Stereoselective effects of ketamine on dopamine,serotonin and noradrenaline release and uptake in rat brain slices

Ketamine (2-(2-chlorophenyl)-(1-methylamino)-cyclohexanone) is a rapid-acting dissociative general anaesthetic whose hallucinogenic properties have made it a popular drug of abuse. Ketamine comprises two optical isomers, with differing pharmacology. In the present study, the effects of (+)- and (-)-ketamine on stimulated efflux and reuptake of dopamine (DA), noradrenaline (NA) and serotonin (5-HT) were compared in isolated superfused slices of the rat caudatoputamen (CPu), ventral bed nucleus of the stria terminalis (BSTV) or dorsal raphe nucleus (DRN), respectively. Monoamine efflux was elicited by local electrical stimulation (20 pulses, 100 Hz trains) at tungsten microelectrodes and measured at adjacent carbon fibre microelectrodes using fast cyclic voltammetry (FCV). In CPu (+)-ketamine increased stimulated DA efflux and slowed DA reuptake in a concentration-dependent manner (25-200 microM). At 100 microM (+)-ketamine increased DA efflux by 109+/-20% (mean+/-S.E.M., n=13) of control values after 30 min (P<0.001 versus control) and prolonged uptake half-time (t(1/2)) by 76+/-38% (n=9, P<0.001) of control. In contrast (-)-ketamine (100 microM) had no effect on DA efflux or uptake. In DRN, both isomers (100 microM) increased stimulated 5-HT efflux. (-)-Ketamine had a larger effect (P<0.001), an 88+/-15% increase in 5-HT efflux (n=9) versus 46+/-10% (n=8) for the (+)-isomer. The isomers had similar effects on 5-HT uptake, increasing t(1/2) by approximately 200%. No evidence of stereospecificity was seen in BSTV: both isomers had small effects (+)- and (-)-ketamine (100 microM) increasing NA efflux by 43+/-10% (n=7, P<0.001) and 29+/-8% (n=7, P<0.001), respectively. The isomers also had identical effects on NA uptake, each increasing uptake t(1/2) by approximately 100%. In summary, our data show that the optical isomers of ketamine have strikingly different stereospecificity for the monoamine systems and one might predict, therefore, a different psychotomimetic potential.     

Differential modulation of alpha2-adrenoceptor subtypes in rat kidney by chronic desipramine treatment.

The profile of [3H]RX821002 (2-methoxy idazoxan) binding to alpha2-adrenoceptor subtypes in rat kidney membranes was evaluated in controls and after chronic treatment with desipramine (10 mg/kg, i.p., every 12 h, 7 days) or clorgyline (2 mg/kg, i.p., every 24 h, 21 days). [3H]RX821002 recognized with high affinity (Kd=1.5+/-0.2 nM in controls) a single and saturable population of binding sites (Bmax=57+/-5 fmol/mg protein in controls). The competitions by (-)-adrenaline, the alpha2B-adrenoceptor selective drug ARC239 (2-[2-[4-(o-methoxyphenyl)-piperazin-1-yl]-ethyl]-4,4-dimethyl-1,3 (2H,4H)-isoquinolindione) and the alpha2A-adrenoceptor selective drug BRL44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidaz ole) suggested the existence of both alpha2A- and alpha2B-adrenoceptors together with a non-adrenoceptor binding site. After chronic desipramine but not after chronic clorgyline treatments, the density (Bmax) of alpha2-adrenoceptors was increased (46%). In the presence of ARC239 (50 nM), the density of alpha2A-adrenoceptors increased (44%) in the desipramine-treated group without changes in the clorgyline-treated group. Conversely, in the presence of BRL44408 (100 nM), the density of alpha2B-adrenoceptors was not affected by the treatments. The selective upregulation of the alpha2A-adrenoceptor subtype following chronic desipramine administration is compatible with a differential location and function of the alpha2-adrenoceptor subtypes in the rat kidney.     

Incremental Changes in Extracellular Noradrenaline Availability in the Frontal Cortex Induced by Naturalistic Environmental Stimuli: A Microdialysis Study in the Freely Moving Rat

Abstract: Changes in levels of extracellular noradrenaline (NA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the frontal cortex, induced by exposure of unrestrained, conscious rats to novel environments, were compared using in vivo microdialysis. NA efflux increased when rats were transferred to a novel cage, but this was not significant when compared with either basal efflux or with changes after equivalent handling in their home cage. Increasing the intensity of illumination of the novel cage by fivefold significantly increased NA efflux with respect to basal efflux but not handled controls. However, a sustained and significant increase in NA efflux (cf. basal efflux or handled controls) was found when an unfamiliar conspecific was also present in the novel cage. In all cases, basal efflux was restored within 1 h of returning rats to their home cage. Neither handling nor environmental stimuli described above affected DOPAC efflux. 5-HIAA efflux was increased (cf. basal) in the presence of an unfamiliar conspecific, but this increase was no greater than that in handled rats. It is concluded that different naturalistic stimuli cause incremental changes in the levels of extracellular NA in the frontal cortex; these changes affect both phasic and tonic components of the response.     

NOX2-dependent ROS is required for HDAC5 nuclear efflux and contributes to HDAC4 nuclear efflux during intense repetitive activity of fast skeletal muscle fibers

Reactive oxygen species (ROS) have been linked to oxidation and nuclear efflux of class IIa histone deacetylase 4 (HDAC4) in cardiac muscle. Here we use HDAC-GFP fusion proteins expressed in isolated adult mouse flexor digitorum brevis muscle fibers to study ROS mediation of HDAC localization in skeletal muscle. H(2)O(2) causes nuclear efflux of HDAC4-GFP or HDAC5-GFP, which is blocked by the ROS scavenger N-acetyl-l-cysteine (NAC). Repetitive stimulation with 100-ms trains at 50 Hz, 2/s ("50-Hz trains") increased ROS production and caused HDAC4-GFP or HDAC5-GFP nuclear efflux. During 50-Hz trains, HDAC5-GFP nuclear efflux was completely blocked by NAC, but HDAC4-GFP nuclear efflux was only partially blocked by NAC and partially blocked by the calcium-dependent protein kinase (CaMK) inhibitor KN-62. Thus, during intense activity both ROS and CaMK play roles in nuclear efflux of HDAC4, but only ROS mediates HDAC5 nuclear efflux. The 10-Hz continuous stimulation did not increase the rate of ROS production and did not cause HDAC5-GFP nuclear efflux but promoted HDAC4-GFP nuclear efflux that was sensitive to KN-62 but not NAC and thus mediated by CaMK but not by ROS. Fibers from NOX2 knockout mice lacked ROS production and ROS-dependent nuclear efflux of HDAC5-GFP or HDAC4-GFP during 50-Hz trains but had unmodified Ca(2+) transients. Our results demonstrate that ROS generated by NOX2 could play important roles in muscle remodeling due to intense muscle activity and that the nuclear effluxes of HDAC4 and HDAC5 are differentially regulated by Ca(2+) and ROS during muscle activity.     

Effects of Milnacipran and Pindolol on Extracellular Noradrenaline and Serotonin Levels in Guinea Pig Hypothalamus

Abstract: Milnacipran, a dual noradrenaline (NA) and serotonin (5-hydroxytryptamine, 5-HT) uptake inhibitor, increased extracellular levels of NA and 5-HT in hypothalamus of freely moving guinea pigs as measured by microdialysis. The basal levels of both monoamines, which were tetrodotoxin sensitive, were increased in a dose-dependent manner and to a similar extent after the intraperitoneal administration of milnacipran (10 and 40 mg/kg i.p.). Levels of the NA metabolite 4-hydroxy-3-methoxyphenylglycol (MHPG) were decreased by milnacipran at 10 and 40 mg/kg i.p., whereas those of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) showed no effect. Subcutaneous injection of 5-HT_1A and β-adrenergic receptor antagonist (−)-pindolol alone, at 10 mg/kg, had no effect on the extracellular levels of NA or 5-HT. The concomitant administration of (−)-pindolol (10 mg/kg s.c.) with milnacipran (10 mg/kg i.p.) increased severalfold the effect of milnacipran on the extracellular levels of NA and 5-HT. These results indicate that milnacipran, by blocking the uptake of NA and 5-HT, increases virtually equipotently the extracellular levels of NA and 5-HT, confirming previous in vitro studies. In addition, the antagonism of 5-HT_1A autoreceptors by (−)-pindolol potentiates the action of milnacipran on both NA and 5-HT systems, without modifying the ratio of these activities.     

Noradrenaline Release from Cultured Mouse Postganglionic Sympathetic Neurons

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.     

Increased vasodilator responsiveness to BRL 34915 in spontaneously hypertensive versus normotensive rats: contrast with nifedipine

The blood pressure-lowering potency and activity of BRL 34915, a new vasodilator and putative stimulator of potassium efflux from vascular smooth muscle, was investigated in conscious spontaneously hypertensive rats (SHR) and normotensive rats (NTR) after intravenous administration and compared with that of the calcium channel blocker, nifedipine. In SHR, BRL 34915 (3-100 micrograms/kg) or nifedipine (10-3000 micrograms/kg) produced similar reductions in mean arterial pressure of 58 +/- 3% and 55 +/- 3%, respectively. BRL 34915 (ED30% = 13.8 micrograms/kg) was 15.3 times more potent than nifedipine (ED30% = 207 micrograms/kg) in SHR. In contrast, only a 1.7-fold difference in potency was observed in NTR between BRL 34915 (ED30% = 123 micrograms/kg) and nifedipine (ED30% = 182 micrograms/kg). The potency ratio (ED30% NTR/ED30% SHR) for BRL 34915 was 8.83 whereas nifedipine had a ratio of 0.88, reflecting the greater responsiveness of the SHR to BRL 34915. Systemic hemodynamics were monitored in anesthetized SHR and NTR to determine the basis for the reductions in blood pressure. BRL 34915 (3-100 micrograms/kg iv) lowered mean arterial pressure in both groups solely by decreasing total peripheral vascular resistance, since no changes in cardiac output were observed. Relaxation responses were also obtained in phenylephrine-contracted isolated aortic strips from both strains of rat to ascertain whether differences in responsiveness existed at this level of the vasculature. No significant difference in the potency of BRL 34915 (3-10 microM) as a vasodilator was found in aortas from SHR or NTR. These results indicate that, unlike nifedipine, BRL 34915 is a more potent vasodepressor agent in SHR than in NTR and suggests that the potassium efflux stimulator may preferentially relax resistance vessels in the hypertensive rat.     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

Kinetic and pharmacological properties of cloned human equilibrative nucleoside transporters, ENT1 and ENT2, stably expressed in nucleoside transporter-deficient PK15 cells. Ent2 exhibits a low affinity for guanosine and cytidine but a high affinity for inosine

We stably transfected the cloned human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) into nucleoside transporter-deficient PK15NTD cells. Although hENT1 and hENT2 are predicted to be 50-kDa proteins, hENT1 runs as 40 kDa and hENT2 migrates as 50 and 47 kDa on SDS-polyacrylamide gel electrophoresis. Peptide N-glycosidase F and endoglycosidase H deglycosylate hENT1 to 37 kDa and hENT2 to 45 kDa. With hENT1 being more sensitive, there is a 7000-fold and 71-fold difference in sensitivity to nitrobenzylthioinosine (NBMPR) (IC(50), 0.4 +/- 0.1 nM versus 2.8 +/- 0.3 microM) and dipyridamole (IC(50), 5.0 +/- 0.9 nM versus 356 +/- 13 nM), respectively. [(3)H]NBMPR binds to ENT1 cells with a high affinity K(d) of 0.377 +/- 0.098 nM, and each ENT1 cell has 34,000 transporters with a turnover number of 46 molecules/s for uridine. Although both transporters are broadly selective, hENT2 is a generally low affinity nucleoside transporter with 2.6-, 2.8-, 7. 7-, and 19.3-fold lower affinity than hENT1 for thymidine, adenosine, cytidine, and guanosine, respectively. In contrast, the affinity of hENT2 for inosine is 4-fold higher than hENT1. The nucleobase hypoxanthine inhibits [(3)H]uridine uptake by hENT2 but has minimal effect on hENT1. Taken together, these results suggest that hENT2 might be important in transporting adenosine and its metabolites (inosine and hypoxanthine) in tissues such as skeletal muscle where ENT2 is predominantly expressed.     

Changes in the mitochondrial calcium influx and efflux properties are responsible for the decline in sperm calcium during epididymal maturation

This study was undertaken to determine the role of calcium ion, a key regulator of the intensity and form of motility in mature demembranated sperm, in the development of motility during passage through the bovine epididymis. Cellular calcium levels in bovine caput and cauda epididymal spermatozoa were measured with three different techniques. 45Ca2+ uptake measurements revealed that net calcium uptake and Ca2(+)-Ca2+ exchange in caput spermatozoa were about 2 to 3 times higher than in caudal spermatozoa. Intracellular free calcium determination with the calcium fluorophore Fura 2 showed that the levels were 6 times higher in caput spermatozoa. The values for caput and caudal sperm were 875 +/- 55 nM (n = 15) and 155 +/- 6 nM (n = 24), respectively. Total cellular calcium levels quantitated by atomic absorption were 626 +/- 30 (n = 48) and 304 +/- 19 (n = 46) ng/10(8) sperm in caput and caudal epididymal sperm, respectively. At least one of the reasons for the high calcium content of caput epididymal sperm is the result of a higher rate and extent of mitochondrial calcium accumulation in caput compared to caudal sperm. Mitochondrial calcium uptake rates measured in digitonin permeabilized cells revealed uptake rates 2- to 3-fold higher in caput compared to caudal sperm. However, mitochondrial calcium efflux rates were identical in caput and caudal epididymal sperm. The efflux rates in both cell types were unaffected by external sodium levels but were found to be proportional to pH. Alkalinization or acidification of internal pH of intact sperm resulted in a corresponding lowering or elevation of cytoplasmic free calcium levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and release effects of diethylpropion and its metabolites with biogenic amine transporters

Three metabolites of diethylpropion (1), (±)-2-ethylamino-1-phenyl-propan-1-one (2), (1R,2S)-(−)-N,N-diethylnorephedrine (3a) and (1S,2R)-(−)-N,N-diethylnorephedrine (3b) were synthesized. Their uptake and release effects with biogenic amine transporters were evaluated. A major finding of this study is that the in vivo activity of diethylpropion on biogenic amine transporters is most likely due to metabolite 2 as diethylpropion (1) and the metabolites 3a and 3b showed little or no effect in the assays studied. These studies also revealed that 2 acted as a substrate at the norepinephrine (IC₅₀=99 nM) and serotonin transporters (IC₅₀=2118 nM) and an uptake inhibitor at the dopamine transporter (IC₅₀=1014 nM). The potent action of 2 at the NE transporter supports the hypothesis that amphetamine-type subjective effects may be mediated in part by brain norepinephrine.     

Sodium flux through the sodium channels of axon membrane fragments isolated from lobster nerves

The efflux of 22Na from vesicles formed by axolemma fragments isolated from lobster nerves was studied in the presence and in the absence of drugs having well-known action on the sodium channels. The vesicles were equilibrated 12-14 h at 4 degrees C with 22Na in lobster solution containing 1 mM ouabain. Afterwards the suspension was divided: one portion was used as control and the others were treated with veratrine (0.025-0.50 mg/ml), tetrodotoxin (1-2,000 nM) in the presence of veratrine, or tetrodotoxin alone. After 3 h at 20-22 degrees C, the suspensions were diluted into nonradioactive solutions and the 22Na efflux followed by a rapid filtration technique. The results revealed that veratrine increases the efflux rate and the additional application of tetrodotoxin abolishes it, e.g., 0.50 mg of veratrine/ml increases the rate, expressed in 10(-2) min(-1), from 0.59 +/- 0.04 (mean +/- SEM; n = 13) to 0.86 +/- 0.05 (n = 13), and the addition of 100 nM tetrodotoxin diminishes it to 0.48 +/- 0.07 (n = 4). This increase and diminution are statistically significant (P less than 0.005), but this is not the case between the control and the veratrine plus tetrodotoxin values (P greater than 0.05). 50% of the diminution is produced by 11.9 +/- 2.4 nM tetrodotoxin. Tetrodotoxin alone produces a slight diminution of the 22Na efflux. Batrachotoxin (0.50 muM) has an action similar to veratrine's. These findings are considered evidence of the presence of functioning sodium channels in the isolated axolemma fragments.     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

A role for the magnetic field in the radiation-induced efflux of calcium ions from brain tissue in vitro

Two independent laboratories have demonstrated that electromagnetic radiation at specific frequencies can cause a change in the efflux of calcium ions from brain tissue in vitro. In a local geomagnetic field (LGF) at a density of 38 microTesla (microT), 15- and 45-Hz electromagnetic signals (40 Vp-p/m in air) have been shown to induce a change in the efflux of calcium ions from the exposed tissues, whereas 1- and 30-Hz signals do not. We now show that the effective 15-Hz signal can be rendered ineffective when the LGF is reduced to 19 microT with Helmholtz coils. In addition, the ineffective 30-Hz signal becomes effective when the LGF is changed to +/- 25.3 microT or to +/- 76 microT. These results demonstrate that the net intensity of the LGF is an important variable. The results appear to describe a resonance-like relationship in which the frequency of the electromagnetic field that can induce a change in efflux is proportional to a product of LGF density and an index, 2n + 1, where n = 0,1. These phenomenological findings may provide a basis for evaluating the apparent lack of reproducibility of biological effects caused by low-intensity extremely-low-frequency (ELF) electromagnetic signals. In future investigations of this phenomenon, the LGF vector should be explicitly described. If the underlying mechanism involves a general property of tissue, then research conducted in the ambient electromagnetic environment (50/60 Hz) may be subjected to unnoticed and uncontrolled influences, depending on the density of the LGF.     

Aluminum exchange between citrate and human serum transferrin and interaction with transferrin receptor 1

The kinetics and thermodynamics of Al(III) exchange between aluminum citrate (AlL) and human serum transferrin were investigated in the 7.2-8.9 pH range. The C-site of human serum apotransferrin in interaction with bicarbonate removes Al(III) from Al citrate with an exchange equilibrium constant K1 = (2.0 +/- 0.6) x 10(-2); a direct second-order rate constant k1 = 45 +/- 3 M(-1) x s(-1); and a reverse second-order rate constant k(-1) = (2.3 +/- 0.5) x 10(3) M(-1) x s(-1). The newly formed aluminum-protein complex loses a single proton with proton dissociation constant K1a = (15 +/- 3) nM to yield a first kinetic intermediate. This intermediate then undergoes a modification in its conformation followed by two proton losses; first-order rate constant k2 = (4.20 +/- 0.02) x 10(-2) s(-1) to produce a second kinetic intermediate, which in turn undergoes a last slow modification in the conformation to yield the aluminum-loaded transferrin in its final state. This last process rate-controls Al(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau3(-1) = (6 +/- 1) x 10(-5) x s(-1). The affinities involved in aluminum uptake by serum transferrins are about 10 orders of magnitude lower than those involved in the uptake of iron. The interactions of iron-loaded transferrins with transferrin receptor 1 occur with average dissociation constants of 3 +/- 1 and 5 +/- 1 nM for the only C-site iron-loaded and of 6.0 +/- 0.6 and 7 +/- 0.5 nM for the iron-saturated ST in the absence or presence of CHAPS, respectively. No interaction is detected between receptor 1 and aluminum-saturated or mixed C-site iron-loaded/N-site aluminum-loaded transferrin under the same conditions. The fact that aluminum can be solubilized by serum transferrin in biological fluids does not necessarily imply that its transfer from the blood stream to cytoplasm follows the receptor-mediated pathway of iron transport by transferrins.     

Glucose uptake and metabolic stress in rat muscles stimulated electrically with different protocols.

In the present study, the relationship between the pattern of electrical stimulation and glucose uptake was investigated in slow-twitch muscles (soleus) and fast-twitch muscles (epitrochlearis) from Wistar rats. Muscles were stimulated electrically for 30 min in vitro with either single pulses (frequencies varied between 0.8 and 15 Hz) or with 200-ms trains (0.1-2 Hz). Glucose uptake (measured with tracer amount of 2-[(3)H]deoxyglucose) increased with increasing number of impulses whether delivered as single pulses or as short trains. The highest glucose uptake achieved with short tetanic contractions was similar in soleus and epitrochlearis (10.9 +/- 0.7 and 12.0 +/- 0.8 mmol x kg dry wt(-1) x 30 min(-1), respectively). Single pulses, on the other hand, increased contraction-stimulated glucose uptake less in soleus than in epitrochlearis (7.5 +/- 1.1 and 11.7 +/- 0.5 mmol x kg dry wt(-1) x 30 min(-1), respectively; P < 0.02). Glucose uptake correlated with glycogen breakdown in soleus (r = 0.84, P < 0.0001) and (epitrochlearis: r = 0.91, P < 0.0001). Contraction-stimulated glucose uptake also correlated with breakdown of ATP and PCr and with reduction in force. Our data suggest that metabolic stress mediates contraction-stimulated glucose uptake.     

Beta(3)-adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6-8-week-old male Sprague-Dawley rats. In outside-out patches (high K(+), no Ca(2+) in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance (gamma) of 16 pS (24 degrees C) in modified Ringer's was active at a density of 0.5/microm(2). It was blocked by TEA (IC(50)=1.5 mM). A Ca(2+)-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/microm(2) in inside-out patches ([Ca(2+)](i)=1.2 mM). gamma was 28 pS (24 degrees C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 microM propranolol even 1 microM NA did not induce any activity. The alpha-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The beta(3)-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that beta-receptor activation opens NSC-channels thus allowing for Na(+) entry into white adipocytes.     

Effects of Angiotensin II on [3H]Noradrenaline Release and Phosphatidylinositol Hydrolysis in the Parietal Cortex and Locus Coeruleus of the Rat

Angiotensin II (ANGII) (3-100 nM) facilitated the potassium-evoked (22.5 mM) release of [3H]-noradrenaline ([3H]NA) from slices of parietal cortex in a concentration-dependent manner, but did not significantly alter the release of [3H]NA evoked in a similar manner from locus coeruleus slices. The facilitatory action of ANGII was blocked by saralasin (0.1-3 microM). Neither nimodipine (10-30 microM) nor phenylmethylsulphonyl fluoride (1 mM) altered either [3H]NA release or the facilitatory action of ANGII in the parietal cortex. Carbachol (0.01-3 mM) and raised potassium (22.5 mM), but not ANGII (3-100 nM), stimulated the production of inositol phosphates in parietal cortex slices. The potassium-evoked increase in inositol phosphate production was unaffected by ANGII (3-100 nM). In the locus coeruleus, ANGII (3-100 nM) did not stimulate inositol phosphate production. The mechanism underlying the ANGII facilitation of [3H]NA release from the parietal cortex does not appear to involve either nimodipine-sensitive calcium channels, or, as far as we have been able to determine, the release of calcium from intracellular stores following the breakdown of phosphoinositides.     

Vasoconstrictor-mediated release of lactate from the perfused rat hindlimb.

The effects of different vasomodulators on lactate release by the constant-flow-perfused rat hindlimb were examined and compared with that by perfused mesenteric artery, incubated preparations of aortas, soleus and epitrochlearis muscles, and perifused soleus muscles. Infusion of vasopressin (0.5 nM), angiotensin II (5 nM), norepinephrine (50 nM), and methoxamine (10 microM) into the hindlimbs of 180- to 200-g rats increased the perfusion pressure by 112-167% from 30.4 +/- 0.8 mmHg, O2 consumption by 26-68% from 6.4 +/- 0.2 mumol.g-1 x h-1, and lactate efflux by 148-380% from 5.41 +/- 0.25 mumol.g-1 x h-1. Hindlimbs of 100- to 120-g rats responded similarly to angiotensin II. Isoproterenol (1 microM) had no effect on O2 uptake or perfusion pressure but increased lactate release by 118%. Nitroprusside (0.5 mM) markedly inhibited the vasoconstrictor-mediated increases in lactate release, perfusion pressure, and O2 consumption by the hindlimb but had no effect on isoproterenol-mediated lactate efflux. Serotonin (6.7 microM) increased lactate release from the perfused mesenteric artery by 120% from 5.48 mol.g-1 x h-1. Lactate release by incubated aorta was increased by angiotensin II (50 nM), isoproterenol (1 microM), and mechanical stretch. The increase mediated by angiotensin II was blocked by glycerol trinitrate (2.2 microM), which had no effect on lactate release by isoproterenol. Neither angiotensin II (5 nM) nor vasopressin (0.5 nM) increased lactate release from incubated soleus and epitrochlearis muscles; however, lactate release was increased by isoproterenol, and this increase was unaffected by glycerol trinitrate (2.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential

The principal aim of the present study was to investigate the effects of variation in proton gradient and membrane potential on the transport of glycyl-L-glutamine (Gly-Gln) by renal brush border membrane vesicles. Under our conditions of transport assay, Gly-Gln was taken up by brush border membrane vesicles almost entirely as intact dipeptide. This uptake was mediated by two transporters shared by other dipeptides and characterized as the high affinity (Kt = 44.1 +/- 11.2 microM)/low capacity (Vmax = 0.41 +/- 0.03 nmol/mg protein/5 s) and low affinity (Kt = 2.62 +/- 0.50 mM)/high capacity (Vmax 4.04 +/- 0.80 nmol/mg protein/5 s) transporters. In the absence of a pH gradient, only the low affinity system was operational, but with a reduced transport capacity. Imposing a pH gradient of 1.6 pH units increased the Vmax of both transporters. Kinetic analysis of the rates of Gly-Gln uptake as a function of external pH revealed Hill coefficients of close or equal to 1, indicating that transporters contain only one binding site for the interaction with external H+. The effects of membrane potential on Gly-Gln uptake were investigated with valinomycin-induced K+ diffusion potentials. The velocity of the high affinity system but not of the low affinity system increased linearly with increasing inside-negative K+ diffusion potentials (p less than 0.01). The Kt of neither system was affected by alterations in either pH gradient or membrane potential. We conclude that (a) the high affinity transporter is far more sensitive to changes in proton gradient and membrane potential than the low affinity transporter and (b) in the presence of a pH gradient, transport of each dipeptide molecule requires cotransport of one hydrogen ion to serve as the driving force.