branches, and the formation of compensatory moyamoya collateral vessels. Homozygous mutations in GUCY1A3 have been reported as a cause of MMD and achalasia. Probands (n = 96) from unrelated families underwent sequencing of GUCY1A3. Functional studies were performed to confirm the pathogenicity of identified GUCY1A3 variants. Two affected individuals from the unrelated families were found to have compound heterozygous mutations in GUCY1A3. MM041 was diagnosed with achalasia at 4 years of age, hypertension and MMD at 18 years of age. MM149 was diagnosed with MMD and hypertension at the age of 20 months. Both individuals carry one allele that is predicted to lead to haploinsufficiency and a second allele that is predicted to produce a mutated protein. Biochemical studies of one of these alleles, GUCY1A3 Cys517Tyr, showed that the mutant protein (a subunit of soluble guanylate cyclase) has a significantly blunted signaling response with exposure to nitric oxide (NO). GUCY1A3 missense and haploinsufficiency mutations disrupt NO signaling leading to MMD and hypertension, with or without achalasia.

Pilot study of chronic maternal hyperoxygenation and effect on aortic and mitral valve annular dimensions in fetuses with left heart hypoplasia.

OBJECTIVE: Acute maternal hyperoxygenation (AMH) results in increased fetal left heart blood flow. Our aim was to perform a pilot study to determine the safety, feasibility and direction and magnitude of effect of chronic maternal hyperoxygenation (CMH) on mitral and aortic valve annular dimensions in fetuses with left heart hypoplasia (LHH) after CMH. METHODS: Gravidae with fetal LHH were eligible for inclusion in a prospective evaluation of CMH. LHH was defined as: sum of aortic and mitral valve annuli Z-scores < -4.5, arch flow reversal and left-to-right or bidirectional atrial level shunting without hypoplastic left heart syndrome or severe aortic stenosis. Gravidae with an affected fetus and with ≥ 10% increase in aortic/combined cardiac output flow after 10 min of AMH at 8 L/min 100% fraction of inspired oxygen were offered enrollment. Nine gravidae were enrolled from February 2014 to January 2015. The goal therapy was ≥ 8 h daily CMH from enrollment until delivery. Gravidae who were cared for from July 2012 to October 2014 with fetal LHH and no CMH were identified as historical controls (n = 9). Rates of growth in aortic and mitral annuli over the final trimester were compared between groups using longitudinal regression. RESULTS: There were no significant maternal or fetal complications in the CMH cohort. Mean gestational age at study initiation was 29.6 ± 3.2 weeks for the intervention group and 28.4 ± 1.8 weeks for controls (P = 0.35). Mean relative increase in aortic/combined cardiac output after AMH was 35.3% (range, 18.1-47.9%). Median number of hours per day on CMH therapy was 9.3 (range, 6.5-14.6) and median duration of CMH was 48 (range, 33-84) days. Mean mitral annular growth was 0.19 ± 0.05 mm/week compared with 0.14 ± 0.05 mm/week in CMH vs controls (mean difference 0.05 ± 0.05 mm/week, P = 0.33). Mean aortic annular growth was 0.14 ± 0.03 mm/week compared with 0.13 ± 0.03 mm/week in CMH vs controls (mean difference 0.01 ± 0.03 mm/week, P = 0.75). More than 9 h CMH daily (n = 6) was associated with better growth of the aortic annulus in intervention fetuses (0.16 ± 0.03 vs 0.08 ± 0.02 mm/week, P = 0.014). CONCLUSIONS: CMH is both safe and feasible for continued research. In this pilot study, the effect estimates of annular growth, using the studied method of delivery and dose of oxygen, were small. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling.

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.

The effect of pre-operative counselling on smoking patterns in patients undergoing forefoot surgery.

BACKGROUND: Smoking contributes to higher surgical complication rates. Previous studies assessing smoking cessation interventions examined the provision of comprehensive packages. The use of surgery as an incentive to complement brief advice has not been fully evaluated. METHODS: Smokers were counselled and referred to their general practitioners for specific cessation strategies. Smoking status was recorded prior to surgery, on admission and in post-operative clinics. A telephone survey at a mean of 12 months post-operation ascertained long-term behavioural changes. RESULTS: Ninety-seven patients underwent surgery with twenty-five recorded as smokers. Sixteen stopped smoking pre-operatively; a further four reduced their intake, as a direct consequence of counselling. No patients were previously aware of the detrimental effects of smoking associated with foot surgery. CONCLUSIONS: Surgery provides an incentive for smoking cessation, maintained post-operatively. Although forefoot fusions and arthrodeses were used in our study, the results are transferable to other branches of orthopaedic surgery.

Synaptic vesicle recycling. The Ferrari of endocytosis?

After neurotransmission, neurons internalize and recycle the membrane components of synaptic vesicles remarkably quickly; they may have a 'rebuilt, turbo-charged' endocytic engine to achieve these speeds.

Interactions of spectrin in hereditary elliptocytes containing truncated spectrin beta-chains.

An abnormal spectrin, in which one subunit is truncated, has been detected in a large German family. The inheritance is autosomal dominant. The affected members of the family suffer in widely varying degree from a microcytic hemolytic anemia. The red cell morphology varies correspondingly from smooth elliptocytes to predominantly poikilocytes. The abnormal spectrin makes up approximately 30% of the total and is almost entirely present as the dimer. The truncated chain is not phosphorylated by the endogenous cAMP-independent kinase, and it has been identified as a chain of beta-type, using monoclonal antibodies. Because a univalent terminal spectrin alpha-chain fragment will bind to normal dimers with an association constant lower by only a factor of two than that for the self-association of the dimers, it would be expected that the mutant dimers (alpha beta') would readily enter into an association with normal (alpha beta) dimers to give alpha 2 beta beta' tetramers (though not with each other). In dilute solution this is indeed observed, and the diminution in tetramer concentration when 30% of normal spectrin is replaced by alpha beta' dimers, amounts to only a small proportion. Moreover, in the membrane skeleton, if there is pairwise apposition of dimer units, only 9% of pairings will be between units that cannot associate. We have shown that the failure of alpha beta' dimers to enter into heterologous associations in situ is not due to the elimination of the ankyrin binding site near the truncated end of the beta-chain: this site is fully functional, as judged by rebinding to spectrin-depleted vesicles. When the spectrin is extracted from the membrane in the cold, the material released initially consists almost entirely of alpha beta' dimers; when the spectrin of normal membranes is partly dissociated to dimers in situ by warming at low ionic strength, extraction in the cold then leads similarly to much more rapid release of the dimer than of the tetramer. The similar rates of liberation of normal and abnormal dimer make it unlikely that the interaction of the latter with the membrane is in any way defective. When mixtures of alpha beta and alpha beta' dimers are bound to spectrin-depleted inside-out membrane vesicles from normal cells and tetramers are allowed to form by equilibration at 30 degrees C, the proportion of the abnormal species appearing in the tetramer is much lower than would be expected on a statistical basis. The relation of the self-association equilibrium on the membrane to that of spectrin in dilute solution is analyzed.

The influence of EDTA on adenylate cyclase activity in membranes from rat and mouse myocardium.

Inclusion of EDTA in the homogenization buffer of both mouse and rat myocardium profoundly alters the properties of the adenylate cyclase complex. EDTA leads to an increase in the Vmax for adenylate cyclase activity due to all of the following agents: isoproterenol, Gpp[NH]p, forskolin and Mg2+. For forskolin and Mg2+, the EDTA-associated increase in Vmax is not accompanied by a change in sensitivity to activation. However, EDTA is associated with enhanced sensitivity to activation by isoproterenol and increased sensitivity to the effect of Mg2+ on isoproterenol-dependent adenylate cyclase activity. A result of greater isoproterenol-dependent adenylate cyclase activity, due to the presence of EDTA, is an attenuated synergistic contribution of Gpp[NH]p. Changes in stimulatable adenylate cyclase activity as a result of EDTA occurs in concert with effects of cholera toxin upon ADPribosylation of the guanine nucleotide regulatory protein, Ns. Substantial auto-ADP-ribosylation occurs in a cholera toxin-sensitive 42 kDa band in membranes prepared in the presence of EDTA. In addition, cofactor and substrate requirements in the cholera toxin-dependent ADP-ribosylation reaction depend on the method of membrane preparation. The results suggest that the integrity of the adenylate cyclase complex depends in part on the attention given to proteolysis that may be activated during the course of homogenization.

Verapamil ameliorates clinical, pathologic and biochemical manifestations of experimental chagasic cardiomyopathy in mice.

The influence of long-term verapamil administration on the consequences of Trypanosoma cruzi infection in mice was studied with regard to animal mortality, morbidity, myocardial pathologic features and myocardial beta-adrenergic adenylate cyclase activity. Verapamil administration dramatically decreased the mortality rate from 60% to 6% during the 70 day period of infection. Three clinical stages of infection were evident. In the acute stage (17 days after infection with maximal parasitemia), verapamil treatment not only decreased the incidence of myocardial disease (fibrosis and inflammation), but also protected myocardial beta-adrenergic adenylate cyclase activity. In addition, there was no increase in total body weight, which was regarded as an index of right-sided heart failure. In the subacute stage (30 to 60 days after infection), administration of verapamil continued to decrease myocardial disease and preserve beta-adrenergic adenylate cyclase activity. In addition, verapamil ameliorated the morbidity and mortality associated with this stage of infection. The chronic stage of infection was characterized by a decrease in myocardial disease and in beta-adrenergic adenylate cyclase activity. Thus, independent of the state of infection, long-term verapamil treatment enhanced beta-adrenergic adenylate cyclase activity. In addition, verapamil ameliorated the morbidity associated with infection. Although the relation among these various effects of verapamil in the setting of T. cruzi infection remains to be determined, collectively the results suggested that verapamil administration attenuated the consequences of T. cruzi infection.

Release of guanosine triphosphate binding protein alpha subunits from mouse myocardial membranes: basic properties and their alterations in acute murine Chagas disease.

OBJECTIVE: The aim was to investigate the arrangement of heterotrimeric alpha beta gamma G proteins in myocardial membranes using GTP gamma S dependent release characteristics of their alpha subunits in acute murine Chagas disease. METHODS: The properties of GTP gamma S dependent alpha subunit release were monitored immunochemically as well as by cholera toxin and pertussis toxin catalysed [32P]ADP ribosylation. RESULTS: GTP gamma S, as opposed to other nucleotides, caused optimal and virtually instantaneous release of soluble 40 kDa [32P]ADP ribosylated protein in pertussis toxin treated membranes. When determined immunochemically, infection decreased both the sensitivity to GTP gamma S dependent release of alpha i subunits and appeared to facilitate the appearance of GTP gamma S dependent release of alpha i3. GTP gamma S also caused the release of soluble 45 and 40 kDa proteins as detected by cholera toxin-[32P]ADP ribosylated membranes and immunochemical analysis. With regard to cholera toxin-[32P]ADP ribosylated Gs substrates sensitive to GTP gamma S dependent release, infection (1) decreased the amount of 45 kDa alpha s protein, (2) increased the amount of 40 kDa protein, and (3) enhanced sensitivity to GTP gamma S. In contrast, there was no effect of infection on the magnitude or sensitivity to GTP gamma S dependent release of immunochemical alpha s. CONCLUSIONS: The diverse characteristics of GTP gamma S dependent release of the very similar alpha subunits from myocardial membranes and their unique sensitivity to infection with T cruzi suggest that these very similar proteins are arranged within the plasma membrane in such a manner as to modify their biochemical behaviour.

The Influence of Lumbar Spinal Subtype on Lumbar Intervertebral Disc Degeneration in Young and Middle-Aged Adults.

STUDY DESIGN: Retrospective cohort study. OBJECTIVE: To identify whether an in vivo correlation exists between lumbar spinal subtype (LSS) and lumbar disc degeneration (LDD) in young adults. SUMMARY OF BACKGROUND DATA: Lumbar disc degeneration has largely been ascribed to biomechanical and structural alterations to the disc, which are attributed to aging and pathological physical loading. Sagittal alignment in the asymptomatic spine has also been considered. A biomechanical study by Roussouly and Pinheiro-Franco proposed level-specific patterns in LDD. To date, no in vivo correlation between the LSS and LDD has been established. METHODS: The authors screened 608 consecutive patients over 5.3 years. Lumbar spinal subtype and pelvic parameters were collected from standing lumbar radiographs and were categorized using the classification of Roussouly and Pinheiro-Franco. Lumbar disc degeneration at all lumbar intervertebral levels was classified using criteria of Pfirrmann et al. A stratified disc degeneration score was derived for each patient. Lumbar disc degeneration in type I, II, and IV LSS was compared using chi-square test. Pelvic incidence was correlated with stratified disc degeneration score using Spearman R, to determine whether a high PI was protective against LDD. Statistical significance was accepted at p < .05. RESULTS: A total of 139 patients were included, with 91 females and a mean age of 32.6 years (range, 13-49 years). For LSS grades I to IV, there were 10 (7.3%), 43 (30.9%), 50 (35.9%), and 36 (25.9%) patients, respectively. The proportion of high-grade (Pfirrmann grades IV and V) LDD increased distally toward the lower intervertebral levels, affecting 2.88%, 2.9%, 5%, 9.4%, 33.1%, and 54% of discs at each sequential lumbar level from T12-L1 to L5-S1, respectively. Age but not gender was statistically significant for higher-grade LDD (p < .0001 and p = .442, respectively). Pelvic incidence across all LSS grades was not significantly correlated with stratified disc degeneration score (Spearman R = 0.0933; p = .335). No LSS (type I-IV) reached statistical significance for a specific pattern of LDD. CONCLUSIONS: In this study, LSS was not statistically significantly correlated with LDD, nor was a high pelvic incidence protective against LDD.

Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators.

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP3) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP3 receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace 3H-InsP3 from its receptor and that they differ substantially in their affinities for the InsP3 receptor (IC50 = 6.5-137 microM). Recombinant type 1 InsP3 receptors expressed in Sf9 cells were used to examine 3H-InsP3 binding in cytosol-like medium: both fura-2 (IC50 = 796 +/- 86 microM) and Ca Green-5N (IC50 = 62 +/- 7 microM) completely inhibited the binding, but only in their Ca(2+)-free forms. Similar results were obtained with type 3 InsP3 receptors. We conclude that many Ca2+ indicators in their Ca(2+)-free forms compete with InsP3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.

Rapid purification of clathrin-coated vesicles by free-flow electrophoresis.

Free-flow electrophoresis was successfully used as the final step in the purification of clathrin-coated vesicles from bovine brain. Based on biochemical analysis, the material obtained in this way was found to be of equal purity with respect to the protein composition and lipid content as that purified by the previously widely used methods of permeation chromatography on controlled pore glass or Sephacryl S-1000. However, as judged by electron microscopy, the electrophoretically purified coated vesicles contained less smooth membranes than the coated vesicle preparations that had been obtained by permeation chromatography. Free-flow electrophoresis offers considerable advantages in speed of purification, in the total amount of material processed and in flexibility of operation. Analysis of the electrophoretic mobility of purified coated vesicles showed that this is governed by the coat proteins rather than by the vesicle contained therein. A shift in electrophoretic mobility of purified coated vesicles was obtained by the binding of coat protein specific monoclonal antibodies. This raises the possibility of purifying subpopulations of coated vesicles with respect to coat protein composition.

Estrogen uncouples beta-adrenergic receptor from the stimulatory guanine nucleotide-binding protein in female rat hypothalamus.

The responsiveness of adenylyl cyclase to beta-adrenergic receptor stimulation was investigated in membranes prepared from hypothalamus-preoptic area and cortex of ovariectomized female rats injected with oil vehicle or estradiol benzoate 24 or 48 h before death. Membranes from the hypothalamus-preoptic area of ovariectomized animals displayed a concentration-dependent stimulation of adenylyl cyclase when incubated with the beta-adrenergic receptor agonist, isoproterenol (10(-7)-10(-5) M). This response was suppressed in membranes from estrogen-treated animals. The effect of estrogen was observed 48 h, but not 24 h, after hormone administration. In addition, estrogen had no measurable effect on hypothalamic adenylyl cyclase activation by either GTP (10(-8)-10(-5) M) or forskolin (10(-8)-10(-6) M), on beta-adrenergic receptor density, or on antagonist binding affinity measured with the beta-adrenergic antagonist [125I]iodocyanopindolol. Analysis of isoproterenol displacement of iodocyanopindolol binding revealed that estrogen reduced agonist binding affinity in hypothalamus-preoptic area membranes. In membranes from ovariectomized controls, high affinity agonist binding to the beta-adrenergic receptor was apparent and was abolished by guanine nucleotides. However, membranes from estradiol-treated rats demonstrated only low affinity agonist binding that was unaffected by guanine nucleotides. Estradiol did not detectably alter concentrations of either cholera or pertussis toxin substrates in hypothalamus-preoptic area membranes. These data indicate that estrogen promotes a stable time-dependent desensitization of beta-adrenergic receptor activation of adenylyl cyclase in hypothalamus and preoptic area by uncoupling the receptor from the guanine nucleotide-binding protein, G8.

Opposing influences of dexamethasone and retinoic acid on adenylate cyclase activity in ROS 17/2.8 cells.

Exposure of ROS 17/2.8 cells to dexamethasone (DEX) or retinoic acid (RA) increases and decreases, respectively, adenylate cyclase activity (ACA) in response to isoproterenol, forskolin, guanylylimidodiphosphate, or NaFl. Despite dramatic changes in ACA, there were no significant changes in levels of cholera toxin- or pertussis toxin (PT)-dependent ADP-ribosylation of membranes prepared from cells after DEX or RA exposure as compared to controls. Similarly, immunochemical detection of alpha S, alpha i1-3, and alpha O, as well as Northern blot analysis of messenger RNA for each of the respective GTP binding proteins, also failed to demonstrate an influence of DEX or RA when contrasted with controls. In a novel use of the cyc- reconstitution assay, wherein the influence of inhibitory guanine nucleotide binding proteins in the extracts of control, DEX-, and RA-treated membranes is removed by a previous 24-h incubation with PT in the intact cell, we demonstrate that this PT treatment markedly enhances ACA in the cyc- reconstitution assay for all three preparations, but that the fold-increase due to PT-treatment is greatest in RA-treated cells. The greater magnitude of the effect of PT on RA-treated ROS 17/2.8 cells, in the absence of any obvious quantitative changes in the levels of the PT substrates, suggests that the effect of RA on ROS 17/2.8 cells appears to be an augmentation of the influence of inhibitory guanine nucleotide binding proteins, ultimately leading to reduced ACA.