Krishnamurti Dakshinamurti

Neurobiology of pyridoxine

The diversity of biochemical reactions involving the coenzymatic forms of pyridoxine (vitamin B6) is well recognized. There are over 100 pyridoxal phosphate (PLP) dependent enzymes. Most are involved in catabolic reactions of various amino acids. The crucial role played by pyridoxine in the nervous system is evident from the fact that the putative neurotransmitters, dopamine (DA), norepinephrine (NE), serotonin (5-HT), y-aminobutyric acid (GABA), and taurine as well as the sphingolipids and polyamines are synthesized by PLP-dependent enzymes. Considerable variation exists in the affinities of different apoenzymes for PLP. This explains the observed differential susceptibility of various PLP enzymes to decrease during the progression of pyridoxine deficiency.’ Of the pyridoxine enzymes, three, namely, glutamic acid decarboxylase, 5-hydroxytryptophan decarboxylase, and ornithine decarboxylase, are crucial and can explain most of the neurological defects of pyridoxine deficiency in animals.*~-’ This presentation will focus on the role of pyridoxine in the control of hypothalamo-pituitaryend organ systems, melatonin synthesis, and convulsive seizure activity. The enzyme L-aromatic amino acid decarboxylase (EC 4.1.1.28), which lacks substrate specificity, has been considered to be involved in the formation of the catecholamine as well as ~ero tonin .~ This has been suggested to be a single protein entity, based on immunological evidence of Christenson et al.4 However, the recent demonstration by Ando-Yamamoto et aL4 of immunological cross-reactivity of dihydroxyphenylalanine (DOPA) decarboxylase and histidine decarboxylase using antibodies against these two enzymes suggests the presence of similar antigenic recognition sites inside the native molecules of the decarboxylases that are exposed when the enzymes are denatured. There are many differences in the optimal conditions for enzyme activity, including kinetics, affinity for PLP, activation and inhibition by specific chemicals, and regional differences in the distribution of DOPA and 5hydroxytryptophan (5-HTP) decarboxylases.6-8 During the course of purification of DOPA decarboxylase from bovine striatum, there was a preferential enrichment of

Sympathetic stimulation and hypertension in the pyridoxine-deficient adult rat.

Pyridoxal phosphate is the coenzyme of various decarboxylases involved in the formation of monoamine neurotransmitters such as gamma-aminobutyric acid, serotonin, dopamine, and norepinephrine. Adult male Sprague-Dawley rats placed on a pyridoxine-deficient diet for 8 weeks showed significant hypertension compared with pyridoxine-supplemented controls. Hypothalamic contents of pyridoxal phosphate, gamma-aminobutyric acid, and serotonin in the pyridoxine-deficient rats were significantly lower than those in pyridoxine-supplemented controls. Hypertension was associated with sympathetic stimulation. Treatment of pyridoxine-deficient rats with a single dose of pyridoxine (10 mg/kg body weight) reversed the blood pressure to normal levels within 24 hours, with concomitant restorations of hypothalamic serotonin and gamma-aminobutyric acid as well as the return of plasma norepinephrine and epinephrine to normal levels. Also, pyridoxine treatment reversed the hypothalamic hypothyroidism observed in pyridoxine-deficient rats. These results indicate an association between pyridoxine deficiency and sympathetic stimulation leading to hypertension.

Transcriptional regulation of liver phosphoenolpyruvate carboxykinase by biotin in diabetic rats

Rat liver phosphoenolpyruvate carboxykinase (PEPCK) activity was followed over a time period of 5h following administration of biotin to streptozotocin-induced diabetic rats. In parallel with the decrease in enzyme activity liver PEPCK mRNA decreased by 85% at 3 h after injection of biotin to diabetic rats. There was no significant change in the accumulation of kidney PEPCK mRNA. Parallel studies with insulin indicated that biotin had a regulatory effect similar to that of insulin on liver PEPCK mRNA. The administration of biotin did not change the insulin status of the diabetic rat indicating that biotin did not act via insulin. The transcriptional activity of the hepatic PEPCK gene, as measured by nuclear run-on assay, was decreased by 57% within 30 min of biotin administration. The results suggest that biotin regulates hepatic, but not renal, PEPCK mRNA concentration at the transcriptional level in diabetic rats.

Domoic acid induced seizure activity in rats

Domoic acid, in increasing doses (10-300 pmol), was microinjected into the hippocampal CA3 region of rats. All rats consistently exhibited generalized bilateral electrical seizure discharge activity at 100 pmol of domoic acid. Seizure latency varied inversely with the dose of domoic acid in the range tested. Local hippocampal administration of gamma-aminobutyric acid (GABA) resulted in neuronal recovery from domoic acid-induced seizures. The seizure activity of domoic acid might be the result of decreased GABAergic inhibition.

Neuroprotective actions of pyridoxine.

Electroencephalographic recordings in cerebral cortex of mice given a single sub-convulsive dose of domoic acid exhibited typical spike and wave discharges. Administration of the anti-epileptic drugs sodium valproate, nimodipine, or 5 alpha-pregnan 3 alpha-ol-20-one as well as pyridoxine simultaneously with or after domoic acid treatment resulted in significantly less spike and wave activity. Administration of these same drugs 45 min prior to the administration of domoic acid also significantly reduced EEG background. Mechanistically, sodium valproate and pyridoxine significantly attenuated domoic acid-induced increase in levels of glutamate, increase in levels of calcium influx, decrease in levels of gamma-aminobutyric acid and increase in levels of the protooncogenes c-fos, jun-B and jun-D. In hippocampal cells, domoic acid-induced increases in glutamate and calcium influx were significantly decreased by pyridoxal phosphate or nimodipine. Similarly in neuroblastoma-glioma hybrid cells (NG 108/15), pyridoxine attenuated domoic acid-induced increases in glutamate, influx of extracellular calcium, and enhanced induction of oncoproteins regardless of whether cells were undifferentiated, differentiated or de-differentiated. Pyridoxine has anti-seizure and neuroprotective actions mediated through mechanisms similar to those targeted by current therapeutic strategies.

Acetyl-CoA carboxylase from rat adipose tissue

Abstract Rat epididymal adipose tissue acetyl-CoA carboxylase activity has been shown to respond to various dietary treatments. Feeding a high dextrose diet increases the enzyme activity. In biotin-deficient rats the enzyme activity is reduced to less than a sixth of the levels found in control animals fed the deficient diet supplemented with biotin. Adipose tissue acetyl-CoA carboxylase is inhibited by both malonyl-CoA and coenzyme A. Unlike the liver enzyme, the adipose tissue carboxylase in not inactivated by cold treatment.

The effect of vitamin B6 on the systolic blood pressure of rats in various animal models of hypertension.

Objective To investigate whether a dietary supplement of vitamin B6 could attenuate the elevation of systolic blood pressure (SBP) in Zucker obese or spontaneously hypertensive rats, or rats ingesting sucrose. Methods Zucker obese rats (fa/fa), Sprague-Dawley rats with sucrose-induced elevation of SBP, spontaneously hypertensive rats (SHRs) and their corresponding controls were tested for the effects of vitamin B ingestion in different ways: (1) vitamin B6 was included as a supplement (five times the normal intake) from the start of the experiment until the development of hypertension; (2) vitamin B6 supplement was removed from the diet of Zucker obese and Zucker lean control groups after 16 weeks on the dietary treatments; and (3) a diet deficient in vitamin B6 was instituted in SHRs and control Wistar-Kyoto (WKY) rats. The SBP of rats in all groups was monitored in the conscious animal by tail-cuff plethysmography. The effects of the various treatments on the uptake of calcium by caudal artery segments were examined. Results Fale Zucker obese rats (fa/fa) of age 6 weeks fed a commercial rat chow developed hypertension in 3-4 weeks, whereas their lean controls (Fa/Fa) did not. The inclusion of a vitamin B6 supplement (five times the normal intake) resulted in a complete attenuation of the hypertension in the obese strain. Removal of the vitamin B6 supplement from the diet of these obese rats resulted in the return of hypertension within 2 weeks. Similar changes in SBP were also observed in the Zucker lean controls treated with vitamin B6. The ingestion of sucrose by male Sprague-Dawley rats resulted in modest elevation of SBP that was attenuated by the inclusion of the vitamin B6 supplement in their diet. In contrast, there was no response to the inclusion or removal of dietary vitamin B6 supplement in the SHRs. However, the WKY control rats responded to both these conditions in a similar manner to that seen in the Sprague-Dawley strain. Increased peripheral resistance resulting from increased permeability of vascular smooth muscle plasma membrane to Ca2+ is thought to be one of the mechanisms of hypertension. Changes in SBP correlated with changes in the uptake of calcium by caudal artery segments in all the groups studied. The Zucker obese and sucrose-induced hypertensive rats have abnormalities in carbohydrate metabolism. The vitamin B6 supplement decreased the random or fasting blood glucose levels in the Zucker obese and sucrose-fed rats respectively. Conclusion This is the first observation that animal models of hypertension can be classified on the basis of their response to a vitamin B6 supplement. On this basis, the etiology of hypertension in SHRs is quite distinct from that in Zucker obese rats and in rats ingesting sucrose.

Requirement for Biotin and the Function of Biotin in Cells in Cultureaa

The requirement of biotin in the culture medium has been established for HeLa cells, human fibroblasts, baby hamster kidney cells (BHK) as well as polyoma-transformed BHK cells. Growth, viability, biotin content and the activities of biotin-dependent enzymes were used as criteria. Cells in culture appear to bind and internalized avidin as well as the avidin-biotin complex. Avidin seems to mimic a natural ligand that could be the biotin-binding protein of serum. Additions of serum fatty acids, aspartate or asparagine to the culture medium do not supplant the biotin requirement. Nuclear fractions of cells contain significant biotin without the ability to fix carbon dioxide. A biotin-containing protein has been isolated from rat liver nuclei. The incorporation of amino acids into cellular protein is decreased in biotin-deficient HeLa cells. The proportion of active ribosomes is also decreased in these cells. Supplementation of the medium of deficient cells with biotin results in stimulation of protein synthesis. It is suggested that biotin might subserve a function in cells in addition to its role as the prosthetic group of biotin-enzymes.

Neuroendocrinology of pyridoxine deficiency

Dihydroxyphenylalanine decarboxylase and 5-hydroxytryptophan decarboxylase respectively have high and low affinities for pyridoxal phosphate. In the pyridoxine-deficient animal, hypothalamic serotonin content is significantly reduced without any change in catecholamine levels. Hypothalamic neurotransmitters affect the hypothalamo-pituitary-end organ axes. Specifically, the decrease in hypothalamic serotonin in the pyridoxine-deficient rat results in tertiary hypothyroidism. In addition, pineal function is affected in deficient animals due to decreased synthesis of melatonin.

Neurobiology of Pyridoxinea

The diversity of biochemical reactions involving the coenzymatic forms of pyridoxine (vitamin B6) is well recognized. There are over 100 pyridoxal phosphate (PLP) dependent enzymes. Most are involved in catabolic reactions of various amino acids. The crucial role played by pyridoxine in the nervous system is evident from the fact that the putative neurotransmitters, dopamine (DA), norepinephrine (NE), serotonin (5-HT), y-aminobutyric acid (GABA), and taurine as well as the sphingolipids and polyamines are synthesized by PLP-dependent enzymes. Considerable variation exists in the affinities of different apoenzymes for PLP. This explains the observed differential susceptibility of various PLP enzymes to decrease during the progression of pyridoxine deficiency.’ Of the pyridoxine enzymes, three, namely, glutamic acid decarboxylase, 5-hydroxytryptophan decarboxylase, and ornithine decarboxylase, are crucial and can explain most of the neurological defects of pyridoxine deficiency in animals.*~-’ This presentation will focus on the role of pyridoxine in the control of hypothalamo-pituitaryend organ systems, melatonin synthesis, and convulsive seizure activity. The enzyme L-aromatic amino acid decarboxylase (EC 4.1.1.28), which lacks substrate specificity, has been considered to be involved in the formation of the catecholamine as well as ~ero tonin .~ This has been suggested to be a single protein entity, based on immunological evidence of Christenson et al.4 However, the recent demonstration by Ando-Yamamoto et aL4 of immunological cross-reactivity of dihydroxyphenylalanine (DOPA) decarboxylase and histidine decarboxylase using antibodies against these two enzymes suggests the presence of similar antigenic recognition sites inside the native molecules of the decarboxylases that are exposed when the enzymes are denatured. There are many differences in the optimal conditions for enzyme activity, including kinetics, affinity for PLP, activation and inhibition by specific chemicals, and regional differences in the distribution of DOPA and 5hydroxytryptophan (5-HTP) decarboxylases.6-8 During the course of purification of DOPA decarboxylase from bovine striatum, there was a preferential enrichment of