Prostaglandin F2 alpha fails to alter the number of beta adrenoceptive specific binding sites in metestrous rat uteri but diminishes noradrenaline tissue uptake

3H-dihydroalprenolol (DHA) -- receptor binding was studied in membrane preparations from metestrous uterine tissue, both in presence and absence of exogenous prostaglandin (PG) F2 alpha at 10(-9) M. In addition, the uptake of 3H-noradrenaline (NA) by uterine segments from estrous and metestrous rats and the influences of PGF2 alpha (10(-9) M), cocaine (10(-5) M) corticosterone (5.10(-5) M), normetanephrine (10(-6) M) or acetylsalicylic acid (ASA: 10(-4) M), were explored. The Scatchard analysis of experimental data with 3H-DHA with or without added PGF2 alpha indicates the existence of a single class of high affinity receptors and no differences were found, in presence of PGF2 alpha, regarding the control dissociation constant or the control maximal sites of specific binding. On the other hand, the uptake of 3H-NA by uterine segments at metestrus was significantly greater than at estrus. In metestrous uteri PGF2 alpha (10(-9) M) reduced significantly NA uptake. ASA enhanced NA uptake by uteri from estrous rats, an influence prevented by PGF2 mu. In uterine segments isolated at estrus, cocaine, corticosterone and normetanephrine failed to alter 3H-NA uptake, whereas in preparations isolated at metestrus, corticosterone and normetanephrine reduced the uptake, but cocaine did not evoke any influence. Results are discussed in terms of previous findings documenting an amplification of the negative inotropic influence of NA mediated by the activation of beta-adrenoceptors, both in estrous or in metestrous preparations incubated with PGF2 alpha. Such previous findings cannot be explained by changes in the number of NA receptors or by a greater affinity of tissue receptors for the agonist, but rather by differences in NA uptake controlling its effective concentration at the biophase, near receptor sites. Interrelationships along sex hormones (estradiol), prostaglandins (PGF2 alpha) and catecholamines (NA) in uteri, are also discussed.     

Actions of 6-hydroxydopamine quinones on catecholamine neurons.

—The effect of the para-(PQ) and the ortho-(OQ) quinones of 6-hydroxydopamine (6-OH-DA) on transmitter uptake-storage mechanisms of catecholamine neurons in mouse and rat has been investigated. After the administration of PQ and OQ there was a dose-dependent and long-lasting disappearance of noradrenaline (NA) nerve terminals as demonstrated by fluorescence histochemistry and a reduction of the in vitro uptake of [³H]NA in mouse atrium and iris. These effects could be completely counteracted by blockade of the ‘membrane pump’ transport mechanism with desipramine, while monoamine oxidase inhibition, by nialamide and administration of ascorbic acid potentiated the effects produced by the two quinones. The results obtained after PQ and OQ were largely identical with those seen after administration of 6-OH-DA, well-known for its neurotoxic action on catecholamine neurons. It is therefore concluded that PQ and OQ are able to produce an acute and selective degeneration of NA nerve terminals similar to that of 6-OH-DA. The results obtained after intraventricular injection of the quinones into rat brain were also in agreement with this view. Neonatal administration of PQ or OQ to mice caused a permanent and marked decrease in [³H]NA uptake in the cerebral cortex and the spinal cord, whereas the uptake was markedly increased in the pons-medulla, similar to that seen after 6-OH-DA. The PQ and the OQ were equally potent in most experiments although clearly less potent than 6-OH-DA itself. The quinones were also found to be equally or slightly less potent than 6-OH-DA in affecting [³H]NA uptake and retention in vitro in atrium and cerebral cortex from untreated mice. It may be concluded that PQ and OQ exert their neurotoxic action on NA neurons after transition to 6-OH-DA, after a rapid extraneuronal equilibration. 6-OH-DA thus formed can thereafter be taken up and accumulated intraneuronally by use of the ‘membrane pump’ and the specific degenerative action is elicited. The lower neurotoxic potency of the quinones may be attributed to their known ability to undergo covalent binding with proteins and/or formation of 5,6-dihydroxyindole.     

Release of Purines, Noradrenaline, and GABA from Rat Hippocampal Slices by Field Stimulation

Labelled adenine, noradrenaline (NA), and gamma-aminobutyric acid (GABA) were taken up by the transversely cut hippocampal slice. [3H]NA and [14C]GABA were retained as such, [3H]- (or [14C]-) adenine mainly as adenine nucleotides. There was a spontaneous overflow of all three types of compounds ranging from 0.1 (GABA) to 0.21 (NA) %/min. The rate of [3H]NA overflow increased rapidly during electrical field stimulation. The release rate was well maintained over a 15-min period. The rate of [14C]GABA release also increased rapidly but it was not maintained over a 15-min period even if uptake and/or metabolism was inhibited by nipecotic acid (1 mM) and aminooxyacetic acid (AOAA, 0.1 mM). The bulk of the purines was released after the stimulation period. For all compounds the amounts released were frequency- and calcium-dependent. At a frequency of 3 Hz a 10 V stimulation was sufficient to cause a maximal [3H]NA release and 20 V to cause maximal [14C]GABA release, but 14C-purine release was increased further by increasing the voltage to 40 V. The evoked purine release was inhibited by a nucleoside uptake inhibitor (dipyridamole). On stimulation of [3H]NA-labelled slices the released radioactivity was composed of greater than 95% unchanged NA. The specific activities of NA in the slice and in the superfusate were practically identical. In [3H]adenine-labelled slices the released radioactivity was composed of adenosine, inosine, and hypoxanthine, but the activity in the slice of ATP, ADP, and AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of uptake and retrograde axonal transport of noradrenaline in sympathetic neurons in culture: reserpine-resistant large dense-core vesicles as transport vehicles

The uptake and retrograde transport of noradrenaline (NA) within the axons of sympathetic neurons was investigated in an in vitro system. Dissociated neurons from the sympathetic ganglia of newborn rats were cultured for 3-6 wk in the absence of non-neuronal cells in a culture dish divided into three chambers. These allowed separate access to the axonal networks and to their cell bodies of origin. [3H]NA (0.5 X 10(-6) M), added to the axon chambers, was taken up by the desmethylimipramine- and cocaine-sensitive neuronal amine uptake mechanisms, and a substantial part was rapidly transported retrogradely along the axons to the nerve cell bodies. This transport was blocked by vinblastine or colchicine. In contrast with the storage of [3H]NA in the axonal varicosities, which was totally prevented by reserpine (a drug that selectively inactivates the uptake of NA into adrenergic storage vesicles), the retrograde transport of [3H]NA was only slightly diminished by reserpine pretreatment. Electron microscopic localization of the NA analogue 5-hydroxydopamine (5-OHDA) indicated that mainly large dense-core vesicles (700-1,200-A diam) are the transport compartment involved. Whereas the majority of small and large vesicles lost their amine dense-core and were resistant to this drug. It, therefore, seems that these vesicles maintained the amine uptake and storage mechanisms characteristic for adrenergic vesicles, but have lost the sensitivity of their amine carrier for reserpine. The retrograde transport of NA and 5-OHDA probably reflects the return of used synaptic vesicle membrane to the cell body in a form that is distinct from the membranous cisternae and prelysosomal structures involved in the retrograde axonal transport of extracellular tracers.     

Biological activity, binding, and metabolic fate of Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 with the F1 variant of B16 melanoma cells

The alpha-MSH (alpha-melanocyte-stimulating hormone) agonist, Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 (hereafter called ND4-11 alpha-MSH), is at least 10-fold more potent than alpha-MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C is maximal at 0-30 min, 22 fmol/10(6) cells, but declines to 40% of this value at 4 hr. in the presence of 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/10(6) cells at 5 min to 4.6 fmol/10(6) cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4-11 alpha-MSH in the medium but there was evidence of intracellular degradation of (3H)ND4-11 alpha-MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr. The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4-11 alpha-MSH at 4 degrees C was constant from 4 hr to 24 hr. Under these conditions, there was no time-dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4-11 alpha-MSH binding to F1 variant cells at 4 degrees C demonstrated that there were approximately 4500 receptors per cell and an association constant of 17.1 nM-1. These results are consistent with a process of (3H)ND4-11 alpha-MSH binding to its receptor followed by internalization of the receptor-hormone complex and then intracellular degradation of the hormone.     

Spatiotemporal interaction of alpha(2) autoreceptors and noradrenaline transporters in the rat locus coeruleus: implications for volume transmission

We investigated the roles of alpha(2) autoreceptors and noradrenaline (NA) transporters on NA efflux and uptake in the rat locus coeruleus after electrical stimulation. NA efflux was evoked by various trains (50 pulses, 10-500 Hz) and measured by fast cyclic voltammetry. NA efflux and uptake half-time (t(1/2)) were stimulus-dependent, ranging from 43 +/- 3 nM and 2.45 +/- 0.21 s, respectively, with 500-Hz stimuli to 127 +/- 11 nM and 4.41 +/- 0.34 s, respectively, with 100-Hz trains. Based on these data, we calculate that each transporter removes 0.19 NA molecules from the extracellular space every second, a velocity compatible more with transporter-than channel-mode conduction. Dexmedetomidine (10 nM) decreased NA efflux by approximately 30% on stimulations of < or =1 s in duration. BRL 44408 (1 microM) increased NA efflux on stimuli of > or =2 s (by up to 92 +/- 16%). Desipramine (50 nM) increased NA efflux on stimuli of > or =1 s (by 113 +/- 24%) but slowed NA uptake on all stimuli. When given together, the effects of desipramine and BRL 44408 were additive at stimuli of >or =1 s but showed potentiation on shorter trains. There was a significant time delay for the elevation of NA efflux by blockade of uptake (0.79 s) or autoreceptors (1.14 s), suggesting that both are located extrasynaptically and that NA must diffuse through the extracellular space to these structures. We suggest that released NA may interact with alpha(2) autoreceptors and NA transporters as far as 10 microm from the release sites, an action compatible with a volume transmission role of NA in the locus coeruleus.     

Contraction of various zones of the rat testicular capsule in vitro induced by noradrenaline

The contractions induced by Noradrenaline (NA) on several preparations of the testicular capsule of the rat have been studied. Increasing concentrations of NA were added both in a single and cumulative way to the whole capsule and to some portions: the mediastinal and non mediastinal portions and to the anterior and posterior portions. All the preparations show concentration-dependent responses to NA except the non mediastinal portion. The maximal amplitude of the contraction was induced in the mediastinal portion, where the greatest amount of contractile cells exists. The NA dose that induces the maximal effect (Emax) is different in each preparation. A lower pD2 is obtained in the cumulative curves when they are compared with the non cumulative curves. In both concentration-response curves a desensitizing effect appears, when 3 X 10(-6)M of NA is reached in the cumulative ones and with higher NA doses in the non cumulative ones. Repeated single doses of NA (10(-5)M) also show desensitization. This effect is independent of the resting time between consecutive doses and it is not modified by propranolol (10(-6)M), normetanephrine (10(-5)M) or TMB-8 (10(-5)M), but it is decreased by imipramine (3 X 10(-8)M) and indomethacin (3 X 10(-6)M). This suggests that neural uptake and release of prostaglandins might be involved in this desensitization.     

Niacin deficiency increases spontaneous and etoposide-induced chromosomal instability in rat bone marrow cells in vivo

Poly(ADP-ribose) polymerase (PARP) binds to DNA single and double strand breaks and uses NAD in the synthesis of poly(ADP-ribose) (pADPr). Niacin deficiency in rats decreases bone marrow NAD(+) and limits pADPr synthesis in response to DNA damage, while pharmacological supplementation with nicotinic acid (NA) increases bone marrow NAD(+) and pADPr. The purpose of this study was to determine if niacin status alters the extent of DNA damage and chromosomal instability before and after treatment with the chemotherapy drug etoposide (ETO). Genotoxicity was evaluated using the comet, micronucleus and sister chromatid exchange (SCE) assays. Male Long-Evans rats were fed niacin deficient (ND), or pair-fed (PF) niacin replete (30mg niacin/kg) or NA supplemented (4g niacin/kg) diets for 3 weeks. Rats were gavaged with ETO (1-25mg/kg) suspended in corn oil or an equal volume of vehicle (CON). Comet analysis demonstrated that ETO-induced DNA damage (mean tail moment (MTM) and proportion of cells with significant damage) was greater in bone marrow cells from ND rats, compared to PF or NA rats. Surprisingly, niacin deficiency alone caused 6.2- and 2.8-fold increases in spontaneous micronucleus formation and SCE frequency, respectively. As expected, ETO treatment increased the level of micronuclei (MN) and SCEs in all diet groups; however, the absolute increases were greater in ND bone marrow. These data show that niacin is required for the maintenance of chromosomal stability and may facilitate DNA repair in vivo, in a tissue that is sensitive to niacin depletion and impaired pADPr metabolism. Pharmacological intakes of niacin do not appear to be further protective compared to adequate intakes. Niacin supplementation may help to protect the bone marrow cells of cancer patients with compromised nutritional status from the side effects of genotoxic chemotherapy drugs.     

Involvement of noradrenaline transporters in S-nitrosocysteine-stimulated noradrenaline release from rat brain slices: existence of functional Na(+)-independent transporter activity

Noradrenaline (NA) can be released by both exocytosis and by the membrane transporter responsible for transmitter uptake. Previously, we reported that S-nitrosocysteine (SNC), an S-nitrosothiol, stimulated [3H]NA release from the rat hippocampus. In this study, we investigated the involvement of the NA transport system in SNC-stimulated NA release from rat brain (cerebral cortex and hippocampus) slices. [3H]NA release by SNC in normal Na(+) (148 mM)-containing buffer from both slices was slightly, but significantly, inhibited by 1 microM desipramine, an NA transporter inhibitor. [3H]NA release in low Na(+) (under 14 mM)-containing buffer was inhibited by over 50% by desipramine. [3H]NA release by tyramine from both slices in normal and low Na(+) buffer was almost completely inhibited by desipramine. [3H]NA uptake into cerebral cortical slices was observed in low Na(+) buffer at 20-30% of normal Na(+) buffer levels. [3H]NA uptake in both normal and low Na(+) buffers was inhibited by desipramine and by SNC. Although [3H]NA uptake in normal Na(+) buffer was almost completely inhibited by 500 microM ouabain, the uptake in low Na(+) buffer was resistant to ouabain. These findings suggest the existence of a functional Na(+)-independent NA transport system and that SNC stimulates NA release at least partially via this system in brain slices.     

Inhibitory effects of cis- and trans-resveratrol on noradrenaline and 5-hydroxytryptamine uptake and on monoamine oxidase activity

This study investigated for the first time the potential effects of cis- and trans-resveratrol (c-RESV and t-RESV) on noradrenaline (NA) and 5-hydroxytryptamine (5-HT) uptake by synaptosomes from rat brain, on 5-HT uptake by human platelets, and on monoamine oxidase (MAO) isoform activity. Both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the uptake of [3H]NA and [3H]5-HT by synaptosomes from rat brain and the uptake of [3H]5-HT by human platelets. In both experimental models, t-RESV was slightly more efficient than c-RESV. Furthermore, in synaptosomes from rat brain, the RESV isomers were less selective against [3H]5-HT uptake than the reference drug fluoxetine (0.1-30 microM). On the other hand, both c-RESV and t-RESV (5-200 microM) concentration-dependently inhibited the enzymatic activity of commercial (human recombinant) MAO isoform (MAO-A and MAO-B) activity, c-RESV being slightly less effective than t-RESV. In addition, both RESV isomers were slight but significantly more selective against MAO-A than against MAO-B. Since the principal groups of drugs used in the treatment of depressive disorders are NA/5-HT uptake or MAO inhibitors, under the assumption that the RESV isomers exhibit a similar behaviour in humans in vivo, our results suggest that these natural polyphenols may be of value as structural templates for the design and development of new antidepressant drugs with two important biochemical activities combined in the same chemical structure: NA/5-HT uptake and MAO inhibitory activity.     

Ventral Horn Neuropeptides Modulate the Release of Noradrenaline from Tissue Slices of Rat Brainstem and Ventral Thoracic Spinal Cord

The release of endogenous noradrenaline (NA) from slices of adult rat brainstem and ventral thoracic spinal cord was investigated using a fixed-volume incubation technique and HPLC with electrochemical detection. Incubation with potassium (15-50 mM) produced a dose-related increase in basal NA release that was calcium dependent. The potassium-evoked release of NA from spinal cord or brainstem slices was potentiated according to dose by preincubation with either (a) the selective alpha 2-adrenoceptor antagonist idazoxan (10(-6)-10(-4) M) or (b) the thyrotrophin-releasing hormone (TRH) analogue RX 77368 (pGlu-His-3,3'-dimethyl ProNH2; 10(-5) and 10(-4) M). Incubation of spinal cord slices with the NA uptake inhibitor maprotiline (1 microM) enhanced the effect of idazoxan but inhibited that of RX 77368. The effects of RX 77368 and potassium alone (15 mM) on NA release from both spinal cord and brainstem slices were reduced to basal levels with tetrodotoxin (10(-7) M). Similarly, preincubation of spinal cord, but not brainstem, slices with the insect neuropeptide proctolin (10(-4) M) significantly attenuated the potassium- or RX 77368-induced release of NA, whereas substance P (3 X 10(-5) and 1 X 10(-4) M) had no effect on either tissue. These results suggest that changes in NA release in the spinal cord and brainstem may mediate some of the actions of neuropeptides in ventral spinal cord, although the peptides may not be acting directly on the noradrenergic nerve terminals in these tissues.     

Reduced plasma FFA availability increases net triacylglycerol degradation, but not GPAT or HSL activity, in human skeletal muscle

Intramuscular triacylglycerols (IMTG) are proposed to be an important metabolic substrate for contracting muscle, although this remains controversial. To test the hypothesis that reduced plasma free fatty acid (FFA) availability would increase IMTG degradation during exercise, seven active men cycled for 180 min at 60% peak pulmonary O(2) uptake either without (CON) or with (NA) prior ingestion of nicotinic acid to suppress adipose tissue lipolysis. Skeletal muscle and adipose tissue biopsy samples were obtained before and at 90 and 180 min of exercise. NA ingestion decreased (P < 0.05) plasma FFA at rest and completely suppressed the exercise-induced increase in plasma FFA (180 min: CON, 1.42 +/- 0.07; NA, 0.10 +/- 0.01 mM). The decreased plasma FFA during NA was associated with decreased (P < 0.05) adipose tissue hormone-sensitive lipase (HSL) activity (CON: 13.9 +/- 2.5, NA: 9.1 +/- 3.0 nmol.min(-1).mg protein(-1)). NA ingestion resulted in decreased whole body fat oxidation and increased carbohydrate oxidation. Despite the decreased whole body fat oxidation, net IMTG degradation was greater in NA compared with CON (net change: CON, 2.3 +/- 0.8; NA, 6.3 +/- 1.2 mmol/kg dry mass). The increased IMTG degradation did not appear to be due to reduced fatty acid esterification, because glycerol 3-phosphate activity was not different between trials and was unaffected by exercise (rest: 0.21 +/- 0.07; 180 min: 0.17 +/- 0.04 nmol.min(-1).mg protein(-1)). HSL activity was not increased from resting rates during exercise in either trial despite elevated plasma epinephrine, decreased plasma insulin, and increased ERK1/2 phosphorylation. AMP-activated protein kinase (AMPK)alpha1 activity was not affected by exercise or NA, whereas AMPKalpha2 activity was increased (P < 0.05) from rest during exercise in NA and was greater (P < 0.05) than in CON at 180 min. These data suggest that plasma FFA availability is an important mediator of net IMTG degradation, and in the absence of plasma FFA, IMTG degradation cannot maintain total fat oxidation. These changes in IMTG degradation appear to disassociate, however, from the activity of the key enzymes responsible for synthesis and degradation of this substrate.     

CHANGES IN CENTRAL NORADRENALINE NEURONSADMINISTRATION AFTER SYSTEMIC 6-HYDROXYDOPAMINE ADMINISTRATION

—Intravenous injection of a large dose of 6-hydroxydopamine (100 mg/kg) to adult rats caused a significant and long-lasting reduction (about 30 per cent) of the in oirro uptake of [³H]NA in the cerebral cortex and spinal cord, while no changes were seen in the hypothalamus. The endogenous NA in whole brain was similarly reduced (about 20 per cent). Fluorescence histochemistry revealed catecholamine accumulations which are degenerative signs, induced by 6-hydroxydopamine, in axons of the dorsal NA bundle innervating the cerebral cortex. It is concluded that the blood–brain barrier in adult rats is not completely protective with respect to the neurotoxic action of systemically injected 6-hydroxydopamine, which can produce degeneration of a significant number of NA nerve terminals in the cerebral cortex and spinal cord. Previous studies have shown that 6-hydroxydopamine caused a permanent and selective degeneration of a large number of central NA nerve terminals when injected systemically up to 1 week after birth, due to an incompletely developed blood-brain barrier. This barrier for 6-hydroxydopamine develops between the 7th and 9th day after birth (Sachs , 1973). In the present study 6-hydroxydopamine was found to cause a small transient reduction in [³H]NA uptake in cerebral cortex of rats between 9 and 28 days of age, while in older rats the damage produced by 6-hydroxydopamine was long-lasting. Thus, the NA nerves ascending to the cerebral cortex seem to possess a regenerative capacity to a 6-hydroxydopamine-induced degeneration up to about 28 days postnatally, but which later disappears or is markedly retarded.     

Plant-Derived Neurotoxic Amino Acids (β-N-Oxalylamino-l-Alanine and β-N-Methylamino-l-Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.     

Adaptation of influenza A viruses to cells expressing low levels of sialic acid leads to loss of neuraminidase activity

Influenza A viruses possess two virion surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA binds to sialyloligosaccharide viral receptors, while the NA removes sialic acids from the host cell and viral sialyloligosaccarides. Alterations of the HA occur during adaptation of influenza viruses to new host species, as in the 1957 and 1968 influenza pandemics. To gain a better understanding of the contributions of the HA and possibly the NA to this process, we generated cell lines expressing reduced levels of the influenza virus receptor determinant, sialic acid, by selecting Madin-Darby canine kidney cells resistant to a lectin specific for sialic acid linked to galactose by alpha(2-3) or alpha(2-6) linkages. One of these cell lines had less than 1/10 as much N-acetylneuraminic acid as its parent cell line. When serially passaged in this cell line, human H3N2 viruses lost sialidase activity due to a large internal deletion in the NA gene, without alteration of the HA gene. These findings indicate that NA mutations can contribute to the adaptation of influenza A virus to new host environments and hence may play a role in the transmission of virus across species.     

Neuroprotective Effects of Some Monoamine Oxidase-B Inhibitors Against DSP-4-Induced Noradrenaline Depletion in the Mouse Hippocampus

Abstract: DSP-4 [ N -(2-chloroethyl)- N -ethyl-2-bromobenzylamine], a selective noradrenaline (NA) uptake blocker, is capable of inducing long-lasting depletion of NA in some noradrenergic axon terminals and of subsequently causing cell death to NA neuronal cell bodies in rodents. R (−)-Deprenyl, a selective monoamine oxidase (MAO)-B inhibitor, has been shown to be capable of protecting animals against this DSP-4-induced neuronal degeneration. Its action, however, has been claimed to be unrelated to the inhibition of MAO-B activity but rather due to competition for the NA uptake sites. The effects of several types of MAO inhibitors against DSP-4 toxicity, MAO-B activity both in vivo and in vitro, and NA uptake into the hippocampus have been assessed. N -(2-Hexyl)- N -methylpropargylamine (2-HxMP), a potent MAO-B inhibitor, for example, exerts no appreciable effect on NA uptake but is quite potent in counteracting the NA-depleting effect of DSP-4. Such results rule out the possibility that the neuroprotective effect of the MAO-B inhibitors is due mainly to their effect on NA uptake. The in vitro inhibition of MAO-B activity seems to correlate positively with their neuroprotective effects against DSP-4. In comparison to the MAO-B inhibitors, NA uptake blockers, such as desipramine and S (+)-deprenyl, exhibit relatively low efficacy in protecting the NA axon terminals from the effects of DSP-4-induced damage. The restoration of hippocampal NA levels is significantly enhanced with repeated treatments of R (−)-deprenyl or 2-HxMP even at very low doses following the DSP-4 insult. This suggests that in addition to neuroprotection, these MAO-B inhibitors may rescue some of the noradrenergic axon terminals damaged by DSP-4.     

Presynaptic alpha2-receptors regulate reverse Na+/Ca2+-exchange and transmitter release in Na+-loaded peripheral sympathetic nerves

Electrical depolarisation-(2 Hz, 1 ms)-induced [3H]noradrenaline ([3H]NA) release has been measured from the isolated main pulmonary artery of the rabbit in the presence of uptake blockers (cocaine, 3 x 10(-5) M; corticosterone, 5 x 10(-5) M). Substitution of most of the external Na+ by Li+ (113 mM; [Na+]0: 25 mM) slightly potentiated the axonal stimulation-evoked release of [3H]NA in a tetrodotoxin (TTX, 10(-7) M) sensitive manner. The reverse Na+/Ca2+-exchange inhibitor KB-R7943 (3 x 10(-5) M) failed to inhibit the stimulation-evoked release of [3H]NA, but increased the resting outflow of neurotransmitter. The 'N-type' voltage-sensitive Ca2+-channel (VSCC) blocker omega-conotoxin (omega-CgTx) GVIA (10(-8) M) significantly and irreversibly inhibited the release of [3H]NA on stimulation (approximately 60-70%). The 'residual release' of NA was abolished either by TTX or by reducing external Ca2+ from 2.5 to 0.25 mM. The 'residual release' of NA was also blocked by the non-selective VSCC-blocker neomycin (3 x 10(-3) M). Correlation was obtained between the extent of VSCC-inhibition and the transmitter release-enhancing effect of presynaptic alpha2-receptor blocker yohimbine (3 x 10(-7) M). When the release of [3H]NA was blocked by omega-CgTx GVIA plus neomycin, yohimbine was ineffective. Inhibition of the Na+-pump by removal of K+ from the external medium increased both the resting and the axonal stimulation-evoked release of [3H]NA in the absence of functioning VSCCs (i.e., in the presence of neomycin and after omega-CgTx treatment). Under these conditions the stimulation-evoked release of NA was abolished either by TTX or by external Ca2+-removal (+1 mM EGTA). Similarly, external Li+ (113 mM) or the reverse Na+/Ca2+ exchange blocker KB-R7943 (3 x 10(-5) M) significantly inhibited the stimulation-induced transmitter release in 'K+-free' solution. KB-R7943 decreased the resting outflow of NA as well. Under conditions in which the Na+-pump was inhibited in the absence of functioning VSCCs, yohimbine (3 x 10(-7) M) further enhanced the release of neurotransmitter, while l-noradrenaline (l-NA, 10(-6) M), an agonist of presynaptic alpha2-receptors, inhibited it. The yohimbine-induced enhancement of NA-release was abolished by Li+-substitution and significantly inhibited by KB-R7943 application. It is concluded that after blockade of VSCCs brief depolarising pulses may reverse Na+/Ca2+-exchange and release neurotransmitter in Na+-loaded sympathetic nerves. Further, similar to that of VSCCs, the reverse Na+/Ca2+-exchange may also be regulated by presynaptic alpha2-receptors.     

Topological mapping of the active sites of cytochromes P4502B1 and P4502B2 by in situ rearrangement of aryl-iron complexes.

Cytochrome P4502B1 reacts with phenylhydrazine or phenyldiazene to give an iron-phenyl complex that oxidatively rearranges in situ to the two N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A (NA) and D (ND) [Swanson, B. A., Dutton, D. R., Lunetta, J. M., Yang, C. S., & Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19258-19264]. The conclusion that the active site of cytochrome P4502B1 is open above pyrrole rings A and D but not B and C is extended here by studies with larger arylhydrazines. The N-arylprotoporphyrin IX standards required for product identification were obtained by reaction of the arylhydrazines with equine myoglobin. Cytochrome P4502B1 aryl-iron complex formation followed by oxidative shift of the aryl group produces the following N-aryl-protoporphyrin IX NA:ND regioisomer ratios: phenylhydrazine (39:61), 3,5-dimethylphenylhydrazine (29:71), 4-tert-butylhydrazine (25:75), 2-naphthylhydrazine (less than 2:greater than 98), and 4-(phenyl)phenylhydrazine (87:13). Electron-withdrawing substituents (as in 3,5-dichlorophenyl) prevent the aryl group shift. The increase in the proportion of the ND regioisomer with increasing bulk of the aryl group suggests that the region over pyrrole ring A is more sterically encumbered than that over pyrrole ring D. The regiospecificity is reversed, however, with 4-(phenyl)phenylhydrazine, which primarily gives the NA regioisomer. This reversal suggests that the active site has a sloping roof that is higher over pyrrole ring A than pyrrole ring D and that provides a larger steric barrier to the shift of tall aryl moieties than the barrier over pyrrole ring A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extraneuronal effects of 6-hydroxydopamine and extraneuronal uptake of noradrenaline

Summary 6-hydroxydopamine (6-OHDA) was shown to cause ultrastructural changes in adrenocortical cells of lizards and rats. These changes comprised the formation of dense bodies with lamellar and crystalloid patterns, a decrease in the number of mitochondria and structural alterations of mitochondria. Alterations in adrenocortical cells of lizards and rats differed in both qualitative and quantitative aspects. Adrenomedullary cells were not affected as a rule. Only in young animals did 6-OHDA cause deposits of an electrondense material in medullary cells.An attempt was made to obtain information on amine uptake into cortical cells using the Falck-Hillarp technique to analyse the in-vivo and in-vitro uptake of noradrenaline (NA) into the adrenal cortex in adult rats. Extraneuronal uptake into heart and spleen was studied as well. Our results suggest that NA is taken up into cortical cells, particularly into nuclei, after exposure to 10^-4 gm/ml in-vitro indicating that uptake of 6-OHDA is also likely. Investigations using labelled 6-OHDA are required for further elucidating its extraneuronal uptake.Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/3) and a Research Fellowship of the University of Melbourne to K.U.