Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy.

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl--dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector.

Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.

Purinergic modulation of glutamate release under ischemic-like conditions in the hippocampus.

The aim of the present study was to explore whether endogenous activation of different purine receptors by ATP and adenosine contributes to or inhibits excess glutamate release evoked by ischemic-like conditions in rat hippocampal slices. Combined oxygen-glucose deprivation (OGD) elicited a substantial, [Ca(2+)](o)-independent release of [(3)H]glutamate, which was tetrodotoxin (1 microM)-sensitive and temperature-dependent. The P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 0.1-10 microM), and the selective P2X(7) receptor antagonist Brilliant Blue G (1-100 nM), decreased OGD-evoked [(3)H]glutamate efflux indicating that endogenous ATP facilitates ischemia-evoked glutamate release. The selective A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.1-250 nM) and the selective A(2A) receptor antagonists 4-(2-[7-amino-2-)2-furyl(triazolo-[1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385, 0.1-20 nM) and 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261, 2-100 nM) decreased OGD-evoked [(3)H]glutamate efflux, indicating that endogenous adenosine also facilitates glutamate release under these conditions. The effect of DPCPX and ZM241385 was reversed, whereas the action of P2 receptor antagonists was potentiated by the selective ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL67156, 50 microM). The binding characteristic of the A(2A) ligand [(3)H]CGS21680 to hippocampal membranes did not change significantly in response to OGD. Taken together these data suggest that while A(1) receptors might became desensitized, A(2A) and P2X receptor-mediated facilitation of glutamate release by endogenous ATP and its breakdown product adenosine remains operational under long-term OGD. Therefore the inhibition of P2X/A(2A) receptors rather than the stimulation of A(1) adenosine receptors could be an effective approach to attenuate glutamatergic excitotoxicity and thereby counteract ischemia-induced neurodegeneration.

Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

Recombinant human tissue non-specific alkaline phosphatase successfully counteracts lipopolysaccharide induced sepsis in mice.

Sepsis is a life threatening condition that arises when the body's response to an infection injures its own tissues and organs. Sepsis can lead to shock, multiple organ failure and death especially if not recognized early and treated promptly. Molecular mechanisms underlying the systemic inflammatory response syndrome associated with sepsis are still not completely defined and most therapies developed to target the acute inflammatory component of the disease are insufficient. In this study we investigated a possibility of combating sepsis in a mouse model by intravenous treatment with recombinant human tissue non-specific alkaline phosphatase (rhTNAP) derived from transgenic rabbit milk. We induced sepsis in mice by intraperitoneal injection of LPS and three hours later treated experimental group of mice by intravenous injection with rhTNAP derived from transgenic rabbits. Such treatment was proved to be physiologically effective in this model, as administration of recombinant rhTNAP successfully combated the decrease in body temperature and resulted in increased survival of mice (80 % vs. 30 % in a control group). In a control experiment, also the administration of bovine intestinal alkaline phosphatase by intravenous injection proved to be effective in increasing survival of mice treated with LPS. Altogether, present work demonstrates the redeeming effect of the recombinant tissue non-specific AP derived from milk of genetically modified rabbits in combating sepsis induced by LPS.

Structure of the rabbit kappa-casein encoding gene: expression of the cloned gene in the mammary gland of transgenic mice.

The rabbit kappa-casein (kappa-Cas) encoding gene has been isolated as a series of overlapping DNA fragments cloned from a rabbit genomic library constructed in bacteriophage lambda EMBL3. The clones harboured the 7.5-kb gene flanked by about 2.1 kb upstream and 9 kb downstream sequences. The cloned gene is the most frequently occurring of two kappa-Cas alleles identified in New Zealand rabbits. Comparison of the corresponding domains in rabbit and bovine kappa-Cas shows that both genes comprise 5 exons and that the exon/intron boundary positions are conserved whereas the introns have diverged considerably. The first three introns are shorter in the rabbit, the second intron showing the greatest difference between the two species: 1.35 kb instead of 5.8 kb in the bovine gene. Repetitive sequence motives reminiscent of the rabbit C type repeat and the complementary inverted C type repeat were identified in the fourth and first introns, respectively. Transgenic mice were produced by microinjecting into mouse oocytes an isolated genomic DNA fragment which contained the entire kappa-Cas coding region, together with 2.1-kb 5' and 4.0-kb 3' flanking region. Expression of transgene rabbit kappa-Cas mRNA could be detected in the mammary gland of lactating transgenic mice and the production of rabbit kappa-Cas was detected in milk using species-specific antibodies. The cloned gene is thus functional.

Polymorphic insertions/deletions of both 1550nt and 100nt in two microsatellite-containing, LINE-related intronic regions of the rabbit kappa-casein gene.

The most frequent allele of the rabbit kappa-casein (kappa-Cas)-encoding gene (A allele) has previously been shown to possess two sequences similar to those found in the 5' end of long interspersed repeated elements (LINE). Part of an inverted rabbit LINE is present in the first intron and part of a direct rabbit LINE in the fourth intron. We describe herewith a less frequent allele (B allele) that lacks both 100bp in the first intron and 1550bp in the fourth intron. It was not possible to identify any allele exhibiting only one of the deletions in a population of 55 rabbits. The 100bp present in the first intron of the A allele, but absent from the B allele, are located at the 5' end of the inverse complementary LINE and include the poly (T) track of the LINE. The 1550bp present in the fourth intron of the A allele, but absent from the B allele, include the entire direct LINE sequence. Therefore, the B allele only possesses one partial LINE sequence that is located in the first intron and is truncated when compared to the copy found in the first intron of the A allele. The B allele might thus be more recent than the A allele. Differences between the sequences of transcripts corresponding to each allele are limited to two silent mutations and three modifications in the 3' UTR. In the mammary glands of lactating rabbits, which are homozygous for both alleles, kappa-Cas mRNA accumulate to similar levels and are translated into identical kappa-Cas that are secreted at similar concentrations into milk.

Differential mechanisms involved in the effect of nicotinic agonists DMPP and lobeline to release [3H]5-HT from rat hippocampal slices.

In the present study we investigated the effect of different nicotinic agonists (dimethylphenyl-piperazinium-iodide (DMPP), (-)nicotine, cytisine, (-)-lobeline, and (-)epibatidine) and antagonists (mecamylamine and dihydro-beta-erythroidine) on the release of [3H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [3H]5-HT from the hippocampus; other nicotinic agonists, such as (-)-nicotine, cytisine, and (-)-epibatidine had no effect. Unlike lobeline-induced release of [3H]5-HT, the effect of DMPP (10 and 40 microM) was antagonized by mecamylamine (20 and 10 microM). The effect of DMPP was [Ca2+]o-independent. In experiments carried out at 7 degrees C, i.e. the membrane carrier proteins are inhibited and the release by lobeline was abolished while the DMPP-induced release of 5-HT was rather potentiated. It is proposed that the effect of DMPP and lobeline, to enhance the release of [3H]5-HT from the hippocampus, was mediated by two different mechanisms. While DMPP-induced 5-HT release can be linked to a non-classical nAChR activation ([Ca2+]o-independence), the effect of lobeline was likely mediated by uptake carriers.

Ischemic-like condition releases norepinephrine and purines from different sources in superfused rat spleen strips.

Transmitters and cotransmitters of the sympathetic nervous system are involved in the regulation of a variety of immune cell functions. However, it is not entirely clear what stimuli lead to the release of these molecules in immune organs. In this study, we investigated whether local ischemia can cause the parallel release of norepinephrine and its cotransmitter, ATP, in the spleen. Ischemic-like conditions, simulated by transient (15 min) O(2) and glucose deprivation, elicited a reversible increase in the release of both norepinephrine and purines from superfused spleen strips preloaded with [3H]norepinephrine or [3H]adenosine. HPLC analysis of the released tritium label revealed a net increase in the amount of ATP, ADP, AMP, adenosine, inosine, hypoxanthine and xanthine in response to ischemic-like condition. Selective O(2) or glucose deprivation, and Ca(2+)-free conditions differentially affected the outflow of [3H]norepinephrine and [3H]purines, indicating that they derived from different sources. The ABC transporter inhibitors glibenclamide (100 microM) and verapamil (100 microM) as well as low-temperature inhibited [3H]purine release evoked by ischemic-like conditions. Surgical denervation of the spleen reduced endogenous catecholamine content and [3H]norepinephrine uptake of the spleen, but not that of [3H]adenosine. In summary, these results demonstrate the release of norepinephrine and purines in response to an ischemic-like condition in an immune organ. Although both could provide an important source of extracellular catecholamines and purines involved at various levels of immunomodulation, the source and mechanism of norepinephrine and purine efflux seem different.

Evidence that ATP released from the postsynaptic site by noradrenaline, is involved in mechanical responses of guinea-pig vas deferens: cascade transmission.

The release of endogenous ATP and [3H]noradrenaline, and the mechanical response of the guinea-pig vas deferens to field stimulation of its motor nerves were examined using a perfusion system. The release of ATP at rest was 0.83 +/- 0.13 pmol/g per min, and ATP released by field stimulation (8 Hz, 480 shocks) was 5.47 +/- 1.23 pmol/g. The evoked release was completely inhibited when Ca2+ was removed and 1 mM EGTA was added, or by 1 microM tetrodotoxin. The release of ATP and [3H]noradrenaline in response to field stimulation was constant with an S2/S1 ratio of 1.10 +/- 0.11 for ATP and 0.92 +/- 0.03 for [3H]noradrenaline, respectively (where S1 and S2 are stimulation periods). Prazosin (1 microM), a potent alpha 1-adrenoceptor antagonist, significantly reduced the stimulation-evoked release of ATP by 75% and significantly reduced both mechanical twitch and tonic responses, but enhanced the release of [3H]noradrenaline. This finding indicates that there is an alpha 1-adrenoceptor-mediated release of endogenous ATP. However, the prazosin-insensitive portion of ATP release (25%) is considered to be of presynaptic origin. The stimulation of alpha 1-adrenoceptors by 1-noradrenaline or methoxamine in concentrations ranging from 10 to 100 microM resulted in a concentration-dependent release of ATP and a biphasic contraction of the vas deferens: a twitch response was followed by a tonic contraction. Prazosin (1 microM) completely prevented the effect of 1-noradrenaline or methoxamine on both ATP release and mechanical response. When Ca2+ was omitted and EGTA (1 mM) was added, 1-noradrenaline was still able to release ATP but failed to produce contraction. Nifedipine, a Ca-channel and ATP receptor antagonist, reduced the twitch contraction and enhanced the release of ATP from muscle in response to noradrenaline administration. This finding indicates that the release of ATP from the muscle is not linked to mechanical contraction. When the vas deferens was made deficient in noradrenaline by 6-hydroxydopamine pretreatment (100 + 250 mg/kg, i.p.), electrical field stimulation failed to release [3H]noradrenaline and ATP. Under these conditions, exogenous 1-noradrenaline was much more effective in releasing ATP from the smooth muscle, and producing twitch responses, followed by a tonic contraction. After reserpine pretreatment (2 x 5 mg/kg, i.p.), the field stimulation-evoked release of ATP and both phases of contraction were markedly reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

Non-synaptic release of [3H]noradrenaline in response to oxidative stress combined with mitochondrial dysfunction in rat hippocampal slices.

Brain ischemia is frequently associated with oxidative stress in the reperfusion period. It is known that noradrenaline (NA) is released in excess under energy deprivation by the sodium-dependent reversal of the monoamine carrier. However, it is not known how oxidative stress affects NA release in the brain alone or in combination with energy deprivation. As a model of oxidative stress, the effect of H(2)O(2) (0.1-1.5 mM) perfusion was investigated in superfused rat hippocampal slices. It elicited a dose-dependent elevation of the release of [(3)H]NA and its tritiated metabolites as well as a simultaneous drop in the tissue energy charge. Mitochondrial inhibitors, i.e. rotenone (10 microM), and oligomycin (10 microM) in combination, also decreased the energy charge, but they had only a mild effect on [(3)H]NA release. However, when H(2)O(2) was added together with oligomycin and rotenone their effect on [(3)H]NA release was greatly exacerbated. H(2)O(2) and mitochondrial inhibitors also induced an increase in [Na(+)](i) in isolated nerve terminals, and their effect was additive. The effect of H(2)O(2) on tritium release was temperature-dependent. It was also attenuated by the glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM) and (+/-)-2-amino-5-phosphonopentanoic acid (10 microM), by the nitric oxide synthase inhibitors, N omega-nitro-L-arginine methyl ester (100 microM), or 7-nitroindazole (50 microM) and by the vesicular uptake inhibitor tetrabenazine (1 microM). Our results suggest that oxidative stress releases glutamate followed by activation of postsynaptic ionotropic glutamate receptors that trigger nitric oxide production and results in a flood of NA from cytoplasmic stores. The massive elevation of extracellular NA under conditions of oxidative stress combined with mitochondrial dysfunction may provide an additional source of highly reactive free radicals thus initiating a self-amplifying cycle leading to neuronal degeneration.

Analysis of high intracellular [Na+]-induced release of [3H]noradrenaline in rat hippocampal slices.

Our aim was to investigate the mechanisms involved in the high intracellular sodium-induced transmitter release in the CNS through the characterisation of the veratridine-evoked (40 microM) noradrenaline release from rat hippocampal slices. The response to veratridine was completely inhibited by tetrodotoxin (1 microM), indicating that the effect is due to the activation of sodium channels. Omission of Ca2+ from the superfusion fluid inhibited the veratridine-evoked release by 72%, showing that the majority of release results from external Ca2+-dependent exocytosis. The residual Ca2+-independent release was not blocked by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (100 microM) suggesting that intracellular Ca2+ stores are not involved in this component of veratridine effect. The noradrenaline uptake blockers, desipramine (10 microM) and nisoxetine (10 microM), inhibited the external Ca2+-independent release by 50 and 46%, respectively, indicating that the release partly originates from the reversal of transporters (carrier-mediated release). In contrast to uptake blockers, lowering the temperature, another possibility to inhibit transporter function, completely inhibited the effect of veratridine in the absence of Ca2+. Further experiments revealed that low temperature (20 and 12 degrees C) reduces the veratridine-induced increase of intracellular sodium concentration ([Na+]i) in rat cortical synaptosomes (68 and 78% inhibition, respectively). The clinical relevance of our data is that during ischemia a massive release of transmitters occurs mainly due to the elevation of [Na+]i, which contributes to the development of ischemic brain injury. Our results show that low temperature may be a better therapeutic approach to the treatment of ischemia because it has a dual action on this process. Firstly, it inhibits the function of uptake transporters and hence reduces the carrier-mediated outflow of transmitters. Secondly, it inhibits the sodium influx and therefore prevents the unwanted elevation of [Na+]i. Our data also suggest that veratridine stimulation can be a suitable model for ischemic conditions.

Dopamine is taken up from the circulation by, and released from, local noradrenergic varicose axon terminals in zona glomerulosa of the rat: a neurochemical and immunocytochemical study.

The effect of supramaximal electric field stimulation on [3H]dopamine (DA) release by rat adrenal capsule-glomerulosa preparations was studied using a micro-volume perfusion system. When the tissues were preloaded with [3H]DA, a considerable amount of [3H]DA and [3H]noradrenaline (NA) were released in response to field stimuli. Reserpinization, calcium removal or tetrodotoxin blocking of Na+ influx all completely inhibited the stimulation-evoked release of DA/NA, indicating that the radioactivity released is of neuronal and vesicular origin. In the adrenal cortex, a substantial proportion of tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactive nerve fibres and varicosities were observed around the zona glomerulosa. DA-containing nerves were not seen in the adrenal cortex; however, the same immunocytochemical procedures clearly demonstrated dopaminergic nerve cells and fibres in the substantia nigra and the striatum respectively, and cells of the adrenal medulla. Like the NA release from noradrenergic varicosities in the zona glomerulosa, the DA release from noradrenergic endings is not subject to negative feedback modulation through DA2 receptors since apomorphine, a DA2-receptor agonist, and sulpiride, a selective DA2-receptor antagonist, failed to affect the release. After in-vivo i.v. administration of [3H]DA, the glomerulosa content of DA and NA and the in-vitro release of [3H]DA and [3H]NA of zona glomerulosa both increased, indicating that the local varicose axon terminals were able to accumulate DA from the circulation, convert it into NA and release it in response to neural activity. This local arrangement of noradrenergic axon terminals, able to take up DA from the circulation and release it or convert it into NA, provides the possibility of a fine tuning of local circulation and aldosterone synthesis in the zona glomerulosa.

Hormonal and metabolic responses during psychosocial stimulation in aggressive and nonaggressive rats.

The physiological effects of a psychosocial threat (the mere presence of a potentially antagonist individual in the home cage) were studied in aggressive and nonaggressive rats. Aggressive animals spent a significantly longer time with the investigation of the opponent compared with the nonaggressive group. An increase in plasma epinephrine and corticosterone was noticed both in aggressive and nonaggressive animals. Ir beta-endorphin increased significantly only in nonfighters. Glycemia was slightly larger in nonaggressives, while lactaemia increased in both groups. The possibility is discussed that differences in psychosocial stress response may be involved in the regulation of behavior in a real encounter.

Implication of ionotropic glutamate receptors in the release of noradrenaline in hippocampal CA1 and CA3 subregions under oxygen and glucose deprivation.

A strong linkage between adrenergic and glutamatergic systems exists in the CNS but it is still unclear whether the excessive release of noradrenaline under ischemic conditions is modulated by excitatory amino acids. We studied the effect of selective glutamate receptor antagonists on the release of [3H]noradrenaline evoked by glucose and oxygen deprivation in hippocampal CA1, CA3 and dentate gyrus subregions. The release of glutamate, aspartate and GABA was measured by HPLC. Omission of oxygen and glucose increased the release of [3H]noradrenaline as well as the release of amino acids. Maximum effect on noradrenaline release was observed in CA1 region. The relative increase of the release after 30 min energy deprivation (R(2)) versus the basal release under normal conditions (R(1)), i.e. the R(2)/R(1) ratio was 7.1+/-1.0, 3.87+/-0.4 and 3.26+/-0.27 for CA1, CA3 and dentate gyrus, respectively. The [3H]noradrenaline outflow in response to glucose and oxygen deprivation was abolished at low temperature, but not by Ca(2+) removal, suggesting a cytoplasmic release process. In CA1 and CA3 [3H]noradrenaline release was significantly attenuated by MK-801, an NMDA receptor antagonist. The AMPA receptor antagonist GYKI-53784 had no effect in CA3, but partly reduced noradrenaline release in CA1. Our results suggest that ionotropic glutamate receptors seem to be implicated in the massive cytoplasmic release of noradrenaline in CA1 what may contribute to its selective vulnerability.

Effect of hypoxia and glucose deprivation on ATP level, adenylate energy charge and [Ca2+]o-dependent and independent release of [3H]dopamine in rat striatal slices.

Release of [3H]dopamine ([3H]DA) from rat striatal slices kept under hypoxic or/and glucose-free conditions was measured using a microvolume perfusion method. The corresponding changes in nucleotide content were determined by reverse-phase high-performance liquid chromatography (RPHPLC). The resting release of [3H]DA was not affected by hypoxia, but under glucose-free conditions massive [Ca2+]o-independent release of [3H]DA was observed. Hypoxia reduced the energy charge (E.C.) and the total purine content from 19.36 +/- 4.15 to 6.98 +/- 1.83 nmol/mg protein. Glucose deprivation by itself, or in combination with hypoxia, markedly reduced the levels of adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The E.C under glucose-free conditions was significantly reduced from 0.73 +/- 0.04 to 0.44 +/- 0.20. When the tissue was exposed to hypoxic and glucose-free conditions for 18 min the level of ATP was reduced to 3.15 +/- 0.11 nmol/mg protein. However, when the exposure time was 30 min the ATP level was further reduced to 1.11 +/- 0.37 nmol/mg protein. The resting release was enhanced in a [Ca2+]o-independent manner, but there was no release in response to stimulation, and tetrodotoxin did not affect the enhanced resting release, indicating that the release was not associated with axonal activity. Similarly, 50 microM ouabain, inhibitor of Na+/K(+)-activated ATPase, enhanced the release of [3H]DA at rest in a [Ca2+]o-independent manner. It seems very likely that the reduced ATP level under glucose-free conditions leads to an inhibition of the activity of Na+/K(+)-ATPase that results in reversal of the uptake processes and in [Ca2+]o-independent [3H]DA release from the axon terminals.

Expression of active human blood clotting factor VIII in mammary gland of transgenic rabbits.

Human clotting factor VIII is probably the largest protein to be expressed to date in the mammary gland of a transgenic animal, and it requires extensive posttranslational modification to achieve full biological activity. The mammary gland specific construct mWAP-hFVIII-MT-I was injected into the pronuclei of rabbit zygotes, and three transgenic offspring were obtained. Founder 385 showed germ-line transmission of a single integrated copy, and a homozygous line was established from this animal. The rhFVIII was transcribed and translated exclusively in the mammary gland. The activity of rhFVIII in the rabbit milk ranged from 5 to 8% of that found in normal human plasma. Results indicate the suitability of the transgenic rabbit mammary gland for rhFVIII production.

Dopamine, as well as norepinephrine, is a link between noradrenergic nerve terminals and splenocytes.

The effect of supramaximal electric field stimulation on 3H released from rat spleen strips was studied after loading with either [3H]dopamine ([3H]DA) or [3H]norepinephrine ([3H]NE). In some experiments, [3H]DA and [3H]NE stored in the tissue or released in response to electrical stimulation were separated from their tritiated metabolites using HPLC followed by radiochemical detection. The stimulation-evoked release of 3H after loading with either derivative was subject to negative feedback modulation through alpha2-adrenergic, D2-dopamine and muscarinic acetylcholine receptors, and could be prevented by either calcium removal or tetrodotoxin blocking of Na+ influx, indicating its neuronal and vesicular origin. After the separation of radioactive metabolites by HPLC, both the tissue loaded with [3H]DA and the fractions collected during electrical stimulation contained a considerable amount of [3H]NE, providing evidence that the neurons it originated from were adrenergic in function. [3H]DA was also released during electrical stimulation. Since the spleen does not receive dopaminergic innervation, it was concluded that the noradrenergic axon terminals in the spleen were able to take up DA, convert it in part into NE, and release it as both DA and NE in response to neural activity. The ratio of [3H]DA and [3H]NE in the spleen loaded with [3H]DA was found to be dependent on both temperature and time of loading, and could be modulated by various drugs such as desmethylimipramine, a NE uptake blocker, and disulfiram or fusaric acid, dopamine beta-hydroxylase inhibitors. The phenomenon may reveal a new mechanism by which immunocytes in the spleen can be regulated by the neuroendocrine system.

Age-dependent changes of presynaptic neuromodulation via A1-adenosine receptors in rat hippocampal slices.

The presynaptic neuromodulation of stimulation-evoked release of [3H]-acetylcholine by endogenous adenosine, via A1-adenosine receptors, was studied in superfused hippocampal slices taken from 4-, 12- and 24-month-old rats. 8-Cyclopentyl-1,3-dimethylxanthine (0.25 microM), a selective A1-receptor antagonist, increased significantly the electrical field stimulation-induced release of [3H]-acetylcholine in slices prepared from 4- and 12-month-old rats, showing a tonic inhibitory action of endogenous adenosine via stimulation of presynaptic A1-adenosine receptors. In contrast, 8-cyclopentyl-1,3-dimethylxanthine had no effect in 24-month-old rats. 2-Chloroadenosine (10 microM), an adenosine receptor agonist decreased the release of [3H]-acetylcholine in slices taken from 4- and 12-month-old rats, and no significant change was observed in slices taken from 24-month-old rats. In order to show whether the number/or affinity of the A1-receptors was affected in aged rats, [3H]-8-cyclopentyl-1,3-dimethylxanthine binding was studied in hippocampal membranes prepared from rats of different ages. Whereas the Bmax value was significantly lower in 2-year-old rats than in younger counterparts, the dissociation constant (Kd) was not affected by aging, indicating that the density rather than the affinity of adenosine receptors was altered. Endogenous adenosine levels present in the extracellular space were also measured in the superfusate by high performance liquid chromatography (HPLC) coupled with ultraviolet detection, and an age-related increase in the adenosine level was found. In summary, our results indicate that during aging the level of adenosine in the extracellular fluid is increased in the hippocampus. There is a downregulation and reduced responsiveness of presynaptic adenosine A1-receptors, and it seems likely that these changes are due to the enhanced adenosine level in the extracellular space.

Excessive release of [3H]noradrenaline and glutamate in response to simulation of ischemic conditions in rat spinal cord slice preparation: effect of NMDA and AMPA receptor antagonists.

In the present study we investigated the effects of NMDA and non-NMDA glutamate receptor antagonists on the ischemia-evoked release of [3H]noradrenaline from rat spinal cord slices. An in vitro ischemia model (oxygen and glucose deprivation) was used to simulate the ischemic conditions known to cause neuronal injury. Spinal cord slices were loaded with [3H]noradrenaline and superfused with Krebs solution in a micro-organ bath. Both axonal stimulation and ischemia increased the release of [3H]noradrenaline, but the release in response to glucose and oxygen deprivation was [Ca2+]o independent. Dizocilpine (MK-801), an NMDA receptor antagonist, suppressed the release of [3H]noradrenaline produced by ischemia, while it enhanced the release of [3H]noradrenaline evoked by electrical field stimulation. In contrast, LY300168 (GYKI-53655) [(+/-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylen e-dioxy-5H-2.3-benzodiazepine] and its (-)isomer LY303070 (GYKI-53784) [(-)-3-N-methylcarbamyde-1-(4-aminophenyl)-4-methyl-1.8-methylene- dioxy-5H-2.3-benzodiazepine] AMPA receptor antagonists, had no effect on the release of [3H]noradrenaline evoked by either electrical stimulation or ischemia. Desipramine, a noradrenaline uptake inhibitor, potentiated the release of [3H]noradrenaline evoked by ischemia, while in the absence of [Ca2+]o but under conditions when [3H]noradrenaline release was further increased, it reduced the release. Dizocilpine also decreased glutamate and aspartate release, measured by high performance liquid chromatography, during ischemia. It is concluded that glutamate release and NMDA receptors, but not AMPA receptors, are involved in the acute effect of oxygen and glucose deprivation on the excessive release of noradrenaline and that this release is not related to physiological axonal conduction.