Mechanism of norepinephrine release elicited by Na+-K+ adenosine triphosphatase inhibition in the isolated rat kidney: involvement of voltage-dependent Ca++ channels

The mechanism by which ouabain and Na+ depletion enhance the release of norepinephrine (NE) was investigated in the isolated rat kidney prelabeled with [3H]NE by examining the efflux of tritium elicited by these stimuli during 1) Ca++ depletion and 2) administration of tetrodotoxin, amiloride and Ca++ channel blockers. In kidneys perfused with Tyrode's solution containing low K+ solution (0.54 mM), ouabain (10(-4) M) enhanced tritium efflux markedly by about 20-fold at 30 min. Depletion of Na+ from the perfusion medium also produced an increase in tritium overflow which peaked at 20 min. Administration of tetrodotoxin (0.3 microM) inhibited the effect of ouabain, but not that of Na+ depletion, to increase tritium efflux and perfusion pressure. In contrast, amiloride (180 microM) enhanced the overflow of tritium elicited by ouabain but failed to alter that elicited by Na+ depletion. The rise in perfusion pressure caused by both stimuli was attenuated by amiloride. Omission of Ca++ (1.8 mM) from the perfusion medium inhibited the increase in tritium efflux and perfusion pressure elicited by ouabain and Na+ depletion by 80 and 65%, respectively. The Ca++ channel blockers omega-conotoxin (50 nM), diltiazem (60 microM) and flunarizine (2 microM), but not nifedipine (1.4 microM), inhibited tritium overflow elicited by ouabain. However, nifedipine, diltiazem and flunarizine, but not omega-conotoxin attenuated the tritium overflow elicited by Na+ depletion. The rise in perfusion pressure elicited by ouabain in low K+ and Na+ depletion was inhibited by these Ca++ channel blockers.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors regulating hippocampal norepinephrine release. II. Evidence for functional cooperation and for coexistence on the same axon terminal.

The possible interactions between activation of N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors regulating the release of [3H]norepinephrine [( 3H]NE) have been investigated in superfused synaptosomes from rat hippocampus. NMDA--at a concentration (100 microM) which, in a medium containing 1.2 mM Mg++ ions, did not evoke [3H]NE release--acquired releasing activity in the presence of equimolar concentrations of quisqualic acid (QA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. The [3H] NE release evoked by NMDA plus QA in the presence of Mg++ ions was Ca(++)-dependent, partly tetrodotoxin-sensitive, inhibited by clonidine but insensitive to desipramine. The NMDA receptor antagonists D-2-amino-5-phosphonopentanoic acid (D-AP5) and (+)-5-methyl-10,11-dihydro-5-H-dibenzo[a,d]cycloepten-5,10-imine (MK-801) antagonized the NMDA-induced [3H]NE release in Mg(++)-free medium; the IC50 values amounted, respectively, to 81.4 microM and to 1.11 microM. When NMDA was tested in the presence of QA and Mg++ ions, the affinity of D-AP5 was enormously increased (IC50 = 40 nM; i.e., more than 6 orders of magnitude); the affinity of MK-801 was found to be augmented by 350-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transporter-mediated release: a superfusion study on human embryonic kidney cells stably expressing the human serotonin transporter

HEK 293 cells stably expressing the human serotonin transporter (hSERT) were grown on coverslips, preincubated with [(3)H]5-hydroxytryptamine (5-HT), and superfused. Substrates of the hSERT [e.g., p-chloroamphetamine (PCA)], increased the basal efflux of [(3)H]5-HT in a concentration-dependent manner. 5-HT reuptake blockers (e.g., imipramine, paroxetine) also raised [(3)H]5-HT efflux, reaching approximately one-third of the maximal effect of the hSERT substrates. In uptake experiments, both groups of substances inhibited [(3)H]5-HT uptake. Using the low-affinity substrate [(3)H]N-methyl-4-phenylpyridinium (MPP(+)) to label the cells in superfusion experiments, reuptake inhibitors failed to enhance efflux. Similar results were obtained using human placental choriocarcinoma (JAR) cells that constitutively express the hSERT at a low level. By contrast, PCA raised [(3)H]MPP(+) efflux in both types of cells, and its effect was inhibited by paroxetine. The addition of the Na(+),K(+)-ATPase inhibitor ouabain (100 microM) to the superfusion buffer enhanced basal efflux of [(3)H]5-HT-loaded hSERT cells by approximately 2-fold; the effect of PCA (10 microM) was strongly augmented by ouabain, whereas the effect of imipramine was not. The Na(+)/H(+) ionophore monensin (10 microM) also augmented the effect of PCA on efflux of [(3)H]5-HT as well as on efflux of [(3)H]MPP(+). In [(3)H]5-HT-labeled cells, the combination of imipramine and monensin raised [(3)H]5-HT efflux to a greater extent than either of the two substances alone. In [(3)H]MPP(+)-labeled cells, imipramine had no effect on its own and fully reversed the effect of monensin. The results suggest that the [(3)H]5-HT efflux caused by uptake inhibitors is entirely due to interrupted high-affinity reuptake, which is ongoing even under superfusion conditions.     

LLC-PK(1) cells stably expressing the human norepinephrine transporter: A functional model of carrier-mediated norepinephrine release in protracted myocardial ischemia.

In myocardial ischemia, adrenergic terminals undergo ATP depletion, hypoxia, and intracellular pH reduction, causing the accumulation of axoplasmic norepinephrine (NE) and intracellular Na(+) [via the Na(+)-H(+) exchanger (NHE)]. This forces the reversal of the Na(+)- and Cl(-)-dependent NE transporter (NET), triggering massive carrier-mediated NE release and, thus, arrhythmias. We have now developed a cellular model of carrier-mediated NE release using an LLC-PK(1) cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [(3)H]NE and [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)) in an inward direction. This uptake was abolished by the NET inhibitors desipramine (100 nM) and mazindol (300 nM) and by extracellular Na(+) removal. Na(+)-gradient reversal induced an efflux of (3)H-substrate from preloaded LLC-NET cells. Desipramine and mazindol blocked this efflux. Because of its greater intracellular stability and higher sensitivity to Na(+)-gradient reversal, [(3)H]MPP(+) proved preferable to [(3)H]NE as an NET substrate; therefore, only [(3)H]MPP(+) was used for subsequent studies. The K(+)/H(+) ionophore nigericin (10 microM) evoked a large efflux of [(3)H]MPP(+). This efflux was potentiated by the Na(+),K(+)-ATPase inhibitor ouabain (100 microM), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 microM). In contrast, EIPA failed to inhibit the [(3)H]MPP(+) efflux elicited by the Na(+) ionophore gramicidin (10 microM). Furthermore, [(3)H]MPP(+) efflux induced by the NHE-stimulant proprionate (25 mM) was negatively modulated by imidazoline receptor activation. Our findings suggest that LLC-NET cells are a sensitive model for studying transductional processes of carrier-mediated NE release associated with myocardial ischemia.     

Alpha-2 adrenergic inhibition of Ca(++)-evoked [3H]norepinephrine release from synaptosomes is blocked by depolarization

The Ca(++)-evoked release of [3H]norepinephrine was used in these studies to investigate presynaptic regulation of norepinephrine release. In hippocampal synaptosomes, previously unexposed to Ca++ during isolation and superfusion, 1.25 mM Ca++ evoked a modest (4 to 7% of total stores) release of [3H]norepinephrine with 4.5 mM [K+] present. The alpha-2 adrenergic agonist clonidine inhibited 60% of the Ca(++)-evoked [3H]norepinephrine release. The alpha-2 adrenergic antagonists idazoxan and yohimbine reversed clonidine inhibition of release whereas the alpha-1 antagonist prazosin did not. Increasing the [K+] before Ca++ exposure increased [3H]norepinephrine release, and at 20 [K+] the release increased to over 20% of total stores. However, at [K+] above 9 mM, inhibition of Ca(++)-evoked release by clonidine decreased, and by 20 mM [K+] clonidine no longer inhibited release. Release was unaffected by 5 microM idazoxan or the opiate antagonist naloxone at 15 or 20 mM [K+]. The K+ channel blockers tetraethylammonium (5 mM) and 4-aminopyridine (0.1 mM) increased Ca(++)-evoked release almost 4-fold above control (4.5 mM [K+] present). Neither clonidine nor idazoxan affected Ca(++)-evoked release with the K+ channel blockers present. Therefore, even though K+ channel blockers and 20 mM [K+] increase neurotransmitter release, it is not autoreceptor activation by released endogenous norepinephrine that is responsible for blocking alpha-2 inhibition, but the depolarization produced by these treatments. The 20 mM [K+] blockade of alpha-2 inhibition was decreased by lowering the [Ca++] in the superfusion buffer. Therefore, synaptosomal accumulation of Ca++ may partially explain the loss of alpha-2 inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of the atypical antipsychotic drug, amperozide, on carrier-mediated striatal dopamine release measured in vivo.

The effect of the novel atypical antipsychotic drug, amperozide, on carrier-mediated dopamine efflux was studied using in vivo microdialysis in the striatum of awake-behaving rats. Amperozide was infused directly through the dialysis probe. This local infusion produced a concentration-dependent increase in striatal dopamine overflow. This increase was attenuated when a Ca(++)-free perfusion medium was used. Local infusion of amperozide blocked dopamine efflux after the systemic administration of amphetamine in a concentration-dependent manner. The antagonistic effect of amperozide (50 microM) on amphetamine-induced efflux of dopamine was not attenuated under Ca(++)-free conditions. Similar to its effects on amphetamine-induced dopamine efflux, amperozide (50 microM) attenuated the increase in dopamine overflow produced by ouabain (10 microM) but not veratridine (15 microM). The systemic coadministration of amperozide (10 mg/kg, i.p.) and haloperidol (2 mg/kg, i.p.) increased extracellular dopamine levels in an additive manner when compared to the increases observed after the administration of either drug alone. Overall, these data indicate that amperozide acts on the dopamine transporter to inhibit carrier-mediated release.     

Modulation of the release of [3H]norepinephrine from the base and body of the rat urinary bladder by endogenous adrenergic and cholinergic mechanisms

Modulation of [3H]NE release was studied in rat urinary bladder strips prelabeled with [3H]NE. [3H]NE uptake occurred in strips from the bladder base and body, but was very prominent in the base where the noradrenergic innervation is most dense. Electrical field stimulation markedly increased [3H]NE outflow from the superfused tissue. The quantity of [3H]NE release was approximately equal during three consecutive periods of stimulation. Activation of presynaptic muscarinic receptors by 1.0 microM oxotremorine reduced [3H]NE release to 46% of the control. Atropine (1 microM) blocked the effect of oxotremorine and increased the release to 147% of predrug control levels. Activation of presynaptic alpha-2 adrenoceptors by 1 microM clonidine reduced [3H]NE release to 55% of control. Yohimbine blocked the action of clonidine and increased the release to 148% of control. The release of [3H]NE from the bladder base and body was increased by both 1 microM atropine (to 167% and 174% of control, respectively) and 1 microM yohimbine (to 286% and 425% of control, respectively). Atropine and yohimbine administered in combination had similar facilitatory effects as when administered alone. We conclude that the release of [3H]NE from adrenergic nerve endings in electrically stimulated bladder strips is modulated via endogenous transmitters acting on both muscarinic and alpha-2 adrenergic presynaptic receptors and that the latter provide the most prominent control.     

Stereoselective modulation of ryanodine-sensitive calcium channels by the delta isomer of hexachlorocyclohexane (delta-HCH).

delta-Hexachlorocyclohexane (delta-HCH) is shown to be 30-fold more potent as a positive inotropic agent with rat atrial strips compared with lindane (gamma-HCH). Threshold and ED50 values for enhanced contractile force at a pacing frequency of 0.5 Hz are less than 1 microM and 2.2 microM for delta-HCH and 40 microM and 63 microM for gamma-HCH, respectively. Contracture developed in atria exposed to greater than 4 microM delta-HCH (ED50 = 11 microM) but not in atria exposed to gamma-HCH. Uptake and release of Ca++ measured from actively loaded cardiac sarcoplasmic reticulum (SR) vesicles is measured with antipyrylazo III. Although delta-HCH (30 microM) decreases Ca(++)-dependent ATPase by 20%, it does not significantly alter Ca++ loading in the presence of ruthenium red. Addition of delta-HCH (5-50 microM) after loading is complete causes rapid, dose-dependent release of Ca++ from SR. Ca++ release induced by delta-HCH is markedly stereoselective. Compared with gamma-HCH (50 microM), delta-HCH (50 microM) induces a nearly 20-fold higher initial rate of Ca++ release (4.3 nmol of Ca++/mg/sec). Studies with [3H]ryanodine demonstrate that delta-HCH sharply inhibits Ca(++)- or daunorubicin-activated radioligand binding (IC50 = 37 and 25 microM, respectively, logit slope = 2). Inhibition of [3H]ryanodine-binding by delta-HCH is stereoselective inasmuch as IC50 values for alpha, beta and gamma isomers are greater than 100 microM. The delta-HCH modified Ca++ channel appears to proceed by a noncompetitive mechanism (reducing Bmax in equilibrium experiments) with respect to the conformationally sensitive binding site for [3H]ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine and adenosine triphosphate release in different ratio from guinea pig vas deferens by high potassium chloride, ouabain and monensin

Release of norepinephrine (NE) and ATP from the guinea pig vas deferens evoked by ouabain in combination with monensin or by high KCl was measured by a high-pressure liquid chromatography-ECD and luciferin-luciferase assay, respectively. Ouabain (10-100 microM) induced a concentration-dependent liberation of NE, which was enhanced by 10 microM monensin, a Na+-ionophore. The marked NE release elicited by the combined administration of both the drugs was unaffected by Ca++-removal but was reduced by lowering Na+ from the medium. This NE release in the Ca++-free medium was diminished markedly after treatment with 6-hydroxydopamine or reserpine and in low-temperature (25 degrees C) medium. This release was also decreased by ruthenium red (10-30 microM), an uptake inhibitor of Ca++ to mitochondria, and carbonyl cyanide-m-chlorophenyl hydrazone (10 microM), a metabolic inhibitor. On the other hand, 100 mM KCl caused a moderate, extracellular Ca++-dependent release of NE. ATP-outflow from the tissue evoked by 100 microM ouabain plus 10 microM monensin was almost unaltered by Ca++-removal but was inhibited by 6-hydroxydopamine or prazosin (0.3 microM), whereas release induced by high KCl was reduced by 6-hydroxydopamine and Ca++-free medium but was unaffected by prazosin. ATP/NE ratios at respective maximum effluxes evoked by 100 mM KCl and ouabain plus monensin were 6.59 and 0.22, respectively. These findings suggest that there may be more than one site of corelease for NE and ATP. Ouabain plus monensin seems to produce an extracellular Ca++-independent neuronal release of NE and ATP from the cytoplasmic and vesicular storage sites which predominantly release NE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane events and ionic processes involved in dopamine release from tuberoinfundibular neurons. I. Effect of the inhibition of the Na+,K+-adenosine triphosphatase pump by ouabain

In the present study we investigated the membrane events and the ionic processes which mediate the stimulatory effect of ouabain on the release of endogenous dopamine (DA) and "previously taken-up" [3H]DA release from rat hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. Ouabain (0.1-1 mM) dose-dependently stimulated endogenous DA and "newly taken-up" [3H]DA release. This effect was counteracted partially by nomifensine (10 microM). Removal of Ca++ ions from the extracellular space in the presence of the Ca++-chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid prevented completely ouabain-elicited [3H]DA release. Lanthanum (1 mM) and cobalt (2 mM), two inorganic Ca++-entry blockers, were able to inhibit this stimulatory effect, whereas verapamil (10 microM) and nitrendipine (50 microM), two organic antagonists of the voltage-operated channel for Ca++ ions, failed to affect ouabain-induced [3H]DA release. By contrast, adriamycin (100-300 microM), a putative inhibitor of cardiac Na+-Ca++ antiporter, dose-dependently prevented ouabain-induced [3H]DA release from TIDA neurons. Finally, tetrodotoxin reduced digitalis-stimulated [3H]DA release. In conclusion, these results seem to be compatible with the idea that the inhibition of Na+,K+-adenosine triphosphatase by ouabain stimulates the release of [3H]DA from a central neuronal system like the TIDA tract and that this effect is critically dependent on the entrance of Ca++ ions into the nerve terminals of these neurons. In addition the Na+-Ca++ exchange antiporter appears to be the membrane system which transports Ca++ ions into the neuronal cytoplasm during Na+,K+-adenosine triphosphatase inhibition. The enhanced intracellular Ca++ availability triggers DA release which could occur partially through a carrier-dependent process.     

Mechanism and metabolic disposition of verapamil-evoked overflow of radioactivity from isolated atria preloaded with [3H]norepinephrine

In this study the mechanism and metabolic profile of verapamil-evoked release of radioactivity was investigated in the rat isolated atria preloaded with [3H]norepinephrine [( 3H]NE). Verapamil (10(-7) to 10(-3) M) caused a dose-related increase in the outflow (or the fractional release) of 3H. The fractional 3H-release produced by verapamil was reduced markedly in tissues which had been preloaded with [3H]NE in the presence of cocaine (10 microM) or after pretreatment of animals with reserpine (5 mg/kg i.p., 24 hr before sacrifice). Verapamil-evoked fractional 3H-release was unchanged in the presence of tetrodotoxin (5 X 10(-6) M) or in Ca++-free Krebs' medium containing 2 mM ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid. Whereas greater than 90% of tissue 3H-content consisted of unchanged [3H]NE, 60 to 75% of the spontaneous outflow and the verapamil-evoked overflow consisted of [3H]-3,4-dihydroxyphenylglycol and 2 to 10% was unchanged [3H]NE. When both cocaine (10 microM) and hydrocortisone (28 microM) (uptake-1 and uptake-2 blockers, respectively) were present, although the spontaneous outflow, as well as verapamil-evoked overflow, of radioactivity was increased, the metabolic profiles remained essentially unchanged. The addition of pargyline (10 microM), a monoamine oxidase inhibitor, in addition to the uptake-1 and uptake-2 blockers to the Krebs' solution significantly depressed both the spontaneous outflow and verapamil-evoked overflow of 3H; the verapamil-evoked overflow under this condition, however, consisted of unchanged [3H]NE (greater than 90%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of d-amphetamine and dopamine synthesis inhibitors on dopamine and acetylcholine neurotransmission in the striatum. I. Release in the absence of vesicular transmitter stores

The release of endogenous dopamine (DA) elicited by electrical stimulation and by d-amphetamine (AMPH) from superfused striatal slices of reserpine-pretreated rabbits was examined. Although reserpine pretreatment reduced tissue DA levels by greater than 95%, the basal efflux of DA and the DA metabolite dihydroxyphenylacetic acid (DOPAC) was slightly greater than that observed in untreated slices. DOPAC constituted the large majority of the basal efflux of endogenous compounds. No overflow of endogenous compounds was evoked by electrical stimulation (3 Hz, 3 min) after reserpine pretreatment. Superfusion with alpha-methyl-p-tyrosine (100 microM) abolished the efflux of endogenous DA and DOPAC. AMPH (0.3-10 microM) produced a concentration-dependent increase in the basal efflux of endogenous DA and a concomitant decrease in endogenous DOPAC efflux. The total efflux of endogenous compounds (DA + DOPAC) tended to be decreased by AMPH. No electrically evoked overflow of endogenous compounds was observed in the presence of AMPH. The increase in synaptic DA produced by AMPH was reflected by a concentration-dependent reduction in the electrically evoked overflow of [3H]acetylcholine (ACh). The ability of AMPH to increase DA efflux and inhibit [3H]ACh release was blocked by inhibition of DA synthesis with alpha-methyl-p-tyrosine (100 microM) or by blockade of the DA neuronal uptake carrier with nomifensine (NOM) (10 microM) and was potentiated by inhibition of monoamine oxidase with pargyline (10 microM). NOM also blocked partially the ability of AMPH to reduce endogenous DOPAC efflux. NOM increased the basal efflux of endogenous DA and inhibited electrically evoked [3H]ACh release but these effects were quantitatively much less than those produced by AMPH. NOM had no effect on DOPAC efflux. Pargyline had little effect on endogenous DA efflux or electrically evoked [3H]ACh release but abolished DOPAC efflux and increased tissue DA levels measured at the end of superfusion. When given in combination, NOM and pargyline produced a similar degree of inhibition of [3H]ACh release as AMPH, although the increase in DA efflux produced by this drug combination was less than that produced by AMPH. These results suggest that in the absence of vesicular transmitter stores (reserpine-pretreatment): synthesis provides a continuous supply of DA which is metabolized rapidly within the neuron and is lost as DOPAC; AMPH facilitates the synthesis-dependent efflux of extravesicular DA probably by an accelerated exchange diffusion mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reboxetine: functional inhibition of monoamine transporters and nicotinic acetylcholine receptors

The present study determined whether repeated administration of the antidepressant and selective norepinephrine (NE) uptake inhibitor reboxetine resulted in an adaptive modification of the function of the NE transporters (NETs), serotonin (5-HT) transporters, or dopamine (DA) transporters. Because antidepressants may be effective tobacco smoking cessation agents and because antidepressants have recently been shown to interact with nicotinic acetylcholine receptors (nAChRs), the interaction of reboxetine with nAChRs was also evaluated. Repeated administration of reboxetine (10 mg/kg i.p., twice daily for 14 days) did not alter the potency or selectivity of reboxetine inhibition of [(3)H]NE, [(3)H]DA, or [(3)H]5-HT uptake into striatal or hippocampal synaptosomes (IC(50) values = 8.5 nM, 89 microM, and 6.9 microM, respectively). In a separate series of experiments, reboxetine did not inhibit (K(i) > 1 microM) [(3)H]methyllycaconitine, [(3)H]cytisine, or [(3)H]epibatidine binding to rat whole brain membranes. However, at concentrations that did not exhibit intrinsic activity, reboxetine potently inhibited (IC(50) value = 7.29 nM) nicotine-evoked [(3)H]NE overflow from superfused hippocampal slices via a noncompetitive mechanism. In the latter experiments, the involvement of NET was eliminated by inclusion of desipramine (10 microM) in the superfusion buffer. Reboxetine also inhibited (IC(50) value = 650 nM) nicotine-evoked (86)Rb(+) efflux at reboxetine concentrations that did not exhibit intrinsic activity in this assay. Thus, in addition to inhibition of NET function, reboxetine inhibits nAChR function, suggesting that it may have potential as a smoking cessation agent.     

Effects of L-threo-3,4-dihydroxyphenylserine on efflux of monoamines and acetylcholine in guinea pig brain

The effect of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS) on the release of monoamines and acetylcholine (ACh) was studied in the superfused brain slices of guinea pig. In the tissues preloaded either with [3H]norepinephrine ([3H]NE), [3H]dopamine ([3H]DA), [3H]-5-hydroxytryptamine or [3H]choline, tritium effluxes were estimated in serial fractions of superfusates. L-threo-DOPS produced a concentration-dependent increase in the spontaneous efflux of [3H]NE both in the cortical and hypothalamic slices and to a lesser extent that of [3H]DA in the striatal slices. These effects were still fully detected when slices were superfused with a calcium-free medium or tetrodotoxin (10(-6) M). Carbidopa at 5 X 10(-4) M but not at 10(-4) M significantly depressed the [3H]catecholamine effluxes induced by L-threo-DOPS. L-threo-DOPS produced a minimum increase in the spontaneous efflux of [3H]-5-hydroxytryptamine but not that of [3H]ACh. L-threo-DOPS (5 X 10(-4) M) significantly reduced the [3H]ACh efflux from electrically stimulated striatal slices and this effect was antagonized by an alpha-2 adrenoceptor antagonist yohimbine (10(-6) M) or by a D2 DA receptor antagonist sulpiride (10(-6) M). In vivo, L-threo-DOPS (150 mg/kg i.p.) produced a gradual but long-lasting increase in the efflux of [3H]NE from the parietal cortex of the guinea pig pretreated with carbidopa (20 mg/kg i.p.). In the brain homogenates, L-threo-DOPS (10(-10) to 10(-4) M) itself did not inhibit the bindings of [3H]rauwolscine or [3H]spiperone, specific ligands for labeling alpha-2 and D2 receptors, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the effects of beta-phenylethylamine and d-amphetamine on rat isolated atria

beta-Phenylethylamine (PEA) produced a positive chronotropic effect in the spontaneously beating isolated rat atria. The concentration-response curves to PEA and to d-amphetamine (AMPH) were virtually superimposed and potentiated to the same extent by inhibition of monoamine oxidase with pargyline. As reported for AMPH, the rate accelerating effect of PEA was abolished by pretreatment with reserpine. In the reserpinized tissue, the responses to PEA were partly restored by pargyline pretreatment and fully restored after the incubation with norepinephrine (NE). Repeated exposures to high concentrations of both PEA and AMPH produced tachyphylaxis to low concentrations of the amines but did not modify the endogenous atrial content of NE. in atria labeled with [3H]NE, PEA (6.4 microM) and AMPH (7.9 microM) increased the overflow of tritium mainly as unmetabolized [3H] NE (90%). For both drugs the increase in 3H-transmitter outflow coincided with the positive chronotropic response and it was independent on external Ca++. Cocaine (3.3 microM), which inhibits the uptake mechanism of catecholamines, displaced to the right the concentration-response curves to both PEA and AMPH and reduced the overflow of [3H]NE elicited by either amine. The release of 3H-products elicited by PEA was not modified by drugs that inhibit the uptake mechanism of NE accumulation, such as 26 microM corticosterone or 28 microM hydrocortisone. The results presented show that PEA behaves as AMPH as far as the mechanism of their indirect sympathomimetic effect is concerned. If accepted that PEA and AMPH, due to their high lipid solubility, might freely diffuse into nerves, the antagonism caused by cocaine on the effects of both amines could be interpreted as being the result of the inhibition by this drug of the carrier-mediated efflux of NE.     

An analysis of the action of phorbol 12, 13-dibutyrate on mechanical activity in rat uterine smooth muscle

The effects of phorbol 12, 13-dibutyrate (PDB) on mechanical activity in the pregnant rat uterus were investigated in isolated strips. In Ca(++)-containing solution, PDB (2.5 x 10(-8) to 10(-6) M) increased in a concentration-dependent manner the amplitude of the electrically induced contraction, but had no effect on the resting tension. PDB (10(-7) M) had a dual action, stimulatory then inhibitory, on contractions evoked by K(+)-rich (40 mM K+) solution or oxytocin. The inhibitory effect appeared more rapidly and the percentage of inhibition was increased for 10(-6) M PDB, which in addition abolished completely oxytocin-induced contraction after 20 min of application. PDB also reduced the amplitude of transient contraction evoked by oxytocin in Ca(++)-free solution. In saponin-skinned strips, 10(-7) M PDB increased the contraction induced by pCa ranging from 7 to 6, whereas 10(-6) M PDB reduced all Ca(++)-activated contractions from pCa = 7 to pCa = 5. PDB had no significant effect on the Ca(++)-uptake and the Ca(++)-release mechanisms of the intracellular Ca(++)-store. All of the effects of PDB were antagonized by the addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (2 x 10(-5) M). In addition, the inactive phorbol 13,20-diacetate (10(-8) to 10(-6) M) had no effect on the mechanical activity in uterus. These results suggest the existence of different sites of action of PDB in rat uterus, via the activation of protein kinase C: 1) contractile machinery; 2) potential-dependent Ca channels; and 3) phospholipase C.     

High glutamate concentrations evoke Ca(++)-independent dopamine release from striatal slices: a possible role of reverse dopamine transport.

Glutamate stimulated the efflux of dopamine from slices of rat striatum superfused with a Krebs' bicarbonate buffer containing a physiological concentration of Mg++ (1.2 mM). This effect was observed in the presence of high concentrations of glutamate (3-10 mM), but not at lower concentrations (0.01-1 mM). The response was not accompanied by increased lactate dehydrogenase activity, a measure of glutamate neurotoxicity. At 10 mM, glutamate increased dopamine efflux by more than 9-fold. This was reduced to about 34% of the control response by either the competitive N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid (100 microM) or the noncompetitive N-methyl-D-aspartate receptor antagonist MK 801 [(+)-5-methyl-10,11-dihydro-5H-dibenso[a,d]cyclohepten-5,10- imine hydrogen maleate] (1-10 microM), but was unaffected by a kainate/quisqualate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10-100 microM). Glutamate-stimulated dopamine efflux also was unaffected by tetrodotoxin (0.5 microM), withdrawal of extracellular Ca++ [and addition of 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid] or systemic administration of reserpine (5 mg/kg, 24 hr before the experiment), an inhibitor of the vesicular storage of dopamine. In contrast, nomifensine (10 microM), an inhibitor of high-affinity dopamine transport, reduced glutamate-induced dopamine efflux to 15% of the control response. Moreover, the response to glutamate was blocked by deleting NaCl from the medium. Collectively, these results suggest that, at high concentrations and in the presence of Mg++, glutamate can stimulate the release of dopamine by a mechanism that does not use Ca(++)-dependent exocytosis but instead involves a reversal of the dopamine transport system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thymidine transport and metabolism in choroid plexus: effect of diazepam and thiopental

Choroid plexus contains an active transport (influx) and a facilitated diffusion (efflux) system for nucleosides. The ability of diazepam and thiopental to inhibit active transport or facilitated diffusion of thymidine in choroid plexus was measured in vitro under various conditions. When isolated rabbit choroid plexuses were incubated in artificial cerebrospinal fluid containing 1 microM [3H] thymidine for 10 min at 37 degrees C under 95% O2-5% CO2, diazepam (10 microM) and thiopental (500 microM) doubled the tissue-to-medium ratios of [3H] thymidine from 8 to 15 to 16. These results were not due to metabolism or intracellular binding but rather to inhibition of [3H] thymidine efflux from choroid plexus. Diazepam, unlike thiopental, inhibited [3H] thymidine efflux in a concentration-dependent manner. When isolated choroid plexuses were incubated in artificial cerebrospinal fluid containing low concentrations of [3H] thymidine (6 nM) to allow intracellular conversion of [3H] thymidine into [3H] thymidine phosphates and [3H] DNA, both diazepam (10 microM) and thiopental (500 microM) altered [3H] thymidine accumulation and metabolism consistent with inhibition of facilitated diffusion but not active transport of thymidine. These studies provide evidence that, at toxic but not therapeutic concentrations, diazepam and thiopental alter facilitated nucleoside transport in the choroid plexus.     

Inhibitory effects of amphetamine on potassium-stimulated release of [3H]dopamine from striatal slices and synaptosomes

Amphetamine, 10(-7) M or greater, evoked the release of [3H]dopamine ([3H]DA) and inhibited subsequent K+-evoked [3H]DA release from striatal synaptosomes superfused at a flow rate (1 ml/min) that prevented reuptake. Amphetamine inhibited the K+-evoked release of [3H]DA to a lesser extent in striatal slices or in synaptosomes superfused at a flow rate (0.35 ml/min) that allowed reuptake. The observed decrease in amphetamine inhibition of K+-evoked release was primarily due to amphetamine blocking [3H]DA reuptake. Interneuronal interactions may account for some of the inhibitory effects of amphetamine on K+-evoked release in the slice. Inhibition of K+-evoked release from either slices or synaptosomes was still evident when 10(-6) M amphetamine was removed from the superfusion buffer and the spontaneous release had returned to control levels. The presence of Ca++ during amphetamine exposure was required for subsequent inhibition of K+-evoked release in synaptosomes. Amphetamine in the presence of Ca++ did not affect the subsequent release of [3H]DA evoked by the Ca++ ionophore, A23187. Therefore, amphetamine inhibition of the K+-evoked release of [3H]DA cannot be explained by prior depletion of Ca++-releasable pools. Nifedipine, 1 microM, failed to block either the Ca++-dependent release of [3H]DA or the inhibition of K+-evoked release by amphetamine. However, 1 mM cobalt inhibited the Ca++-dependent release of [3H]DA by amphetamine and antagonized the inhibition of K+-evoked release after amphetamine exposure. This suggests that amphetamine may open voltage-dependent Ca++ channels sensitive to cobalt but not nifedipine. Amphetamine may desensitize these voltage-dependent Ca++ channels and inhibit their activation by K+ depolarization.     

Possible involvement of ATP-sensitive K+ channels in the relaxant response of dog middle cerebral artery to cromakalim

To determine the functions of ATP-sensitive K+ (KATP) channels in cerebral arterial smooth muscle, the effects of cromakalim, an opener of these channels, on tension and 86Rb efflux were investigated in endothelium-removed strips of dog middle cerebral arteries (MCAs). Cromakalim relaxed the strips that were precontracted with 20.9 mM K+ with a small maximum response. The relaxant responses to cromakalim were competitively antagonized by glibenclamide, a blocker of KATP channels. In strips precontracted with 65.9 mM K+, cromakalim failed to relax the strips. The addition of cromakalim to a resting strip caused a dose-dependent relaxation. In the resting strips of MCAs preloaded with 86Rb, cromakalim did not increase the 86Rb efflux. With 42K as the tracer ion, cromakalim still had no effect on the efflux from the resting strips. On the other hand, cromakalim increased the 86Rb and 42K efflux from the strips of dog coronary arteries (CAs). In 20.9 mM K(+)-contracted strips of MCAs, cromakalim significantly decreased the 86Rb efflux. However, after the inactivation of Ca(++)-activated K+ channels by the addition of 1 x 10(-7) M nifedipine to the 20.9 mM K(+)-contracted strips of MCAs, cromakalim produced a small but significant increase in the 86Rb efflux. Similarly, when the resting strips of MCAs were placed in the Ca(++)-free 12 mM-Mg(+)+ solution, cromakalim increased the 86Rb efflux. In 65.9 mM K(+)-contracted strips, cromakalim increased the 86Rb efflux from both arteries. However, the extent of the increase in 86Rb efflux was significantly smaller in the MCA than in the CA.(ABSTRACT TRUNCATED AT 250 WORDS)