Effect of biotin on ribonucleic acid synthesis.

A single injection of biotin to biotin-deficient rats produces a two-fold increase in the incorporation, both in vivo and in vitro of precursors into nucleic acids as early as 2 h after the biotin treatment. The specific activity of the precursor pool is not affected by biotin. Analysis of the polysome profile at various times following biotin treatment and a kinetic study of the effect of excess poly(U) on the incorporation of phenylalanine by cell-free amino acid incorporation experiments indicate a marked decrease in messenger-free ribosomes in rat liver after biotin administration.

The characterization of the uptake of avidin-biotin complex by HeLa cells.

HeLa cells have been shown to internalize the avidin-biotin complex. Adsorptive pinocytosis seems to be the mechanism of this uptake as seen by the requirements of energy and the integrity of the microtubular assembly. Pretreatment of HeLa cells with cycloheximide inhibits uptake and binding of the avidin-biotin complex. Scatchard plot of specific binding of avidin indicates a single type of binding with a Kd of approx. 55 pM with about 21,500 receptors/cell. The lack of inhibition of binding by simple carbohydrates indicates that binding is not through the oligosaccharide chain of avidin.

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.

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.

The relationship between low-calcium-induced increase in systolic blood pressure and vitamin B6.

OBJECTIVE: To investigate the relationship between the increase in systolic blood pressure caused by a low-calcium (0.1%) diet and the vitamin B6 status in the rat. METHODS: Male Sprague-Dawley rats fed a vitamin B6-deficient diet did not show any change in systolic blood pressure (SBP) for the first 4 weeks (prehypertensive phase), but from week 5 there was an increase in SBP lasting until week 10 (hypertensive phase). SBP declined to below normal levels in most rats from week 12 on the vitamin B6-deficient diet (posthypertensive phase). The effect of altering the level of calcium in the diet at different phases of vitamin B6 deficiency was studied. In another experiment the effect on the change in blood pressure induced by the low-calcium diet of increasing the dietary vitamin B6 level to 2.5-, 5- or 10-fold the normal intake was studied. RESULTS: Lowering dietary calcium caused a significant increase in SBP in rats on the vitamin B6-sufficient diet. This occurred during weeks 3 and 4 on the low-calcium diet. Low levels of calcium on the diet potentiated the hypertension induced by the vitamin B6-deficient diet when both deficiencies were present from the beginning of the experiment. Feeding a low-calcium diet during the hypertensive or posthypertensive phase failed to raise the SBP in these rats. Normalizing the vitamin B6 status of posthypertensive vitamin B6-deficient rats restored the ability of low dietary calcium to increase SBP in these rats. Increasing dietary levels of vitamin B6 by itself reduced SBP in normal rats, and attenuated the increase in SBP induced by the low-calcium diet. CONCLUSIONS: Dietary vitamin B6 deficiency and low calcium in the diet seem to share the mechanisms increasing SBP.

Calcium channels in vitamin B6 deficiency-induced hypertension.

OBJECTIVE: To compare the acute hypotensive effects of various calcium channel modulators in vitamin B6-deficient hypertensive (B6DHT) rats. METHODS: Adult male Sprague-Dawley rats were fed a vitamin B6-deficient diet for 7-10 weeks, during which systolic blood pressure (SBP) was measured in the B6DHT and control rats, using tail-cuff plethysmography. The effects of the calcium antagonists nifedipine, verapamil, diltiazem and (-)-202-791 on SBP were determined in conscious B6DHT rats. The effect of the calcium agonist BAY K 8644 on the SBP of rats fed various levels of pyridoxine was also determined. RESULTS: All of the calcium antagonists used were effective in lowering the SBP of the B6DHT rats with the rank order of potency: nifedipine > (-)-202-791 > (+/-)-verapamil > diltiazem. BAY K 8644 elevated the SBP of older rats fed a normal commercially available diet, but had no effect when the vitamin B6 content of their diet was increased or removed for a short period. CONCLUSION: Calcium channel function appears to be related to vitamin B6 status in the rat.

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: Male 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.

Hypothyroidism of hypothalamic origin in pyridoxine-deficient rats.

Pyridoxine-deficient young rats (3 weeks old) had significantly reduced levels of pituitary TSH, serum thyroxine (T4) and tri-iodothyronine (T3) compared with pyridoxine-supplemented rats. The status of the pituitary-thyroid axis of normal, pyridoxine-supplemented and pyridoxine-deficient rats was evaluated by studying the binding parameters of [3H](3-methyl-histidine2)TRH in the pituitary of these rats. The effects of TRH and T4 injections on pituitary TSH and serum TSH, T4 and T3 of these two groups were also compared. The maximal binding of TRH receptors in the pituitary of pyridoxine-deficient rats was significantly higher than that of pyridoxine-supplemented control and normal rats, but there was no change in the binding affinity. Treatment with TRH stimulated TSH synthesis and release. It also increased serum T4 and T3 in both pyridoxine-supplemented and pyridoxine-deficient rats. Treatment with T4 decreased serum and pituitary TSH in both pyridoxine-supplemented and pyridoxine-deficient rats, compared with saline-treated rats. The increased pituitary TRH receptor content, response to TRH administration and the fact that regulation at the level of the pituitary is not affected in the pyridoxine-deficient rat indicates a hypothalamic origin for the hypothyroidism of the pyridoxine-deficient rat.

Thyroid function in pyridoxine-deficient young rats.

In pyridoxine-deficient young rats hypothalamic serotonin was decreased with no changes in the dopamine and noradrenaline content. Serum thyroxine and tri-iodothyronine concentrations were much lower in the deficient rats as compared to pyridoxine-supplemented controls. No significant difference between deficient and control groups in the serum TSH concentration was detected. Highly significant decreases in the content of pituitary TSH and in the number of pituitary thyrotroph secretory granules were found. These results suggest that the hypothyroidism of pyridoxine-deficient young rats might be of hypothalamic origin.

Increased calcium influx in caudal artery of rats made hypertensive with pyridoxine deficiency.

Moderate pyridoxine deficiency in rats has been shown to induce hypertension, which can be corrected by pyridoxine supplementation. In this study, calcium handling by isolated caudal arteries from pyridoxine-deficient and age-matched control rats was evaluated. We found 45Ca influx into the intracellular compartment to be significantly elevated in deficient rats. This increased influx could be attenuated with nifedipine, a calcium channel blocker, in a dose-dependent manner, suggesting alterations in the calcium channels that would make them leaky. The pyridoxine-deficient arterial segments maintained a higher resting tone; removal of extracellular calcium by EGTA or entry blockade by nifedipine decreased the tone significantly in the deficient arteries, compared with little or no effect in controls.

Requirement for biotin and the function of biotin in cells in culture.

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.

Chronic catheterization using vascular-access-port in rats: blood sampling with minimal stress for plasma catecholamine determination.

Chronic catheterization is illustrated using vascular-access-port model SLA where the port is surgically placed subcutaneously on the back of the rat. The catheter is tunnelled to the neck and inserted into the jugular vein. Within 24 h rats showed normal blood pressure and blood samples were collected at intervals with minimal stress to the animals. A comparison of the plasma catecholamine of blood collected from vascular-access-ports with that obtained from decapitation indicates that there was minimal stress to the rats when blood was drawn through the vascular-access-port.

Pineal indoleamine metabolism in pyridoxine-deficient rats.

Pyridoxine deficiency causes physiologically significant decrease in brain serotonin (5-HT) due to decreased decarboxylation of 5-hydroxytryptophan (5-HTP). We have examined the effect of pyridoxine deficiency on indoleamine metabolism in the pineal gland, a tissue with high indoleamine turnover. Adult male Sprague-Dawley rats were fed either a pyridoxine-supplemented or pyridoxine-deficient diet for 8 weeks. Pyridoxine deficiency did not alter the pattern of circadian rhythm of pineal 5-HT, 5-hydroxyindoleacetic acid (5-HIAA), N-acetylserotonin (NAS), and melatonin. However the levels of these compounds were significantly lower in the pineal glands of pyridoxine-deficient animals. Pineal 5-HTP levels were consistently higher in the pyridoxine-deficient animals and a conspicuous increase was noticed at 22.00 h. Increase in pineal NAS and melatonin levels caused by isoproterenol (5 mg/kg at 17.00 h) were significantly lower (P less than 0.05) in the pyridoxine-deficient animals. Treatment of pyridoxine-deficient rats with pyridoxine restored the levels of pineal 5-HT, 5-HIAA, NAS, and melatonin to values seen in pyridoxine-supplemented control animals. These results suggest that 5-HT availability could be an important factor in the regulation of the synthesis of pineal NAS and melatonin.

Hypotensive action of 5-HT receptor agonists in the vitamin B6-deficient hypertensive rat.

Feeding a vitamin B6-deficient diet to rats causes a moderate hypertension. The blood pressure responses to 5-HT1A receptor agonists were studied in conscious vitamin B6-deficient hypertensive rats. They were all effective in lowering blood pressure with the following rank order of potency: 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) > flesinoxan > 5-methylurapidil > urapidil. The putative 5-HT1A receptor antagonist spiroxatrine by itself, did not have any effect on the blood pressure at the doses used (0.01-1 mumol/kg). However, dose dependently, it antagonized the hypotensive effect of flesinoxan and urapidil. The alpha 1-adrenoreceptor antagonist prazosin, on prior treatment, did not change the hypotensive effect of either flesinoxan or urapidil. The alpha 2-adrenoreceptor agonist clonidine dose dependently (0.01-0.1 mumol/kg) reduced blood pressure. This effect of clonidine was unaffected by spiroxatrine, but was dose dependently antagonized by the alpha 2-adrenoreceptor antagonist yohimbine. Binding studies with [3H]8-OH-DPAT indicated that the affinity and Bmax of 5-HT1A receptors was increased in vitamin B6-deficient hypertensive rats. The results suggest that decreased synthesis of 5-HT in brain regions and the consequent alterations in 5-HT receptors in the vitamin B6-deficient rats may be the underlying cause of the hypertension seen in these animals.

Enhancement of high affinity gamma-aminobutyric acid receptor binding in cerebellum of pyridoxine-deficient rat.

The high-affinity of [3H]gamma-aminobutyric acid (GABA) to GABAA receptors and [3H]baclofen to GABAB receptors were studied in the cerebellum of pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding (Bmax) of both GABAA and GABAB receptors with no significant difference in their binding affinities (Kd). The changes observed suggest a supersensitivity of GABAA and GABAB receptors which seems to correlate negatively with the concentration of GABA in the cerebellum of pyridoxine-deficient 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.

Alterations in brainstem alpha 2 adrenoreceptor activity in pyridoxine-deficient rat model of hypertension.

Moderate pyridoxine deficiency in adult male Sprague-Dawley rats results in significant hypertension, associated with a general sympathetic stimulation, including an increase in the turnover of norepinephrine in the heart. Treatment of these rats with pyridoxine reversed blood pressure to normal within 24 h. Treatment of pyridoxine-deficient rats with clonidine or alpha-methyl dihydroxyphenylalanine (alpha-methyl DOPA) also reduced the blood pressure of these animals to normal. There was also a significant increase in the Bmax of high and low affinity [3H]p-amino-clonidine binding to crude synaptosomal membrane preparations of the brain stem of deficient rats indicating chronic underexposure of alpha 2 adrenoreceptors to endogenous norepinephrine.

Consequences of decreased brain serotonin in the pyridoxine-deficient young rat.

The concentrations of serotonin in various brain areas were significantly decreased in the pyridoxine-deficient young rat. There was no change in the concentration of dopamine. Both Bmax and Kd of [3H]serotonin binding to membrane preparations from cerebral cortex were increased in deficiency and were restored to normal upon pyridoxine supplementation. There was no change in [3H]spiroperidol binding to corpus striatal membrane preparations in pyridoxine-deficient rats.

Picrotoxin and pentylene tetrazole induced seizure activity in pyridoxine-deficient rats.

Computerized electroencephalography and thalamic ventro-posterior lateral (VPL) unit activities were recorded from pyridoxine-deficient and pair-fed pyridoxine-supplemented adult male rats. Pyridoxine-deficient animals exhibited slow electroencephalograms (EEG) represented by the dominance of delta activity and reduced seizure thresholds to local (VPL) application of either picrotoxin or pentylene tetrazole. Frequency and amplitude of thalamic VPL unit activity were significantly reduced in pyridoxine-deficient rats as compared to pyridoxine-supplemented controls. Pyridoxine-deficient rats exhibited irregular unit activity with frequent bursts and electrosilent periods in response to local (VPL) picrotoxin or pentylene tetrazole microinjections. They also exhibited severe seizure discharge activity of prolonged duration at any given dose of either picrotoxin or pentylene tetrazole. This was represented by significantly increased burst frequency, burst duration and reduced seizure latencies. Unit activity was transformed into burst discharge activity with intermittent electrosilent zones during picrotoxin or pentylene tetrazole epileptogenesis. Cerebral gamma aminobutyric acid (GABA) level was reduced and glutamate concentration increased in pyridoxine-deficient rats when compared with pyridoxine-supplemented controls. Local (VPL) microinjection of GABA or pyridoxine induced neuronal recovery in both convulsant-treated normal and pyridoxine-deficient rats. Neuronal recovery was however delayed in pyridoxine-deficient rats. Neuronal recovery was associated with a significant increase in EEG background frequency and reduction in delta frequencies in the EEG records of both normal and pyridoxine-deficient rats. Reduced seizure threshold and delayed neuronal recovery are related to the significantly reduced brain regional GABA and elevated glutamate levels in pyridoxine-deficient rats.

Fluorometric assay of B6 vitamers in biological material.

A method for the separation and assay of all B6 vitamers from a single sample of serum is reported. A new reagent system containing sodium glyoxalate, potassium aluminum, sulfate and manganese dioxide was used for quantitative conversion of pyridoxamine and its phosphate to the corresponding aldehydes by a combination of transamination and oxidation reactions.