Inhibition of 3H-1-norepinephrine uptake by ouabain is species dependent

Tissue slice to medium ratios of 3H-1-norepinephrine (3H-1-NE) were used to study the effect of ouabain on uptake of norepinephrine. The effects of ouabain were studied in slices of heart and spleen from three different species: rat, guinea pig, and dog. The drug produced a species as well as a concentration dependent inhibition of norepinephrine uptake in both types of tissue. In order of decreasing sensitivity, the following relationship between species was observed: dogs greater than guinea pigs greater than rats. Since 3H-1-norepinephrine uptake under the present experimental conditions represents uptake into sympathetic nerve terminals, it was concluded that Na+-K+-ATPases of sympathetic nerve terminals have species dependent differences in ouabain sensitivity similar to those of myocardium.     

N-acetyl-aspartylglutamate modulation of N-methyl-D-aspartate-stimulated [3H]norepinephrine release from rat hippocampal slices

The release of preloaded radiolabeled norepinephrine ([3H]NE) from slices of rat hippocampus can be stimulated by excitatory amino acids that interact with the N-methyl-D-aspartate (NMDA) receptor. The acidic dipeptide N-acetyl-L-aspartylglutamate (NAAG) is colocalized with NE in the cell bodies of locus coeruleus (the origin of the noradrenergic projections to the hippocampus) and the hippocampus itself. The function of NAAG in these neurons has not been demonstrated, although evidence exists that it may serve as a neuromodulator in other neuronal pathways. NAAG inhibited the release of [3H]NE stimulated by NMDA and L-glutamate in a concentration-related manner. The maximal inhibition produced by NAAG was about 25% of the control release stimulated by 25 microM NMDA. The effects observed were caused by the intact dipeptide and not the degradation artifacts produced by the enzyme N-acetylated-alpha-linked-acidic dipeptidase because N-acetyl-L-aspartate had no significant effect on the release and L-glutamate was stimulatory. The activity of this enzyme appears to be suppressed under the assay conditions used. Although the addition of glycine did not enhance NMDA-stimulated release, 7-chlorokynurenate and 1-hydroxy-3-amino-pyrrolidone-2 decreased the release in a concentration-dependent manner. Furthermore, the attenuation produced by NAAG plus 7-chlorokynurenate or 1-hydroxy-3-aminopyrrolidone-2 was greater than the inhibitory actions of either glycine antagonist alone. Similarly, NAAG produced additional inhibition over that produced by either of two different voltage-dependent calcium channel blockers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lobeline and nicotine evoke [3H]overflow from rat striatal slices preloaded with [3H]dopamine: differential inhibition of synaptosomal and vesicular [3H]dopamine uptake

Lobeline is currently being developed as a substitution therapy for tobacco smoking cessation. Activation of CNS dopamine (DA) systems results in the reinforcing properties of nicotine. The present study compared the effects of lobeline and nicotine on rat striatum. Both lobeline and nicotine evoked [3H]overflow from striatal slices superfused in the presence of pargyline and nomifensine in the buffer. Marked DA depletion (42-67%) and a concomitant 2-fold increase in dihydroxyphenylacetic acid (DOPAC) in slices superfused with high concentrations (30-100 microM) of lobeline were observed. The effect of nicotine (10 microM) was inhibited in a concentration-dependent manner by mecamylamine (1-100 microM). However, lobeline (0.1-100 microM)-evoked [3H]overflow was calcium-independent, and was not antagonized by mecamylamine (1-100 microM), suggesting a mechanism of action other than stimulation of nicotinic receptors. Lobeline inhibited [3H]DA uptake into synaptosomes (IC50 = 80 +/- 12 microM) and vesicles (IC50 = 0.88 +/- 0.001 microM), whereas nicotine (< or =100 microM) did not inhibit synaptosomal or vesicular [3H]DA uptake. In the absence of pargyline and nomifensine in the buffer, endogenous DA was detected in superfusate only in those slices exposed to the highest concentration (100 microM) of lobeline. However, endogenous DOPAC concentration was increased in a concentration-dependent manner, indicating that lobeline exposure resulted in increased cytosolic DA which was rapidly metabolized to DOPAC. Under these conditions, lobeline (10-100 microM) also significantly depleted (66-85%) DA content; however, no change in DOPAC content was observed. The results suggest that, unlike nicotine, lobeline increases DA release by potent inhibition of DA uptake into synaptic vesicles, and a subsequent alteration in presynaptic DA storage.     

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

Abnormalities intrinsic to skeletal muscle are thought to contribute to decrements in exercise capacity found in individuals with chronic heart failure (CHF). Na+-K+-adenosinetriphosphatase (the Na+ pump) is essential for maintaining muscle excitability and contractility. Therefore, we investigated the possibility that the number and affinity of Na+ pumps in locomotor muscles of rats with CHF are decreased. Myocardial infarction (MI) was induced in 8 rats, and a sham operation was performed in 12 rats. The degree of CHF was assessed approximately 180 days after surgery. Soleus and plantaris muscles were harvested, and Na+ pumps were quantified by using a [3H]ouabain binding assay. At the time of muscle harvest, MI and sham-operated rats were similar in age (458 +/- 54 vs. 447 +/- 34 days old, respectively). Compared with their sham-operated counterparts, MI rats had a significant amount of heart failure, right ventricular-to-body weight ratio was greater (48%), and the presence of pulmonary congestion was suggested by an elevated lung-to-body weight ratio (29%). Left ventricular end-diastolic pressure was significantly increased in the MI rats (11 +/- 1 mmHg) compared with the sham-operated controls (1 +/- 1 mmHg). In addition, mean arterial blood pressure was lower in the MI rats compared with their control counterparts. [3H]ouabain binding sites 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 binding sites was similar for the two groups. The relationship between the reduction in Na+ pump number and the reduced exercise capacity in individuals with CHF remains to be determined.     

Inhibition of [3H]forskolin binding by Gpp[NH]p may reflect adenylyl cyclase coupling to Gi

The effect of the GTP-analogue guanylyl 5'-imidodiphosphate (Gpp[NH]p) on [3H]forskolin binding was studied in rat brain using autoradiography. In the striatum, 100 microM Gpp[NH]p produced a 40% increase in binding, whereas a decrease of about 30% was observed with low Gpp[NH]p concentrations (0.1-1 microM). In the molecular layer of the cerebellum all concentrations of Gpp[NH]p decreased [3H]forskolin binding. The decrease in binding disappeared in both striatum and the molecular layer of cerebellum in sections pretreated with 100 microM N-ethylmaleimide (NEM) for 10 min. NEM pretreatment did not significantly affect the stimulation of [3H]forskolin binding by micromolar concentrations of Gpp[NH]p in the striatum, but reversed the decrease observed in the molecular layer of the cerebellum, to an increase. Based on these data we suggest that the effects of the GTP-analogue Gpp[NH]p on [3H]forskolin binding may involve both Gs and Gi, where a stimulation produces an increase and decrease in binding respectively. The regional effects of Gpp[NH]p may reflect differences in the responsiveness of adenylyl cyclase to Gs and Gi-mediated effects.     

Nicotine-stimulated release of [3H]norepinephrine from fetal rat locus coeruleus cells in culture

Acute nicotine administration stimulated [3H]norepinephrine ([3H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC50 of 0.9 microM, and was abolished by removal of calcium from, or addition of tetrodotoxin (500 nM) to, the assay buffer. Other nicotinic receptor agonists stimulated [3H]NE release, with the rank order of potency being (+)-epibatidine > (-)-nicotine > 1,1-dimethyl-4-phenylpiperazinium (DMPP). Whereas (-)-nicotine and (+/-)-epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (-)-cytisine had no agonist activity. Nicotine-stimulated release of [3H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro-beta-erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an alpha4beta2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA- and nicotine-stimulated [3H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK-801 blockade of nicotine-induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [3H]NE from LC cells and does not act indirectly via activation of glutamate release.     

In vivo uptake of [3H]nimodipine in focal cerebral ischemia: modulation by hyperglycemia

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 +/- 0.11 in normoglycemic rats (n = 12) and 6.57 +/- 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 +/- 0.45) than hyperglycemic (1.40 +/- 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 +/- 22 mL/100 g in normoglycemic rats (n = 8) and 33 +/- 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.     

Uptake of [3H]glycine and [3H]GABA by amacrine cells in the cat retina

After intravitreal injection, [3H]glycine accumulates in 3 distinct subpopulations of amacrine cells in the cat retina whereas [3H]GABA accumulates in 4 different subpopulations. Each labeled cell type can be distinguished on the basis of size and cytologic features. The density of label associated with each subpopulation serves as an additional distinguishing characteristic. [3H]Glycine is concentrated within the outer two-thirds of the inner plexiform layer (IPL). [3H]GABA is localized in two narrow bands in the outer half of the IPL and in a wider band adjacent to the ganglion cell layer.     

Changes in apparent in vivo binding of [3H]raclopride and [3H]N-methylspiperone induced by oxotremorine

The effects of muscarinic agonist, oxotremorine (0.3 mg/kg), and antagonist, scopolamine (0.5 mg/kg), on in vivo [3H]raclopride (RAC) and [3H]N-methylspiperone (NMSP) binding were investigated. Following tracer administration to control or pretreated mice, binding potentials, and the rate constants k3 and k4 were determined by kinetic analysis. Oxotremorine resulted in a 70% increase in striatal RAC binding potential compared with controls. RAC and NMSP showed almost identical decreases in k3 (40%), whereas k4 for RAC was unexpectedly decreased by 64%. Scopolamine resulted in no significant changes in RAC or NMSP binding. These results, in combination with previous data obtained in reserpinized mice, show that 1) competition by endogenous ligand may not be the only factor influencing the magnitude of apparent in vivo receptor binding, and 2) interneuronal communication may be partly mediated by changes in the rates of ligand-receptor binding.     

Developmental alterations in N-methyl-D-aspartate stimulated [3H]norepinephrine release in rat brain cortex and hippocampus

Developmental alterations in N-methyl-D-aspartate (NMDA)-stimulated[3H]norepinephrine release from rat brain cortical and hippocampal slices were studied. NMDA (10-1000 microM) resulted in a concentration-dependent increase in [3H]norepinephrine efflux; maximal responses (% released) in the cortex were: (1.53 +/- 0.12, 3.68 +/- 0.20, 2.94 +/- 0.20, 4.60 +/- 0.28 and 5.28 +/- 0.33) and the hippocampal responses were: (1.90 +/- 0.18, 3.84 +/- 0.23, 3.60 +/- 0.28, 5.16 +/- 0.38 and 5.81 +/- 0.45) at varying postnatal ages (1, 7, 14, 21 and 90 days) respectively. Cortical tissue from 7-day-old pups exhibited a transient increase in maximal efflux and a decrease in EC50. These results indicated that developmental alterations in the NMDA receptor appear to be translated into differences in NMDA stimulated [3H]norepinephrine release.     

Inhibition of [3H]quinpirole binding by a monoamine oxidase inhibitor in subcellular fractions of rat striatum

[3H]Quinpirole is a dopamine agonist with high affinity for D2-like dopamine receptors. A number of non-dopaminergic compounds, most notably monoamine oxidase inhibitors (MAOIs), inhibit the binding of [3H]quinpirole, but not other D2-like agonists and antagonists, in rat striatal membranes by a mechanism that does not involve the enzymatic activity of MAO. To further characterize this novel interaction, the subcellular distribution of spiperone-displaceable, "D2-like" [3H]quinpirole-labeled sites in rat striatum was assessed and compared with the distribution of MAOI-displaceable [3H]quinpirole binding (MQB). "D2-like" [3H]quinpirole binding exhibited similar nanomolar affinity in the crude synaptosomal (P2), crude microsomal (P3), and ribosomal, post-microsomal (P4) fractions. Total binding activity (fmol bound/fraction) of "D2-like" [3H]quinpirole binding was concentrated in the synaptosomal fraction (P2B). The subcellular distribution of MQB paralleled that of "D2-like" [3H]quinpirole binding. This suggests that "D2-like" [3H]quinpirole binding and MQB occur at a common membrane-bound binding site.     

Inhibition of [methyl-3H]diazepam binding to rat brain membranes in vitro by dinatin and skrofulein

Two flavones, 4',5,7-trihydroxy-6-methoxy flavone (dinatin) and 4',5-dihydroxy-6, 7-dimethoxy flavone (skrofulein), were extracted from Artemisia herba alba L. Dinatin and skrofulein inhibited the binding of [methyl-3H]diazepam to rat brain membranes in vitro with IC50 of 1.3 and 23 mumol.L-1, respectively. The GABA-ratios (the ratio of IC50 values in the absence/presence of GABA in the binding assay) were 1.1 and 1.2 for dinatin and skrofulein, respectively. Both flavones induced a slight increase in [35S] TBPS binding. The data suggest that the flavones are antagonists or partial agonists of benzodiazepine receptors.     

High affinity [3H]imipramine and [3H]paroxetine binding sites in suicide brains

Specific binding of [3H]imipramine and [3H]paroxetine was simultaneously examined in human brains (frontal cortex, temporal cortex, cingulate cortex, hypothalamus, hippocampus and amygdala) from 11 controls and 11 depressed suicide victims. A single saturable high affinity site was obtained for both radioligands. Age was not related to significant changes in [3H]imipramine and [3H]paroxetine binding parameters, which indicates the stability of the brain serotonergic system with increasing age. A major finding of the present study concerns the existence of a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in hippocampus from depressed suicides as compared with the control group, without changes in the binding affinity (Kd). In contrast, when [3H]paroxetine was used as radioligand, no changes in either Bmax or Kd were detected in any of the brain regions studied. These findings suggest that [3H]imipramine may be a better marker than [3H]paroxetine when alterations in the presynaptic serotonergic uptake site are to be detected.     

Active [3H]-dopamine uptake displayed by native lymphocyte suspensions is mainly due to contaminating platelets

Kinetic and pharmacologic properties of specific [3H]-dopamine uptake by native human lymphocytes were investigated. Our results suggest that uptake of [3H]-dopamine measured with lymphocytes after separation over Ficoll-Paque or Percoll is mainly caused by platelets which are always part of freshly prepared lymphocyte suspensions. The investigations were extended to well-defined cell lines in order to compare the pharmacological properties of native and immortalized cells regarding the uptake of [3H]-dopamine without any influence of contaminating cells such as platelets. Using the human neuroblastoma cell line IMR32 we demonstrate a GBR-12909 and cocaine-sensitive specific uptake of dopamine, whereas dopamine uptake in platelets is performed by an imipramine-sensitive serotonin transporter. Blood-derived stable cell lines (MOLT-3 and EBV-transformed B-lymphocytes) exhibited no [3H]-dopamine uptake. The view that specific [3H]-dopamine uptake on native human lymphocytes is mainly caused by platelets and not specific for lymphocytes is supported by the finding that homogenous B- and T-lymphoblastoids (MOLT-3 and EBV-transformed B-lymphocytes) exhibited no comparable uptake.     

Laminar distribution of nicotinic receptor subtypes in human cerebral cortex as determined by [3H](-)nicotine, [3H]cytisine and [3H]epibatidine in vitro autoradiography

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.