NADPH oxidase inhibition does not interfere with low PO2 transduction in rat and rabbit CB chemoreceptor cells

The aim of the present work was to elucidate therole of NADPH oxidase in hypoxia sensing and transduction in thecarotid body (CB) chemoreceptor cells. We have studied the effects of several inhibitors of NADPH oxidase on the normoxic and hypoxia-induced release of [³H]catecholamines(CA) in an in vitro preparation of intact CB of the rat and rabbitwhose CA deposits have been labeled by prior incubation with thenatural precursor[³H]tyrosine. It wasfound that diphenyleneiodonium (DPI; 0.2-25 µM), an inhibitor ofNADPH oxidase, caused a dose-dependent release of[³H]CA from normoxic CBchemoreceptor cells. Contrary to hypoxia, DPI-evoked release was onlypartially Ca²⁺ dependent.Concentrations of DPI reported to produce full inhibition of NADPHoxidase in the rat CB did not prevent the hypoxic release response inthe rat and rabbit CB chemoreceptor cells, as stimulation with hypoxiain the presence of DPI elicited a response equaling the sum of thatproduced by DPI and hypoxia applied separately. Neopterin (3-300µM) and phenylarsine oxide (0.5-2 µM), other inhibitors ofNADPH oxidase, did not promote release of[³H]CA in normoxic conditions oraffect the response elicited by hypoxia. On the basis of effects ofneopterin and phenylarsine oxide, it is concluded that NADPH oxidasedoes not appear to play a role in oxygen sensing or transduction in therat and rabbit CB chemoreceptor cells in vitro and, in the context ofthe present study, that DPI effects are not related to NADPH oxidase inhibition.     

Release of dopamine and norepinephrine by hypoxia from PC-12 cells

We examined the effects of hypoxia on the release of dopamine(DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cellsand assessed the involvement ofCa²⁺ and protein kinases instimulus-secretion coupling. Catecholamine release was monitored bymicrovoltammetry using a carbon fiber electrode as well as by HPLCcoupled with electrochemical detection (ECD). Microvoltammetricanalysis showed that hypoxia-induced catecholamine secretion(PO₂ ofmedium ~40 mmHg) occurred within 1 min after the onset of thestimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysisrevealed that, at any level of PO₂, therelease of NE was greater than the release of DA. In contrast, inresponse to K⁺ (80 mM), DA releasewas ~11-fold greater than NE release. The magnitude ofhypoxia-induced NE and DA releases depended on the passage, source, andculture conditions of the PC-12 cells. Omission of extracellularCa²⁺ or addition of voltage-gatedCa²⁺ channel blockers attenuatedhypoxia-induced release of both DA and NE to a similarextent. Protein kinase inhibitors, staurosporine (200 nM) andbisindolylmaleimide I (2 µM), on the other hand, attenuatedhypoxia-induced NE release more than DA release. However, proteinkinase inhibitors had no significant effect onK⁺-induced NE and DA releases.These results demonstrate that hypoxia releases catecholamines fromPC-12 cells and that, for a given change inPO₂, NErelease is greater than DA release. It is suggested that proteinkinases are involved in the enhanced release of NE during hypoxia.

     

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.

     

Stop-flow studies of solute uptake in rat lungs

Stop-flow studies were used to characterize solute uptake inisolated rat lungs. These lungs were perfused at 8 or 34 ml/min for10-28 s with solutions containing¹²⁵I-albumin and two or more ofthe following diffusible indicators: [³H]mannitol,[¹⁴C]urea,³HOH,²⁰¹Tl⁺,or⁸⁶Rb⁺.After this loading period, flow was stopped for 10-300 s and thenresumed to flush out the perfusate that remained in the pulmonary vasculature during the stop interval. Concentrations of²⁰¹Tl⁺and⁸⁶Rb⁺in the venous outflow decreased after the stop interval, indicating uptake from exchange vessels during the stop interval. The amount ofthese K⁺ analogs lost from thecirculation during the stop interval was greater when the intervalswere longer. However, losses of²⁰¹Tl⁺at 90 s approached those at 300 s. Because extraction continued afterthe vasculature had been flushed, vascular levels had presumably fallento negligible levels during the stop interval. By 90 s of stop flow thevascular volume that was cleared of²⁰¹Tl⁺averaged 0.657 ± 0.034 (SE) ml in the experiments perfused at 8 ml/min and 0.629 ± 0.108 ml in those perfused at 34 ml/min. Increases in perfusate K⁺decreased the cleared volumes of²⁰¹Tl⁺and⁸⁶Rb⁺.Uptake of[³H]mannitol,[¹⁴C]urea, and³HOH during the stop intervals wasobserved only when the lungs were loaded at high flow for shortintervals. Decreases in²⁰¹Tl⁺and⁸⁶Rb⁺concentrations in the pulmonary outflow can be used to identify thefraction of the collected samples that were within exchange vessels ofthe lung during the stop interval and may help determine thedistribution of solute and water exchange along the pulmonary vasculature.

     

Accumulation and metabolism of norepinephrine in rat hypothalamus after exhaustive stress

—Exhaustive stress in rats is followed by a temporary reduction of hypothalamic norepinephrine (NE) together with a persistent increase in turnover during recovery. To test for persistent alterations of NE storage and metabolism produced by stress, rats were subjected to 3 h of forced running and were then injected intraventricularly with [³H]NE or [³H]dopamine (DA). The hypothalamus was assayed for [³H]NE and its metabolites at various intervals after injection. The effects of stress were compared with those of reserpine (7·5 mg/kg) or α-methyltyrosine (AMT, 300 mg/kg) pretreatment. It was found that the stress-induced reduction of endogenous NE was not accompanied by a change in the accumulation of exogenous [³H]NE either 10 or 30 min after injection, whereas the NE depletions produced by reserpine or AMT were associated with decreased or increased accumulation, respectively. However, stress did produce an increased accumulation of [³H]NE endogenously synthesized from [³H]DA. These results indicate that exhaustive stress does not adversely affect the storage of NE. They also suggest that stores of NE depleted by stress are replenished chiefly with newly synthesized NE and not through an increased uptake and binding or decreased metabolism of extraneuronal NE. The latter factors may play a role in the maintenance of brain NE stores when biosynthesis is low, i.e. after AMT. The major metabolites of exogenous [³H]NE, at 30 min after injection, were identified as conjugates of 3,4-dihydroxyphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG) in approximately equal amounts. The finding of high levels of conjugated DOPEG confirms a recent report (Slgden and Eccleston , 1971) that this compound is a major metabolite of brain NE. Reserpine produced marked elevations of both conjugates; AMT slightly reduced each. Prior stress increased only conjugated MOPEG, an observation suggesting that CNS levels of this metabolite may reflect NE released by nervous activity.     

[125I]MIBG uptake and release in different regions of the rat brain

Radioiodinated m-iodobenzylguanidine ([¹²⁵I]MIBG) and tritiated norepinephrine ([³H]NE]) uptake and release were compared, in different regions of the brain of the rat. The classification of the regions according to uptake was the same for both tracers: striatum > hypothalamus > hippocampus > cortex > brainstem. Tetrabenazine (TBZ), a granular monoamine uptake inhibitor reduced the uptake in the different regions. The inhibition rate was higher for [³H]NE uptake than for [¹²⁵I]MIBG. The spontaneous release was the same for [¹²⁵I]MIBG and [³H]NE and was the lowest in the striatum. The K⁺ stimulated release of [³H]NE was more complete than the release of [¹²⁵I]MIBG and was the most important in the striatum. From these results, it is inferred that MIBG enters the brain tissue via NE uptake mechanisms. It appears that MIBG is stored in the chromaffin granules, as NE, but also in the cytoplasm. A modified molecule derived from MIBG which would cross the blood-brain barrier, would then appear as a potential scintigraphic marker of monoamine uptake, storage and release.     

Effect of ethanol on [3H]Dopamine release in rat nucleus accumbens and striatal slices

Ethanol (10–200 mM) transiently increased tritium overflow from superfused rat nucleus accumbens slices previously incubated with [³H]dopamine (DA) and [¹⁴C]choline. The effect was greater in striatal tissue and did not appear to be a non-specific membrane effect since [¹⁴C]acetylcholine (ACh) release was not affected. Lack of antagonism by picrotoxin suggested that -aminobutyric acid (GABA) receptors were not involved. Calcium was not a requirement and the DA uptake blocker, nomifensine, was without effect. Ethanol appeared to be causing [³H]DA release into the cytoplasm. K⁺-stimulated release of [³H]DA and [¹⁴C]ACh from nucleus accumbens and striatal slices was not affected. Clonidine-mediated inhibition of the K⁺-evoked release of [³H]DA remained unaltered. Ethanol attenuated the isoproterenol-induced enhancement of [³H]DA release. Ethanol therefore appeared to interact with components of the DA terminal causing a transient increase in the release of neurotransmitter without impairing K⁺-evoked release but apparently interfering with the isoproterenol-induced effect.     

Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs

Zheng, Lu P., Rui Sheng Du, and Barbara E. Goodman.Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs. J. Appl.Physiol. 82(1): 240-247, 1997.We investigatedeffects of acute hyperoxia on solute transport from air space tovascular space in isolated rat lungs. Air spaces were filled withKrebs-Ringer bicarbonate solution containing fluoresceinisothiocyanate-labeled dextran (FD-20; mol wt 20,000) and either²²Na⁺and [¹⁴C]sucrose, orD-[¹⁴C]glucoseandL-[³H]glucose.Apparent permeability-surface area products for tracers over time (upto 120 min) were calculated for isolated perfused lungs from controlrats (room air) and rats exposed to >95%O₂ for 48 or 60 h immediatelypostexposure. After O₂ exposures,mean fluxes for[¹⁴C]sucrose and FD-20were significantly higher than in room-air control lungs. However,amiloride-sensitive Na⁺ and activeD-glucose fluxes were unchangedafter hyperoxic exposure. Therefore, it is unlikely that decreases innet solute transport in this lung-injury model contributed to pulmonaryedema resulting from O₂ toxicity.Increased net solute transport shown to help resolve pulmonary edemaafter acute hyperoxic exposure must therefore begin during the recoveryperiod. In summary, our data show increases in passive solute fluxesbut no changes in active solute fluxes immediately after acutehyperoxic lung injury.

     

Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

Thenotion that intracellular Ca²⁺ (Ca_i²⁺)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa²⁺ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Ca_i²⁺ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca²⁺ concentration([Ca²⁺]_i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca²⁺. We found thatthreshold [Ca²⁺]_i for high extracellularK⁺ (K_e⁺) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high K_e⁺)-inducedrelease of CA. The same agents produced Ca_i²⁺transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Ca_i²⁺responses elicited by high K_e⁺. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca²⁺]_i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Ca_i²⁺ stores do not playa significant role in the instant-to-instant chemoreception process.

     

Decreased [3H]ouabain binding sites in skeletal muscle of rats with chronic heart failure

Pickar, Joel G., John P. Mattson, Steve Lloyd, and TimothyI. Musch. Decreased[³H]ouabainbinding sites in skeletal muscle of rats with chronic heart failure.J. Appl. Physiol. 83(1): 323-329, 1997.Abnormalities intrinsic to skeletal muscle are thought tocontribute to decrements in exercise capacity found in individualswith chronic heart failure (CHF).Na⁺-K⁺-adenosinetriphosphatase(the Na⁺ pump) is essential formaintaining muscle excitability and contractility. Therefore, weinvestigated the possibility that the number and affinity ofNa⁺ pumps in locomotor muscles ofrats with CHF are decreased. Myocardial infarction (MI) was induced in8 rats, and a sham operation was performed in 12 rats. The degree ofCHF was assessed ~180 days after surgery. Soleus and plantarismuscles were harvested, and Na⁺pumps were quantified by using a[³H]ouabain bindingassay. At the time of muscle harvest, MI and sham-operated rats weresimilar in age (458 ± 54 vs. 447 ± 34 days old, respectively).Compared with their sham-operated counterparts, MI rats had asignificant amount of heart failure, right ventricular-to-body weightratio was greater (48%), and the presence of pulmonary congestion wassuggested by an elevated lung-to-body weight ratio (29%). Leftventricular end-diastolic pressure was significantly increased in theMI rats (11 ± 1 mmHg) compared with the sham-operated controls (1 ± 1 mmHg). In addition, mean arterial blood pressure was lower inthe MI rats compared with their control counterparts. [³H]ouabain bindingsites were reduced 18% in soleus muscle (136 ± 12 vs. 175 ± 13 pmol/g wet wt, MI vs. sham, respectively) and 22% in plantaris muscle(119 ± 12 vs. 147 ± 8 pmol/g wet wt, MI vs. sham,respectively). The affinity of these[³H]ouabain bindingsites was similar for the two groups. The relationship between thereduction in Na⁺ pump number andthe reduced exercise capacity in individuals with CHF remains to bedetermined.

     

Dopamine Transporter Loss in 6-OHDA Parkinson’s Model Is Unmet by Parallel Reduction in Dopamine Uptake

The dopamine transporter (DAT) regulates synaptic dopamine (DA) in striatum and modulation of DAT can affect locomotor activity. Thus, in Parkinson’s disease (PD), DAT loss could affect DA clearance and locomotor activity. The locomotor benefits of L-DOPA may be mediated by transport through monoamine transporters and conversion to DA. However, its impact upon DA reuptake is unknown and may modulate synaptic DA. Using the unilateral 6-OHDA rat PD model, we examined [³H]DA uptake dynamics in relation to striatal DAT and tyrosine hydroxylase (TH) protein loss compared with contralateral intact striatum. Despite >70% striatal DAT loss, DA uptake decreased only ∼25% and increased as DAT loss approached 99%. As other monoamine transporters can transport DA, we determined if norepinephrine (NE) and serotonin (5-HT) differentially modulated DA uptake in lesioned striatum. Unlabeled DA, NE, and 5-HT were used, at a concentration that differentially inhibited DA uptake in intact striatum, to compete against [³H]DA uptake. In 6-OHDA lesioned striatum, DA was less effective, whereas NE was more effective, at inhibiting [³H]DA uptake. Furthermore, norepinephrine transporter (NET) protein levels increased and desipramine was ∼two-fold more effective at inhibiting NE uptake. Serotonin inhibited [³H]DA uptake, but without significant difference between lesioned and contralateral striatum. L-DOPA inhibited [³H]DA uptake two-fold more in lesioned striatum and inhibited NE uptake ∼five-fold more than DA uptake in naïve striatum. Consequently, DA uptake may be mediated by NET when DAT loss is at PD levels. Increased inhibition of DA uptake by L-DOPA and its preferential inhibition of NE over DA uptake, indicates that NET-mediated DA uptake may be modulated by L-DOPA when DAT loss exceeds 70%. These results indicate a novel mechanism for DA uptake during PD progression and provide new insight into how L-DOPA affects DA uptake, revealing possible mechanisms of its therapeutic and side effect potential.     

Regional Differences in 5-Hydroxytryptamine and Catecholamine Uptake in Primary Astrocyte Cultures

The uptake of 3H-labelled 5-hydroxytryptamine (5-HT, serotonin) norepinephrine ([3H]NE), and 3,4-dihydroxyphenylethylamine ([ 3H]dopamine, [3H]DA) was studied in primary astrocyte cultures prepared from the cerebral cortex, corpus striatum, and hippocampal regions of neonatal rat brain. Na+-dependent uptake showed marked regional differences. For [3H]5-HT the magnitude of uptake was corpus striatum greater than or equal to cerebral cortex greater than hippocampus, whereas for [3H]NE the order was hippocampus greater than corpus striatum greater than cerebral cortex. For [3H]DA, only the hippocampal cultures showed significant Na+-dependent uptake. [3H]5-HT uptake was specifically inhibited by 10(-7) M fluoxetine whereas [3H]NE uptake was preferentially inhibited by 10(-7) M desipramine. These results may reflect regional brain specialization and/or different developmental patterns of high affinity uptake of serotonin and catecholamines by astrocytes in situ.     

Relationship between [125I]RTI-55-labeled cocaine binding sites and the serotonin transporter in rat placenta

We investigated the characteristics of cocainelike binding sitesin rat placenta using[¹²⁵I]RTI-55.[³H]paroxetine bindingand immunocytochemical staining for serotonin [5-hydroxytryptamine (5-HT)] and for the 5-HT transporterwere also used to obtain evidence for rat placental 5-HT uptake.[¹²⁵I]RTI-55saturation analyses with membranes from normal gestational day 20 placentas yielded curvilinear Scatchard plots that were resolved intohigh- and low-affinity components (mean dissociation constants of 0.29 and 7.9 nM, respectively). Drug competition studies with variousmonoamine uptake inhibitors gave rise to complex multiphasicdisplacement curves, although the results obtained with the selective5-HT uptake inhibitor citalopram suggest that the 5-HT transporter isan important component of placental high-affinity[¹²⁵I]RTI-55 binding.The presence of a rat placental 5-HT uptake system was additionallysupported by the[³H]paroxetine bindingexperiments and by the presence throughout the placenta ofimmunoreactivity for 5-HT and the 5-HT transporter. Immunostaining withboth antibodies was most intense in the junctional zone, whereas thedensity of[¹²⁵I]RTI-55 bindingsites was greater in the placental labyrinth. This discrepancy may bedue to the fact that[¹²⁵I]RTI-55 appearsto be labeling additional cellular components besides the 5-HTtransporter. The presence of cocaine- and antidepressant-sensitive 5-HTtransporters in the placenta has important implications for thepossible effects of these compounds on pregnancy and fetal development.

     

ATP-regulated neuronal catecholamine uptake: a new mechanism

Uptake of the catecholamines (CA), dopamine (DA) and norepinephrine (NE) into synaptosomes prepared from rat and bovine brains was potentiated by ATP (from 0.1 to 5.0 mM) in a dose-dependent manner. Other nucleotides, particularly the nonhydrolyzable ATP analogs beta,gamma-imidoadenosine-5'-triphosphate (AMP-PNP) and beta,gamma-methyladenosine-5'-triphosphate (AMP-PCP) also potentiated [3H]DA and [3H]NE uptake. Several endogenous 5'-nucleotide triphosphates (e.g. GTP, UTP and CTP) potentiated [3H]CA uptake, but were less effective than ATP. Among the ATP metabolites, only ADP potentiated uptake whereas AMP and adenosine did not. [3H]Dopamine uptake measured in Krebs bicarbonate buffer had a Km of 2.1 microM and a Vmax of 163.9 pmol/mg prot./min. In presence of ATP, [3H]DA uptake had much higher affinity (Km = 0.56 microM) and larger capacity (Vmax = 333 pmol/mg prot./min) than uptake in absence of added ATP. Furthermore, [3H]DA uptake in presence of ATP had faster rate of uptake, and was independent of temperature while in absence of added ATP it was temperature-dependent. This ATP-dependent [3H]DA uptake was retained by synaptosomal ghosts that were obtained after lysing the striatal synaptosomes and removing their contents of synaptic vesicles and mitochondria. It is proposed that, in addition to the carrier-mediated (neuronal) uptake of CA, there is neuronal uptake that is regulated by ATP and inhibited by cocaine, which may be more relevant for terminating the synaptic action of CA because of its faster rate of uptake and larger capacity.     

On the significance of perfusion rate in the study of glutamate release from superfused synaptosomes

The effect of perfusion rate on the apparent release of [³H]glutamate from prelabelled and superfused rat cortical synaptosomes was examined. The proportion of tissue [³H]glutamate released in response to a 4 ml depolarizing pulse of 15 mM K⁺ increased almost linearly with perfusion rates from 1 ml min⁻¹ to 10 ml min⁻¹. Release did not increase markedly between 10 ml min⁻¹ and 20 ml min⁻¹. The basal efflux of [³H]glutamate also increased with perfusion rate. The increase in both basal efflux and K⁺-induced release is interpreted as being due to a greater amount of released transmitter avoiding recapture by uptake processes as perfusion rate increases. This is supported by the observation that increasing the potential number of uptake sites in the tissue decreases both the basal and K⁺-evoked release of the transmitter. The significance of this with respect to optimal perfusion rates for studies on the regulation of glutamate release is discussed.     

Characterization of the effects of serotonin on the release of [3H]dopamine from rat nucleus accumbens and striatal slices

The effect of serotonin agonists on the depolarization (K⁺)-induced, calcium-dependent, release of [³H]dopamine (DA) from rat nucleus accumbens and striatal slices was investigated. Serotonin enhanced basal³H overflow and reduced K⁺-induced release of [³H]DA from nucleus accumbens slices. The effect of serotonin on basal³H overflow was not altered by the serotonin antagonist, methysergide, or the serotonin re-uptake blocker, chlorimipramine, but was reversed by the DA re-uptake carrier inhibitors nomifensine and benztropine. With the effect on basal overflow blocked, serotonin did not modulate K⁺-induced release of [³H]DA in the nucleus accumbens or striatum. The serotonin agonists, quipazine (in the presence of nomifensine) and 5-methoxytryptamine, did not significantly affect K⁺-induced release of [³H]DA in the nucleus accumbens. This study does not support suggestions that serotonin receptors inhibit the depolarization-induced release of dopamine in the nucleus accumbens or striatum of the rat brain. The present results do not preclude the possibility that serotonin may affect the mesolimbic reward system at a site which is post-synaptic to dopaminergic terminals in the nucleus accumbens.     

Effects of 5-day hypoxia on cardiac adrenergic neurotransmission in rats

Chronic hypoxiainduces an overall sympathetic hyperactivation associated with amyocardial -receptor desensitization. The mechanismsinvolved in this desensitization were evaluated in 32 male Wistar ratskept in a hypobaric pressure chamber(PO₂ = 40 Torr, atmospheric pressure = 450 Torr) for 5 days. In hypoxic compared with normoxic conditions,plasma norepinephrine (NE) levels were higher (2.1 ± 0.7 vs. 0.6 ± 0.2 ng/ml) with no difference in the plasma epinephrine levels(2.2 ± 0.7 vs. 1.8 ± 0.3 ng/ml). In hypoxia neuronal NE uptakemeasured by [³H]NE wasdecreased by 32% in the right ventricle (RV) and by 35% in the leftventricle (LV), and[³H]mazindol in vitrobinding showed a decrease in uptake-1 carrier protein density by 38%in the RV and by 41% in the LV. In vitro binding assays with[³H]CGP-12177 indicate-adrenoceptor density reduced by 40% in the RV and by 32% in theLV, and this was due to reduced₁-subtype fraction (competitionbinding experiments with practolol). Hypoxia reduced the production ofcAMP induced by isoproterenol (36% decrease in the RV and 41%decrease in the LV), 5'-guanylylimododiphosphate (40% decreasein the RV and 42% decrease in the LV), and forskolin (39% decrease inthe RV and 41% decrease in the LV) but did not alter the effect ofMnCl₂ and NaF. Quantitation ofinhibitory G-protein -subunit by immunochemical analysis showed a46% increase in the cardiac-specific isoformG_i2 inhypoxic hearts. The present data demonstrate that in rats 5-day hypoxialeads to changes in pre- and postsynaptic myocardial adrenergicfunction. The myocardial desensitization associated with both areduction in externalized ₁-adrenoceptor and an increasein inhibitory G-protein subunit may be caused by increased synaptic NElevels due to impaired uptake-1 system.

     

K+ supplementation increases muscle [Na+-K+-ATPase] and improves extrarenal K+ homeostasis in rats

Bundgaard, Henning, Thomas A. Schmidt, Jim S. Larsen, andKeld Kjeldsen. K⁺supplementation increases muscle[Na⁺-K⁺-ATPase]and improves extrarenal K⁺homeostasis in rats. J. Appl. Physiol.82(4): 1136-1144, 1997.Effects ofK⁺ supplementation (~200 mmolKCl/100 g chow) on plasma K⁺,K⁺ content, andNa⁺-K⁺-adeonsinetriphosphatase(ATPase) concentration([Na⁺-K⁺-ATPase])in skeletal muscles as well as on extrarenalK⁺ clearance were evaluated inrats. After 2 days of K⁺supplementation, hyperkalemia prevailed(K⁺-supplemented vs.weight-matched control animals) [5.1 ± 0.2 (SE) vs. 3.2 ± 0.1 mmol/l, P < 0.05, n = 5-6], and after 4 daysa significant increase in K⁺content was observed in gastrocnemius muscle (104 ± 2 vs. 97 ± 1 µmol/g wet wt, P < 0.05, n = 5-6). After 7 days ofK⁺ supplementation, a significantincrease in[³H]ouabain bindingsite concentration (344 ± 5 vs. 239 ± 8 pmol/g wet wt,P < 0.05, n = 4) was observed in gastrocnemiusmuscle. After 2 wk, increases in plasmaK⁺,K⁺ content, and[³H]ouabain bindingsite concentration in gastrocnemius muscle amounted to 40, 8, and 68%(P < 0.05) above values observed inweight-matched control animals, respectively. The latter change wasconfirmed by K⁺-dependentp-nitrophenyl phosphatase activitymeasurements. Fasting for 1 day reduced plasmaK⁺ andK⁺ content in gastrocnemius musclein rats that had been K⁺supplemented for 2 wk by 3.1 ± 0.3 mmol/l(P < 0.05, n = 5) and 15 ± 2 µmol/g wet wt(P < 0.05, n = 5), respectively. After induction of anesthesia, arterial plasma K⁺was measured during intravenous KCl infusion (0.75 mmolKCl · 100 g bodywt¹ · h¹).The K⁺-supplemented fasted groupdemonstrated a 42% (P < 0.05) lower plasma K⁺ rise, associated with asignificantly higher increase inK⁺ content in gastrocnemius muscleof 7 µmol/g wet wt (P < 0.05, n = 5) compared with their controlanimals. In conclusion, K⁺supplementation increases plasmaK⁺,K⁺ content, and[Na⁺-K⁺-ATPase]in skeletal muscles and improves extrarenalK⁺ clearance capacity.

     

Uptake and metabolism of biotin by human peripheral blood mononuclear cells

We studied the uptake of biotin into human peripheral bloodmononuclear cells (PBMC) using[³H]biotin and studiedthe catabolism of biotin in PBMC using[¹⁴C]biotin. Over 30 min, [³H]biotin uptakewas greater at 37°C than at 25°C(K_T = 2.6 ± 0.4 nM, maximal velocity = 2.9 ± 0.2 fmol · 10⁶cells¹ · 30 min¹). Ouabain reduced[³H]biotin uptake to65% of control values, suggesting that biotin uptake is Na-K-ATPasedependent. Unlabeled biotin and biotin analogs reduced the uptake of[³H]biotin to22-70% of control values, suggesting the presence of acompetition for a structurally specific biotin transporter. Whenendocytosis by PBMC was stimulated by various acyl glycerols, [³H]biotin uptake was40-73% of control values; these data are consistent with thehypothesis that stimulated endocytosis reduces biotin transporterdensity on the cell surface. During a 168-h incubation, PBMC did notcatabolize[¹⁴C]biotin.

     

Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue

To investigatethe Ca²⁺-dependent plasticity ofsarcoplasmic reticulum (SR) function in vascular smooth muscle,transient responses to agents releasing intracellularCa²⁺ by either ryanodine(caffeine) orD-myo-inositol1,4,5-trisphosphate [IP₃;produced in response to norepinephrine (NE),5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptorsin rat tail arterial rings were evaluated after 4 days of organculture. Force transients induced by all agents were increased comparedwith those induced in fresh rings. Stimulation by 10% FCSduring culture further potentiated the force andCa²⁺ responses to caffeine (20 mM)but not to NE (10 µM), 5-HT (10 µM), or AVP (0.1 µM). The effectwas persistent, and SR capacity was not altered after reversibledepletion of stores with cyclopiazonic acid. The effects of serum couldbe mimicked by culture in depolarizing medium (30 mMK⁺) and blocked by the additionof verapamil (1 µM) or EGTA (1 mM) to the medium, loweringintracellular Ca²⁺ concentration([Ca²⁺]_i)during culture. These results show that modulation of SR function canoccur in vitro by a mechanism dependent on long-term levels of basal[Ca²⁺]_iand involving ryanodine- but notIP₃ receptor-mediatedCa²⁺release.