Restoration of norepinephrine and reversal of phenotypes in mice lacking dopamine beta-hydroxylase

Mice with a targeted disruption of the dopamine beta-hydroxylase (DBH) gene are unable to synthesize norepinephrine (NE) and epinephrine. These mice have elevated levels of dopamine in most tissues, although the levels are only a fraction of those normally found for NE. It is noteworthy that NE can be restored to normal levels in many tissues after a single injection of the synthetic amino acid precursor of NE, L-threo-3,4-dihydroxyphenylserine (DOPS). In other tissues, NE can be restored to normal levels after multiple injections of DOPS, whereas in the midbrain and cerebellum, restoration of NE is limited to 25-30% of normal. NE levels typically peak approximately 5 h after DOPS administration and are undetectable by 48 h. Epinephrine levels are more difficult to restore. The elevated levels of dopamine fall modestly after injection of DOPS. S(-)-Carbidopa, which does not cross the blood-brain barrier, inhibits aromatic L-amino acid decarboxylase and effectively prevents restoration of NE by DOPS in the periphery, while allowing restoration in the CNS. Ptosis and reductions in male fertility, hind-limb extension, postdecapitation convulsions, and uncoupling protein expression in dopamine beta-hydroxylase-deficient mice are all reversed by DOPS injection.     

Effect of two inhibitors of dopamine beta-hydroxylase on maturation of memory in mice

HL-A antigens of 35 patients with Beh?et's disease and 36 normal Japanese were determined by a standard microlymphocytotoxicity test. The frequency of HL-A5 among patients was significantly higher than the controls. No significant differences were observed in the distribution of other HL-A antigens.     

Electroconvulsive shock and norepinephrine uptake kinetics in the rat brain

1 The effect of isoprenaline on gastric secretion evoked by various means has been studied in conscious rats provided with Pavlov and Heidenhain pouches. 2 Interdigestive acid secretion in the Pavlov pouch was reduced by isoprenaline, whereas pepsin secretion was unaltered. 3 Central vagal stimulation effected by 2-deoxy-D-glucose injection evoked a gastric secretory response that was substantially reduced by isoprenaline. 4 2-Deoxy-D-glucose increased the mobilization of gastric mucosal histamine, an effect that was prevented by isoprenaline. 5 Isoprenaline infusion alone induced a slight increase in histamine mobilization and also a considerable elevation of immunoreactive serum gastrin concentration. 6 The secretory response to food in the Pavlov pouch was almost abolished by isoprenaline. 7 Although the acid response to histamine in the Heidenhain pouch was susceptible to isoprenaline inhibition, that to methacholine was not. 8 Pepsin secretion in the Heidenhain pouch preparation stimulated by histamine or methacholine seemed to be enhanced by isoprenaline.     

Influence of neonatal and adult hyperthyroidism on behavior and biosynthetic capacity for norepinephrine, dopamine and 5-hydroxytryptamine in rat brain

In neonatal rats, administration of l-triiodothyronine (10 mug/100 g/day) for 30 days presented signs of hyperthyroidism which included accelerated development of a variety of physical and behavioral characteristics accompanying maturation. The spontaneous motor activity was increased by 69%. Exposure of developing rats to thyroid hormone significantly increased the endogenous concentration of striatal tyrosine and the activity of tyrosine hydroxylase as well as the levels of dopamine in several brain regions. The concentration of striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid, the chief metabolites of dopamine, was also increased and the magnitude of change was greater than the rise in dopamine. Despite increases in the activity of tyrosine hydroxylase and the availability of the substrate tyrosine, the steady-state levels of norepinephrine remained unaltered in various regions of brain except in cerebellum. Futhermore, neonatal hyperthyroidism significantly increased the levels of midbrain tryptophan and tryptophan hydroxylase activity but produced no change in 5-hydroxytryptamine levels of several discrete brain regions, except hypothalamus and cerebellum where its concentration was slightly decreased. However, the 5-hydroxyindoleacetic acid levels were enhanced in hypothalamus, ponsmedulla, midbrain, striatum and hippocampus. The elevated levels of 5-hydroxyindoleacetic acid did not seem to be due to increased intraneuronal deamination of 5-hydroxytryptamine since monoamine oxidase activity was not affected in cerebral cortex and midbrain of hyperthyroid rats. The data demonstrate that hyperthyroidism significantly increased the synthesis as well as the utilization of catecholamines and 5-hydroxytryptamine in maturing brain. Since the mature brain is known to respond differently to thyroid hormone action than does the developing brain, the effect of L-triiodothyronine treatment on various putative neurohumors also was examined in adult rats. Whereas administration of l-triiodothyronine (10 mug/100 g/day) for 30 days to 120-day-old rats increased the levels of tyrosine by 23% and of tryptophan by 43%, no appreciable change was noted in tryptophan hydroxylase activity. In contrast to neonatal hyperthyroidism, excess of thyroid hormone in adult rats failed to produce any change in motor activity and tended to decrease striatal tyrosine hydroxylase activity only slightly. The concentration of dopamine remained unchanged in all regions of the brain except in midbrain where it rose by 19%. Whereas norepinephrine concentration was altered in hypothalamus, pons-medulla and midbrain, the levels of 5-hydroxytryptamine and its metabolite, 5-hydroxyindoleacetic acid, were significantly decreased in striatum and cerebellum. Since dopaminergic and noradrenergic neurons are the critical components of the motor system, the possibility exists that elevated behavioral activity in young L-triiodothyronine-treated animals might be associated with increased turnover of catecholamines in neuronal tissue.     

Effect of variations in pituitary-adrenal activity on dopamine-beta-hydroxylase activity in various regions of rat brain

Since noradrenergic neurons in the brain appear to inhibit ACTH secretion and dopamine-beta-hydroxylase (DBH) is found in noradrenergic neurons, the effect of variations in pituitary-adrenal activity on the activity of DBH in the brain stem, hypothalamus and hippocampus was determined. There was no significant circadian fluctuation in hypothalamic or brain stem DBH, as measured by the coupled radioenzymatic method of MOLINOFF et. al. [1971]. Pentobarbital and ether anesthesia, injection stress and surgical stress also had no acute effect. There was a decrease in anterior hypothalamic DBH 30 min after immobilization stress. Two days after adrenalectomy, there was a decrease in DBH in the hypothalamus and brain stem. A large dose of corticosterone (B) caused an increase in hypothalamic DBH. However, a smaller dose of B which increased plasma B to values comparable to those produced by endogenous secretion failed to have this effect. The data demonstrate an effect of the adrenal glands on brain DBH activity, but it is as yet uncertain whether this effect is mediated by glucocorticoids.     

Topographic comparison of the expression of norepinephrine transporter, tyrosine hydroxylase and neuropeptide Y mRNA in association with dopamine beta-hydroxylase neurons in the rabbit brainstem

In mammalian species, ovulation occurs following a massive release of hypothalamic gonadotropin-releasing hormone (GnRH). Several chemicals, including norepinephrine (NE) and neuropeptide Y (NPY), are responsible for the initiation and/or magnitude and duration of this pre-ovulatory GnRH surge. In the central nervous system, NE neural cell bodies are located in the brainstem; some are co-localized with NPY neurons and/or co-express the NE transporter (NET) gene which dictates NET protein production. The activity of NET at NE terminals is critical for synaptic NE function. In the rabbit, coitus induces a hypothalamic NE release which precedes the GnRH surge. We hypothesize that the coital stimulus is transmitted to the brainstem and transformed and integrated into GnRH-stimulating signals via NE, NET and/or NPY. However, very little is known about the distribution of cells expressing NET, NPY and tyrosine hydroxylase (TH, the rate-limiting enzyme of NE synthesis) in this species. Therefore, we utilized the sensitive in situ hybridization technique to identify the presence of these messages in conjunction with the location of NE cells, the latter being marked by dopamine beta-hydroxylase (DBH), the specific enzyme for NE synthesis. Three non-mated New Zealand White does were perfused with 4% paraformaldehyde and their brainstems were sectioned at 20-micron thick between 2 mm caudal to the obex and the rostral pons. Serial sections were immunohistochemically stained for DBH and hybridized with rabbit-specific TH and NET cRNAs and a human NPY probe. The data suggest that several DBH-positive areas in the medulla expressed one or more messages, i.e. the lateral tegmentum (A1) and the nucleus of the solitary tract (A2) expressed all three mRNAs, the area postrema (AP) contained NET and TH mRNAs but not NPY cells. In the pons, the locus coeruleus (LC), subnucleus of coeruleus (LCs) and lateral tegmental nuclei (A5) expressed NET and TH mRNAs but contained little or no NPY message. The distribution patterns of TH and NET appeared to be similar in the LC, LCs, A2 and AP.     

Effect of luteinizing hormone-releasing hormone on rat adrenocortical cells

The effects of luteinizing hormone-releasing hormone (LH-RH) on the function of rat adrenal cortex were investigated by using dispersed zona glomerulosa (capsular) and zona fasciculata-reticularis (inner) cells. LH-RH increased basal (but not adrenocorticotropic hormone (ACTH)-stimulated) corticosterone secretion of inner cells, without affecting either aldosterone or corticosterone production by capsular cells. LH-RH markedly raised basal (but not ACTH-enhanced) cyclic-AMP release by inner cells. The corticosterone secretagogue action of LH-RH was abolished by the protein kinase A inhibitor H-89. The conclusion is drawn that LH-RH specifically stimulates adrenal glucocorticoid secretion in rats through the activation of the adenylate cyclase signaling pathway.     

Effect of long-term administration of duloxetine on the function of serotonin and noradrenaline terminals in the rat brain

Duloxetine, an inhibitor of both 5-hydroxytryptamine (5-HT) and noradrenaline (NA) reuptake processes, has been developed as a potential antidepressant drug. The present study was initiated to investigate the functioning of multiple components of the 5-HT and NA systems following the long-term administration of duloxetine. In rats treated for 21 days with duloxetine (20 mg/kg/day), the recovery times of dorsal hippocampus CA3 pyramidal neurons from microiontophoretic applications of 5-HT and NA were significantly increased, indicating ongoing reuptake blockade with the minipump in place delivering the drug. The remaining experiments were performed following a 48-h washout. Electrically evoked release of [3H]5-HT from preloaded slices was enhanced in the midbrain, presumably due to a desensitization of the somatodendritic 5-HT1D and 5-HT1A autoreceptors. In addition, evoked release of [3H]5-HT was increased in the hippocampus, which could have been due to the desensitization of the alpha2-adrenergic heteroreceptors located on the 5-HT terminals. In contrast, there was no change in the evoked release of [3H]5-HT in the frontal cortex despite decreased functioning of the 5-HT transporter found in this brain region. Similar to changes in 5-HT release, electrically evoked release of [3H]NA was enhanced in the hippocampus and frontal cortex of rats treated chronically with duloxetine. These increases in [3H]NA release were most likely due to the desensitization of the alpha2-adrenergic autoreceptor in the hippocampus and to the desensitization of the NA transporter in the frontal cortex, respectively. These data suggest that long-term administration of duloxetine is able to induce changes in the 5-HT and NA systems that lead to enhanced release of both 5-HT and NA in some limbic brain areas. Duloxetine, therefore, may be a useful antidepressant compound.     

Specificity of amphetamine induced release of norepinephrine and serotonin from rat brain in vitro

Incubation of normal rat cortical or brain stem tissue with 3H-NE or 3H-5-HT and subsequent exposure to amphetamine produced a concentration-related release of the transmitters from tissue stores into the incubation media. Although pretreatment with the catecholamine neurotoxin in 6-hydroxydopamine (6-OHDA) reduced the retention of 3H-NE in both of these tissues, the proportion of 3H-NE released by amphetamine was attenuated only in cortical tissue. Pretreatment with the serotonergic neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT) had no effect on the retention or release of 3H-NE in cortical or brain stem tissue. Pretreatment with 5,6-DHT reduced the retention of 3H-5-HT in the cortex and brain stem, but the release of 3H-5-HT was significantly attenuated only in the latter tissue. 6-OHDA pretreatment increased the retention and proportion of cortical 3H-5-HT released by amphetamine but reduced the release of brain stem 3H-5-HT in the absence of an effect on retention. It appears that the in vitro release of 3H-NE from the cerebral cortex occurs primarily from catecholamine and not serotonergic neurons whereas the cortical release of 3H-5-HT is not an event specific to serotonergic nerve terminals. The release of 3H-5-HT from brain stem does not appear to be restricted to the serotonergic cell bodies since its release was attenuated by 5,6-DHT and 6-OHDA.     

Effects of the dopamine D3 antagonist PD 58491 and its interaction with the dopamine D3 agonist PD 128907 on brain dopamine synthesis in rat

The dopamine (DA) D3 receptor antagonist PD 58491 [3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]benzoimidazol++ +-1-yl-butyl]-1H-benzoimidazol-2-yl]phenoxy]propyl]diethylamine] bound with high affinity and selectivity to recombinant human DA D3 versus D2L and D4.2 receptors transfected into Chinese hamster ovary cells: Ki values of 19.5 nM versus 2,362 and >3,000 nM, respectively. In contrast, the putative DA D3 receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D3 versus D2L and D4.2 receptors (91 nM vs. 253 and 193 nM, respectively). In vitro, PD 58491 (1 nM-1 microM) exhibited D3 receptor antagonist activity, reversing the quinpirole (10 nM)-induced stimulation of [3H]thymidine uptake in D3 CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the gamma-butyrolactone-induced increase in DA synthesis (L-3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D2/D3 receptor agonist action at DA autoreceptors. PD 58491 (3-30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D3-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D3 autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Effect of chronic treatment with typical and atypical neuroleptics on the expression of dopamine D2 and D3 receptors in rat brain

The effect of chronic treatment (21 days) with typical and atypical neuroleptics on the expression of striatal and limbic D2 and D3 dopamine receptors was investigated in rat brain by in situ hybridization and receptor autoradiography. Haloperidol and sulpiride increased D2 receptor expression in striatal and limbic areas. In contrast, clozapine had no effect on D2 receptor expression. Haloperidol decreased D3 receptor expression in limbic areas, with the exception of the islands of Calleja where an increase occurred. Sulpiride and clozapine increased D3 receptor expression in limbic and striatal regions but decreased D3 receptor expression in the islands of Calleja. This study demonstrates that chronic treatment with typical and atypical neuroleptics produces different regionally specific changes in limbic and striatal D2 and D3 receptor expression. The alterations in dopamine receptor expression were different for each drug, but a distinction between the effects of atypical and typical neuroleptics could be made. Comparison of mRNA levels in animals which were not withdrawn from drug treatment with those that were withdrawn, demonstrated that some changes in receptor expression occurred during drug treatment, whilst others only manifested when drug treatment had ceased. The different regulation of dopamine D2 and D3 receptor expression by typical and atypical neuroleptics may have relevance to the ability of these drugs to cause extrapyramidal side-effects.     

Adrenal origin of the increase in plasma dopamine beta-hydroxylase and catecholamines induced by hemorrhagic hypotension in dogs

Controlled hemorrhagic hypotension in anesthetized dogs causes progressive increases in dopamine beta-hydroxylase (DBH) and catecholamine (CA) plasma levels and in heart rate. The concentration (units per milliliter) and the calculated total plasma content of DBH activity [(units circulating + reservoir + samples] increased 2.6 and 2.3 times, respectively. A significant positive correlation (P less than .001) was found between the plasma levels of DBH and CA; however, the CA plasma levels increased earlier and were of greater magnitude (10-fold) than those of DBH. These results suggest that CAs are more sensitive indicators of acute changes in adrenergic activity than DBH. Surgical bilateral adrenalectomy completely abolished the increases in circulating CA and DBH levels and in heart rate induced by the hemorrhage, independently of the percentage of blood removed. These results indicate that the adrenal glands contribute almost exclusively to the rise in plasma DBH and CA caused by the bleeding stress and that high circulating CA concentrations seem to account for the tachycardia that accompanies the hemorrhagic hypotension. The infusion of the reservoir blood with a lower DBH and CA content than that present in the animal at that time produced a rapid fall in circulating CA levels (59.2 +/- 8.9 to 10.8 +/- 3.3 ng/ml) and no change in the DBH concentration (5.43 +/- 0.42 and 5.40 +/- 0.53 U/ml). A 38% increase in the calculated total plasma content of DBH occurred with the transfusion. Due to the large size of the DBH molecules, trapping in tissues during the hemorrhagic hypotension period might have occurred. The improvement in the hemodynamic conditions caused by the transfusion would facilitate the washout of the enzyme from the tissues into the circulation.     

Effect of non-catecholic 2-aminotetralin derivatives on dopamine metabolism in the rat striatum

The concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in the striatum of rats after i.p. injection of dipropyl-2-aminotetralin and the four positional isomers of monohydroxy-dipropyl-2-aminotetralin. All compounds except 8-OH dipropylaminotetralin caused a decrease in DOPAC- and HVA-concentrations. In addition, 5-OH-dipropylaminotetralin produced a small elevation in DA concentrations. In contrast, 7-OH dipropylaminotetralin, in doses of 100 mumol/kg and more, decreased DA to 50% and initially increased HVA and DOPAC to about 200%, after which the concentrations of the metabolites fell to 30% or less. The 5-OH isomer was found to be the most potent compound, decreasing HVA concentrations to 70% at a dose of 0.14 mumol/kg. The potencies are compared to those of catechol-group containing DA-agonists such as apomorphine and N,N-dipropyl-5,6-dihydroxy-2-aminotetralin. In addition, a comparison is made with reported behavioural data. It is suggested that the more active N-alkylated 2-aminotetralins have a conformation which corresponds to that of the alpha rotamer of dopamine.     

Monoamines (norepinephrine, dopamine, serotonin) in the rat medial vestibular nucleus: endogenous levels and turnover

The preconcentration of some elements such as Cd, Co, Ni, and V(IV) was modeled in the presence of complexing agents such as citrate and oxalate at high Ca, Mg, and sulfate concentrations on iminodiacetic acid/ethyl cellulose (IDAEC), a chelating cellulose. The effect of the species present in the solution was studied after construction of the species distribution curves using critical, estimated, and measured stability constants. The stability constants of the IDAEC chelates were determined potentiometrically. The constants were calculated or estimated using computer programs. The diagrams calculated in homogeneous media were used for optimization of the flow injection on-line preconcentration for analysis of ultratrace metals in the highly mineralized water "Hunyadi."     

Conditioned adrenocortical steroid elevations in the rat

An illness-induced taste aversion paradigm was used to condition an elevation in plasma corticosterone level. Rats were injected with cyclophosphamide 30 min after consuming a novel saccharin drinking solution. Plasma corticosterone levels were measured before conditioning to determine unconditioned steroid levels and 3 and 6 days after training when conditioned and nonconditioned animals were provided with the saccharin solution or plain water, or were left deprived. The pairing of saccharin and cyclophosphamide was effective in inducing a passive avoidance response. There were no differences between the steroid levels of conditioned and nonconditioned animals supplied with plain water or those that remained deprived, although deprivation increased corticosterone levels. Nonconditioned rats presented with saccharin had steroid levels that did not differ from control values. Conditioned animals presented with saccharin showed an elevation in steroid level which was significantly greater than that observed in any other group. Comparable results were obtained when LiCl was used as the unconditioned stimulus.     

Age-related changes of noradrenergic-NPY interaction in rat brain: norepinephrine, NPY levels and alpha-adrenoceptors

Noradrenergic-neuropeptide Y interaction, which is implicated in different physiological functions, was studied in senescent rats. Norepinephrine (NE) and neuropeptide Y (NPY) levels were measured in brainstem and hypothalamus, and alpha-adrenergic binding was investigated in brainstem in young (4 months) and old (34 months) Wistar rats. NE concentration was the same in senescent rats, whereas NPY concentration was decreased both in brainstem and hypothalamus compared to levels in young rats. [3H]prazosin binding to alpha 1-adrenoceptors was not modified, but [3H]rauwolscine binding to alpha 2-adrenoceptors was altered with age. In fact, the density of alpha 2-adrenoceptors (Bmax) was lower, while the binding affinity (Kd) was increased in old compared to young rats. These results suggest that the decrease of NPY levels could be one of the possible reasons for changes in [3H]rauwolscine binding to alpha 2-adrenoceptors in old rats. The G-protein-adenylate cyclase system, which is impaired in senescent rats, could be involved in the disorganization of noradrenergic-NPY interaction.     

Direct effects of stress on adrenocortical function

The adrenal gland plays a pivotal role in the stress response since this response involves the hypothalamic-pituitary-adrenal axis (HPAA) and the sympatho-adrenomedullary system (SAMS) as its two principal components. An important relation between the immune system and the other stress response systems is also centered on the adrenal gland. It is well known that the cortex secretes glucocorticoids while the medulla secretes epinephrine, two of the major effects of the stress response. Some other aspects, however, also deserve special consideration: The paracrine effects of the cortical secretion on the medullary cells through the special irrigation system of the gland and reciprocally the influence of the medulla upon the cortex, either by direct close contact or by local innervation. The influence of vascular events also needs to be considered as well as the existence of some local hormonal axis such as those resulting from the local production of renin or CRH in adrenal cells. Some other cells such as mast cells, macrophages and endothelial cells seem to play a role in the regulation of the adrenal cortex and hence in the tuning of the stress response. Stressors stimulate the release of CRH from the hypothalamic paraventricular nucleus inducing the secretion of ACTH from the pituitary and that of corticosteroids from the adrenal cortex. Through the activation of the sympathetic system the adrenal can be stimulated even before adequate levels of ACTH are reached. In conditions of chronic stress the adrenal cortex undergoes an adaptation that allows the hypersecretion of glucocorticoids to occur even without the increment of ACTH.     

Effects of calcium and phosphate on catecholamines, ATP and dopamine beta-hydroxylase of chromaffin medullary granules

The penetration into the single layers of human skin in vitro of 17 alpha-estradiol, 17 beta-estradiol and estriol was investigated. The radiolabeled substances were incorporated into 4 standard ointments and into an alcoholic solution mixture. After application to the skin and after different penetration periods, the horny layer was taken off by adhesive tape stripping. The epidermis and the dermis were separated by slicing them down parallel to the skin surface in a freeze microtome. In each single layer, the amount of substance was determined and calculated relative to the applied quantity and in absolute concentrations (mug per tissue weight and molarity). Besides the expected dependence of the penetration on the type of ointment, there is a distinct dependence on the chemical structure: estriol penetrates considerably slower and in less concentrations into the living layers of the human skin than the estradiols. Furthermore, estriol reaches the dermis only in low concentrations so that this substance may be termed epidermotropic. 17 alpha-estriol which has only weak sexhormone properties in humans penetrates as well as the sexhormone 17 beta-estradiol.