Hyperphenylalaninemia and 7-pterin excretion associated with mutations in 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH: analysis of enzyme activity in intestinal biopsies.

Hyperphenylalaninemia, which can cause neurological disorders and mental retardation, results from a mutation in phenylalanine hydroxylase or an enzyme required for biosynthesis or regeneration of its cofactor, tetrahydrobiopterin. The hyperphenylalaninemia variant primapterinuria is characterized by the excretion of 7-biopterin (primapterin). This disorder is thought to be due to a deficiency of 4a-hydroxy-tetrahydrobiopterin dehydratase (pterin-4a-carbinolamine dehydratase), but a lack of tissue activity has not been directly demonstrated. The five mutations so far recognized in patients with primapterinuria are associated with either a single amino acid change or a premature stop codon. Only C81R has been successfully expressed in soluble form, and was found to have 40% of normal activity. Tissues which could be obtained by minimally invasive procedures were analyzed for dehydratase activity. None was detected in normal human white cells or fibroblasts. However, activity was found in intestine of rat, dog, pig, and particularly humans where it was only eight times lower than in liver. Distribution along the length and across the wall of small intestine was relatively uniform. Moreover, the dehydratases from human liver and intestinal mucosa have identical kinetic properties. A biopsy of duodenal mucosa from a patient with homozygous E96K dehydratase had activity of 55 nmol. min(-1)g(-1) mucosa compared to 329 +/- 32 nmol. min(-1)g(-1) tissue in controls (n = 12). The sixfold lower tissue activity of the E96K mutant alone may not be sufficient to account for the biochemical symptoms of primapterinuria in this patient. However, accumulation of a 4a-hydroxy-tetrahydrobiopterin degradation product (a side-chain cyclic adduct), which has been observed in vitro and appears to be a dehydratase inhibitor, may further exacerbate the problem.

Mechanisms underlying depressed Na+/Ca2+ exchanger activity in the diabetic heart.

OBJECTIVES: Depression in Na+/Ca2+ exchanger activity is an important factor in the development of the diabetic cardiomyopathy. Since the mechanism underlying this depression remains unknown, the aim of this study was to determine the contribution of hyperglycemia and insulinopenia towards the observed impairment in Na+/Ca2+ exchanger activity. METHODS: Non-insulin-dependent diabetes was induced in neonatal Wistar rats by injection of 90 mg/kg streptozotocin. Na+/Ca2+ exchange in sarcolemmal vesicles and isolated cardiomyocytes was determined by Na(+)-dependent 45Ca2+ transport. To assess the role of insulin deficiency and hyperglycemia on Na+/Ca2+ exchanger activity, neonatal cardiomyocytes were incubated for 3 days in media containing either 5 mM glucose and 56 U/l insulin (Control), 30 mM glucose and 56 U/l insulin (High glucose) or 5 mM glucose and 0 insulin (Insulin deficiency). Since hyperglycemia has been shown to affect protein kinase C activity, Ca(2+)-dependent isoforms of protein kinase C were examined in non-diabetic and diabetic heart using hydroxylapatite chromatography. Also examined was Na+/Ca2+ exchanger mRNA levels in diabetic and non-diabetic hearts using Northern slot blot analysis. RESULTS: Acute insulin produced a dose-dependent increase in Na+/Ca2+ exchanger activity, which was dramatically attenuated in diabetic membrane. Myocytes incubated in media containing 30 mM glucose exhibited a 33% reduction in Na+/Ca2+ exchanger activity, while insulinopenia reduced activity by 63%. Exchanger mRNA levels of the diabetic heart were normal; however, diabetes was associated with major changes in protein kinase C activity. CONCLUSIONS: Reduced Na+/Ca2+ exchanger activity resulting from diabetes, hyperglycemia or insulinopenia may be related to changes in protein kinase C activity, but is not caused by altered expression of the transporter.

Thyroid hormone regulates Na(+)-Ca2+ exchanger expression during postnatal maturation and in adult rabbit ventricular myocardium.

OBJECTIVES: Expression of the cardiac Na(+)-Ca2+ exchanger (NCX) is high at birth and declines rapidly to adult levels by approximately 21 days in rabbits. The aim was to evaluate the role of thyroid hormone in regulating cardiac NCX expression. METHODS: Adult New Zealand White rabbits were made hypothyroid by treatment with propylthiouracil or hyperthryoid by administration of sigma-thyroxine. Hypothyroidism was induced in immature rabbits by exposure to propylthiouracil from gestational day 25 through the first 21 days after birth. NCX steady-state mRNA levels were quantitated using Northern slot blots with poly(A+) RNA isolated from ventricular myocardium of treated and age-matched euthyroid animals. As a control, steady-state levels of cardiac sarco(endo)plasmic reticulum calcium ATPase (SERCA2a) were measured in each group. Thyroid status was confirmed with serum T4, ventricular weight and body weight measurements. Immunoreactive NCX protein levels were assessed using Western blots. RESULTS: Compared with euthyroid controls, NCX steady-state mRNA levels increased to 189 +/- 20% in hypothyroid adults and decreased to 55 +/- 15% in hyperthyroid adults. Opposite effects were observed for SERCA2a expression (58 +/- 7% in hypothyroidism and 130 +/- 15% in hyperthyroidism). In hypothyroid 21-day-old rabbits, NCX steady-state mRNA levels were elevated to 205 +/- 30% of age-matched euthyroid controls. SERCA2a levels were unaffected in the immature animals, possibly due to inability to reduce thyroid levels sufficiently to affect SERCA2a expression in this model. Changes in NCX mRNA levels produced comparable changes in immunoreactive NCX protein levels. CONCLUSIONS: Thyroid hormone reciprocally regulates NCX and SERCA2a expression in the ventricles of adult rabbits. Hypothyroidism resulted in sustained high levels of NCX expression in 21-day-old rabbits. These results suggest that the postnatal thyroid hormone surge is important for the normal down-regulation of cardiac NCX expression during the first 3 weeks after birth in developing rabbits.

Catalytic characterization of 4a-hydroxytetrahydropterin dehydratase.

The cofactor product of the aromatic amino acid hydroxylases, 4a-hydroxy-6(R)-tetrahydrobiopterin, requires dehydration before tetrahydrobiopterin can be regenerated by dihydropteridine reductase. Carbinolamine dehydration occurs nonenzymatically, but the reaction is also catalyzed by 4a-hydroxytetrahydropterin dehydratase. This enzyme has the identical amino acid sequence to DCoH, the dimerization cofactor of the transcription regulator, HNF-1 alpha. The catalytic activity of rat liver dehydratase was characterized using a new assay employing chemically synthesized 4a-hydroxytetrahydropterins. The enzyme shows little sensitivity to the structure or configuration of the 6-substituent of its substrate, with Km's for 6(S)-methyl, 6(R)-methyl, 6(S)-propyl, and 6(R)-L-erythro-dihydroxypropyl all between 1.5 and 6 microM. Turnover numbers at 37 degrees C are 50-90 s-1 at pH 7.4 and 2.5-3-fold lower at pH 8.4. Both 4a(R)- and 4a(S)-hydroxytetrahydropterins are good substrates. The quinoid dihydropterin products are strong inhibitors of the dehydratase with KI's about one half of their respective Km's, but no inhibition was observed with 7,8-dihydropterins or tetrahydropterins. The enzyme contains no metals and no phosphorus. Reaction mechanisms which involve either acid and/or base catalysis are discussed. 4a-Hydroxy-6(R)-tetrahydrobiopterin was determined not to be a product inhibitor of phenylalanine hydroxylase. It is concluded that the dehydratase (which was found to be 6 microM in rat liver) is essential in vivo to prevent rearrangement of 4a-hydroxy-6(R)-tetrahydrobiopterin and to maintain the supply of tetrahydrobiopterin cofactor for the hydroxylases under conditions where the nonenzymatic rate would be inadequate.

Steady-state mRNA levels of the sarcolemmal Na(+)-Ca2+ exchanger peak near birth in developing rabbit and rat hearts.

To functionally compensate for an underdeveloped sarcoplasmic reticulum in immature cardiomyocytes, it has been proposed that the sarcolemmal Na(+)-Ca2+ exchanger may assume a more predominant role for regulating cytosolic Ca2+. Previous studies using sarcolemma prepared from developing rabbit hearts demonstrated that Na(+)-dependent Ca2+ uptake and exchanger protein content were highest at birth and declined postnatally. To further investigate the significance of the Na(+)-Ca2+ exchanger during normal myocardial development, steady-state mRNA levels of the cardiac Na(+)-Ca2+ exchanger were quantitated by Northern blot and slot-blot analyses using poly(A+) RNA isolated from rabbit and rat ventricles at various fetal and postnatal ages. Northern analyses were performed with a 1.35-kb guinea pig cardiac Na(+)-Ca2+ exchanger cDNA probe. Exchanger mRNA levels were quantitated by densitometric scans of the slot blots, and results were normalized by reprobing the same blots with 32P 5'-end-labeled oligo(dT). In both species, exchanger mRNA levels peaked near birth and declined postnatally. Maximal levels were approximately sixfold greater in the late fetal rabbit (gestational day 29) and eightfold greater in the early newborn rat (postnatal day 1) compared with adults of the respective species. The parallel changes in exchanger mRNA and protein levels suggest that developmental regulation of cardiac Na(+)-Ca2+ exchanger expression involves pretranslational control mechanisms. These results support the concept that during normal cardiac development, Na(+)-Ca2+ exchanger expression is maximal near the time of birth and then declines postnatally as Ca2+ regulation by the sarcoplasmic reticulum reaches functional maturity.