Structural properties of phenylethylamine derivatives which inhibit transport-P in peptidergic neurones.

1. Transport-P is an antidepressant-sensitive, proton-dependent, V-ATPase-linked uptake process for amines in peptidergic neurones of the hypothalamus. It is unusual in its anatomical location in postsynaptic neurones and in that it is activated by its substrate (prazosin). This study examined the structural properties of phenylethylamine derivatives which are substrates for transport-P, as judged by competitive inhibition of the uptake of prazosin 10(-6) M in immortalized hypothalamic peptidergic neurones. 2. A basic amine was essential for activity; absence of the amine or neutralization with a carboxyl group abolished activity. Primary, secondary and tertiary amines were active but quaternary and guanyl amines were inactive. 3. A phenyl group was essential for activity at transport-P. Potency at transport-P was reduced by phenolic hydroxyl groups and enhanced by phenolic halogens. Thus, for maximal potency, the phenyl group should be hydrophobic. Phenolic methoxyl groups had no effect on potency at transport-P. 4. A side chain was necessary for activity at transport-P. Potency at transport-P was reduced by beta-hydroxyl and enhanced by alpha-methyl groups. 5. These findings further distinguish transport-P from other amine uptake processes in the brain.

Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in mice with targeted disruption of the Nurr1 gene.

To ascertain the function of an orphan nuclear receptor Nurr1, a transcription factor belonging to a large gene family that includes receptors for steroids, retinoids, and thyroid hormone, we generated Nurr1-null mice by homologous recombination. Mice, heterozygous for a single mutated Nurr1 allele, appear normal, whereas mice homozygous for the null allele die within 24 h after birth. Dopamine (DA) was absent in the substantia nigra (SN) and ventral tegmental area (VTA) of Nurr1-null mice, consistent with absent tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase, and other DA neuron markers. TH immunoreactivity and mRNA expression in hypothalamic, olfactory, and lower brain stem regions were unaffected. L-Dihydroxyphenylalanine treatments, whether given to the pregnant dams or to the newborns, failed to rescue the Nurr1-null mice. We were unable to discern differences between null and wild-type mice in the cellularity, presence of neurons, or axonal projections to the SN and VTA. These findings provide evidence for a new mechanism of DA depletion in vivo and suggest a unique role for Nurr1 in fetal development and/or postnatal survival.

Visual detection of transport-P in peptidergic neurones.

1. Hypothalamic peptidergic neurones possess an uptake process for amines (transport-P), for which prazosin is a substrate. It is characterized by a paradoxical increase in the accumulation of [3H]-prazosin when the concentration of unlabelled prazosin is increased above 10(-7) M. This increase is due to activation of a proton-dependent, vacuolar-type ATPase-linked pump that is blocked by tricyclic antidepressants. This study utilized a fluorescence method to detect amine uptake in individual cells. 2. Prazosin is fluorescent but most of its emission spectrum is in the ultraviolet range. We therefore used an analogue of prazosin in which the furan ring had been substituted with a fluorescent group, BODIPY FL. This compound's emission maximum is in the green part of the visible spectrum. 3. BODIPY FL prazosin accumulated in immortalised peptidergic neurones and the characteristic emission spectrum of the compound was evident in these cells. Accumulation of BODIPY FL prazosin was saturable and was inhibited by the tricyclic antidepressant desipramine and by unlabelled prazosin. As previously described for prazosin, uptake of BODIPY FL prazosin was blocked by cold temperature and by the organic base chloroquine. Thus, prazosin and BODIPY FL prazosin were accumulated by the same uptake process. 4. BODIPY FL prazosin accumulated in a granular distribution, which is compatible with storage in intracellular vesicles. 5. Hypothalamic cells from foetal rats in primary culture also accumulated BODIPY FL prazosin by a desipramine-sensitive process. Uptake was predominantly in neurones and glial cells did not accumulate the amine. 6. Fluorescent detection provides visual evidence for amine uptake in peptidergic neurones and should enable detailed study of the distribution of this process in the brain.

Functional properties of the uptake of amines in immortalised peptidergic neurones (transport-P).

1. Most neurotransmitters are inactivated by uptake into presynaptic nerve terminals and into glial cells. We recently provided evidence for uptake of amines in postsynaptic neurones. Uptake was evident at nanomolar concentrations of prazosin, but at concentrations of unlabelled prazosin greater than 10(-7) M, there was a further activation of uptake, manifested by a paradoxical increase in accumulation of the radioligand. We have now studied further characteristics of amine uptake in immortalised gonadotrophin-releasing hormone (GnRH) neurones. Control cells included SK-N-SH neuroblastoma cells (which possess presynaptic type amine transporters) and non-neuronal (COS-7) cells. 2. [3H]-prazosin bound to intact GnRH cells and was displaced by unlabelled prazosin in concentrations of 10(-9) to 10(-7) M. However, at higher concentrations of unlabelled prazosin, there was an increase in apparent [3H]-prazosin binding, as we had previously described. This paradoxical increase in accumulation of the radioligand was abolished by desipramine. 3. Desipramine had no effect on the association of prazosin with COS-7 cells. There was no paradoxical increase in accumulation of [3H]-prazosin in COS-7 cells, indicating that this effect requires the presence of a desipramine-blockable uptake process. 4. The increase in binding of the radioligand that was observed in the GnRH cells is not a general property of neuronal transporters; in SK-N-SH cells, there was no increase in accumulation of (-)-[3H]-noradrenaline in the presence of concentrations of unlabelled (-)-noradrenaline greater than 10(-7) M. 5. The uptake of prazosin and the increase in accumulation of [3H]-prazosin were abolished in the cold, indicating that this is an active, energy-requiring process. 6. Desipramine-sensitive uptake of prazosin was demonstrable in the GnRH cells in the absence of sodium. Further, the Na+/K(+)-ATPase inhibitor, vanadate, abolished noradrenaline uptake in SK-N-SH cells but had no effect on prazosin uptake in GnRH cells. Thus, the uptake of prazosin does not derive its energy from the sodium pump. 7. Prazosin uptake was inhibited by the V-ATPase inhibitor bafilomycin A1, the H+/Na+ ionophore, monensin and the organic base, chloroquine, indicating that uptake derives its energy from a proton pump. In contrast to other proton-dependent amine transporters, the uptake of prazosin was unaffected by reserpine. 8. Increasing extracellular pH did not increase the uptake of prazosin into GnRH cells, indicating that it is unlikely to be due to non-specific diffusion and concentration of a lysosomotropic drug into intracellular acidic particles. 9. The uptake of prazosin was unaffected by steroid hormones. 10. In COS-7 cells transfected with alpha 1-adrenoceptor cDNA, [3H]-prazosin was displaced by unlabelled prazosin without causing an increase in binding of the radioligand. This indicated that the increase in accumulation of the radioligand is unlikely to be due simply to some function of alpha 1-adrenoceptors. 11. Thus, peptidergic neurones possess an uptake process with properties that are distinguishable from known amine transporters.

Decreased stress responsivity of central and peripheral catecholaminergic systems in aged 344/N Fischer rats.

We investigated the effects of stress on central and peripheral sympatho-adrenal and sympatho-neural functions in healthy, intact young (3-4 mo) and aged (24 mo) male Fischer 344/N rats. Extracellular fluid (ECF) levels of the catecholamines norepinephrine (NE), dihydroxyphenylglycol (DHPG), methoxyhydroxyphenylglycol (MHPG), and dihydroxyphenylacetic acid (DOPAC) were obtained by microdialysis in the paraventricular nucleus (PVN) of the hypothalamus at baseline and during immobilization (IMMO). The baseline levels of these substances were similar in both age groups, and their concentrations increased significantly in response to IMMO. The IMMO-induced increases of NE and MHPG, however, were significantly smaller in old than in young rats. Plasma levels of the catecholamines NE, DHPG, MHPG, DOPAC, dihydroxyphenylalanine (DOPA), epinephrine (EPI), dopamine (DA), and HVA were also determined in young and old rats during IMMO. Basal levels of these substances were significantly higher in old than in young rats. The magnitude of the IMMO-induced increases in the majority of these compounds however, was significantly smaller in old than in young rats. We conclude that, at the basal state, aging in the Fischer rat is associated with normal PVN ECF, but high plasma catecholamine levels; at stress state, however, old rats have substantially lesser activation of their central and peripheral catecholaminergic systems than young rats.

In vivo hypothalamic release and synthesis of catecholamines in spontaneously hypertensive rats.

Juvenile spontaneously hypertensive rats (SHR) have higher plasma levels of catechols and markedly larger catechol responses to yohimbine than do normotensive Wistar-Kyoto rats, indicating increased sympathoadrenal outflow and increased alpha 2-adrenergic receptor-mediated restraint of peripheral catecholamine release during hypertension development in SHR. Yohimbine-induced catecholamine release and metabolism in the posterolateral hypothalamus of the brain were assessed in juvenile (6 to 7 weeks) and adult (15 to 16 weeks) SHR and Wistar-Kyoto rats. In vivo microdialysis was used to obtain samples for measurements of norepinephrine, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, and dihydroxyphenylacetic acid in conscious animals before and after yohimbine injection (1 mg/kg IV) beginning 24 hours after probe implantation. Catecholamine synthesis was examined from elevations of 3,4-dihydroxyphenylalanine levels after probe perfusion with NSD-1015, an inhibitor of L-aromatic acid decarboxylase. In adults, SHR had higher dialysate norepinephrine (277 +/- 38 versus 181 +/- 35 pg/mL), dihydroxyphenylglycol (3260 +/- 509 versus 2231 +/- 201 pg/mL), methoxyhydroxyphenylglycol (2659 +/- 369 versus 1890 +/- 144 pg/mL), and dihydroxyphenylacetic acid (46,312 +/- 5512 versus 13,187 +/- 1963 pg/mL) levels and markedly larger increases in 3,4-dihydroxyphenylalanine levels after NSD-1015 than Wistar-Kyoto rats. In juveniles, SHR had larger proportionate increments in microdialysate norepinephrine levels after yohimbine than Wistar-Kyoto rats (85% versus 25%). Although juvenile SHR and Wistar-Kyoto rats had similar NSD-1015-elicited increments in 3,4-dihydroxyphenylalanine levels, systemic yohimbine enhanced the NSD-1015-elicited 3,4-dihydroxyphenylalanine elevations in juvenile SHR but not in Wistar-Kyoto rats. These findings suggest augmented norepinephrine release and catecholamine synthesis in the posterolateral hypothalamus of adult SHR and augmented alpha 2-adrenergic receptor restraint of both norepinephrine release and catecholamine synthesis in juvenile SHR.

Zinc, a neurotoxin to cultured neurons, contaminates cycad flour prepared by traditional guamanian methods.

We have used cultured ventral mesencephalic and cerebellar granule cells to test the toxicity of extracts of cycad seeds (genus Cycas) and cycad-derived flours traditionally prepared in Guam. There was no significant difference in the toxicity of extracts prepared from the female gametophyte tissue of C. circinalis, C. revoluta, and C. media, common wheat flour, and 13 of 17 cycad flour samples. However, extracts prepared from 4 of 17 Guamanian flour samples exhibited marked dose-dependent neurotoxicity to mesencephalic and granule cell cultures. There was no correlation between toxicity and 2-amino-3-(methylamino)-propanoic acid (BMAA) content, and the concentration of BMAA in the medium arising from these extracts was far below that required to be neurotoxic. Toxicity of extracts was not blocked by the NMDA receptor antagonist MK-801 or the non-NMDA receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione, indicating that toxicity was not mediated by excitatory amino acid receptors. Analysis of the four toxic processed flour samples indicated high zinc content. Zinc produced a concentration-dependent neurotoxic response in mesencephalic and granule cell cultures that paralleled the calculated concentrations of zinc in the cultures derived from the four toxic flour samples. When sliced C. circinalis gametophyte tissue was "processed" in our laboratory by soaking in a galvanized container, there was a time-dependent increase in zinc content.

Effect of phencyclidine (PCP) on blood pressure and catecholamine levels in discrete brain nuclei.

PCP (10 mg/kg, s.c.) causes hypertension that is associated with decrease or tendency to decrease the levels of epinephrine and norepinephrine in the hypothalamus and the brainstem regions. If this represents a decreased catecholaminergic activity, then this may enhance sympathetic activity and contribute to the PCP-induced hypertension.

Hypothalamic sites for cardiovascular and sympathetic modulation by prostaglandin E2.

Prostaglandin E2 (PGE2, 0.5 - 5 nmol/kg) injected into the lateral cerebral ventricle of the rat increased the systemic blood pressure and heart rate in a dose dependent manner. These effects were accompanied by increases in plasma norepinephrine and epinephrine concentration. Injection of low dose of prostaglandin E2 into discrete hypothalamic nuclei induced a marked increase in heart rate, a moderate increase in the arterial blood pressure and a significant elevation of plasma norepinephrine level. This study suggests a possible central role for PGE2 in modulation of cardiovascular dynamics and sympathetic nervous activity.

Catecholamines and vasopressin in forebrain nuclei of hypertension prone and resistant rats.

Catecholamine and vasopressin content were studied in discrete brain nuclei of the Sabra strain of hypertension prone (SBH) and resistant (SBN) rats. Higher concentrations of dopamine, norepinephrine and epinephrine were observed in the median eminence of SBN compared to SBH or controls (SB) rats. Dopamine and epinephrine levels were higher in the lateral septal nucleus of SBH rats as compared to SBN or SB. Vasopressin content in discrete regions along the hypothalamo-pituitary axis was elevated in both SBH and SBN as compared to SB, but were especially elevated in the SBH group. The catecholamine and vasopressin changes found in SBH are different than those described in other genetically hypertensive rats indicating a difference in either the pathogenesis or central response to hypertension of this strain.

Cardiovascular and sympathetic responses to PGF2 alpha injection into hypothalamic nuclei.

Injection of prostaglandin F2 alpha (1 nmol/rat) into the paraventricular, dorsomedial and posterior hypothalamic nuclei of halothane anesthetized rats elicited rapid increases of heart rate and blood pressure. The injection of this same dose of prostaglandin F2 alpha into the cerebroventricular system or intravenously had no effect on these parameters. The cardiovascular responses observed following prostaglandin F2 alpha injection into these hypothalamic nuclei were accompanied by increases in plasma levels of norepinephrine and epinephrine, with peak levels at the maximal cardiovascular response. This study suggests a possible role for prostaglandin F2 alpha in modulation of the cardiovascular system via specific hypothalamic nuclei.

Mepacrine treatment prevents immobilization-induced desensitization of beta-adrenergic receptors in rat hypothalamus and brain stem.

Forced immobilization is a severe stress in rats which diminishes levels of epinephrine in specific nuclei in the hypothalamus and brain stem, suggesting that release of epinephrine is stimulated to a rate which exceeds the rate of its replacement. In the pineal gland, frog erythrocytes, C6 astrocytoma cells and rat brain, beta-adrenoceptor agonists appear to regulate the number of their receptors. Exposure to high concentrations of an agonist leads to apparent decrease in receptors reflected by a decrease in maximal specific binding of antagonists. The apparent decreases in receptors have been shown to be attended by decreases in physiologic responsiveness. In C6 astrocytoma cells, beta-agonists stimulate methylation of phosphatidylethanolamine to increase formation of membrane phosphatidylcholine which in turn appears to enhance activation of adenyl cyclase. Interference with the metabolism of phospholipids by exposure to phospholipase A2 inhibitor, mepacrine (quinacrine), prevents agonist-induced desensitization of beta-adrenoceptors in astrocytoma cells. In the present study repeated immobilization stress has been found to decrease significantly the number of beta-adrenoceptors in hypothalamus and brain stem while increasing the number of alpha 2-adrenoceptors. The desensitization of beta-adrenoceptors was prevented by treatment with mepacrine.

Hemodynamic and plasma catecholamine responses to hyperthermic cancer therapy in humans.

Cancer patients, treated with hyperthermia (to 41.5 degrees C) under thiopental and fentanyl anesthesia, had smaller increases in heart rate and cardiac index and lesser decreases in mean arterial pressure than those reported in normal volunteers. At basal body temperature anesthesia did not alter catecholamine levels. Increasing body temperature to 39.5 degrees C and 41.5 degrees C resulted in parallel increases in heart rate and cardiac index that were directly related to the increases in plasma norepinephrine levels. At basal temperature cutaneous venous plasma norepinephrine levels exceeded those of arterial; mixed-venous plasma levels were intermediate. At 39.5 degrees C and 41.5 degrees C there were sequential increases in plasma norepinephrine. The increases in mixed-venous and arterial norepinephrine were significantly greater than in cutaneous venous blood. The differential increases in norepinephrine levels in cutaneous venous, mixed-venous, and arterial blood indicate that during hyperthermia sympathetic nerve activity in skin is decreased while that in other areas is increased, suggesting that alterations in sympathetic activity modulate the hemodynamic changes that attend hyperthermia in man.

Glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in discrete nuclei of hypothalamus and substantia nigra.

The distribution of L-glutamate decarboxylase activity (GAD) and gamma-aminobutyric acid (GABA) was investigated in the hypothalamic nuclei and in parts of the substantia nigra in the rat. GAD varied markedly among these areas. The reticular part of the nigra showed the highest activity two-fold higher than any other nucleus. Among the hypothalamic nuclei, a 5-fold difference was found between the poorest and richest nuclei. High GAD was measured in the preoptic, anterior and dorsomedial nuclei. Low activity was found in arcuate and supraoptic nuclei. The lowest GAD activity was measured in the median eminence with only half the activity of the whole brain homogenate. This suggests that GABAergic neurones might not be involved in neuroendocrine regulation at the median eminence level. GABA was determined using the sensitive cycling microassay. The rats were killed by microwaves, procedure which was found to inactivate enzymatic processes within two sec without affecting the level or the distribution of GABA. Postmortem increments during the first 3 min following decapitation ranged up to 5 times the endogenous levels, and were proportional to the GAD activity of the corresponding nucleus. This confirms that GAD is the limiting factor in GABA synthesis and suggests that GABA turnover might be rapid. Endogenous GABA showed a uniform distribution within hypothalamic nuclei and nigra. No relationship appeared between endogenous GABA levels and GAD activities in the various nuclei. These results suggest that while GABA synthesis is likely to occur in non-evenly distributed nerve cells, most GABA may be stored in surrounding cells, presumably glia cells.

Reduction in brain tyrosine hydroxylase activity following acetylcholinesterase blockade in rats.

Activation of cholinergic neurons in the brain is produced by administration of the acetylcholinesterase inhibitors physostigmine and diisopropylfluorophosphate (DFP). This activation has a biphasic effect on tyrosine hydroxylase (EC 4.14.3-) activity. The acute effect of DFP, 1 mg/kg, intraperitoneally, or physostigmine, 0.2 mg/kg, intravenously, or 10 mug, intraventricularly, was a rapid reduction in tyrosine hydroxylase activity in the hypothalamus. The activities of DOPA decarboxylase (EC 4.1.1.28) and dopamine-beta-hydroxylase (EC 1.14.17.1) were not changed. In contrast to the acute effect, chronic administration of physostigmine, 0.2 mg/kg, intravenously, twice daily for 7 days produced an increase in tyrosine hydroxylase activity in the hypothalamus. The rapid acute effects may be due to an allosteric inactivation of tyrosine hydroxylase, while the chronic effects may reflect enzyme induction.