Biomedical subjects

N R Zahniser

Publications and source records attributed to N R Zahniser.

At least 73 records · Page 4Linked to original sources

PubMed1989

Outflow and overflow of picogram levels of endogenous dopamine and DOPAC from rat striatal slices: improved methodology for studies of stimulus-evoked release and metabolism.

An improved method for the rapid and sensitive determination of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in superfusates of single rat striatal slices, using high-performance liquid chromatography (HPLC) coupled with 'coulometric' electrochemical detection (EC), is described. Superfusates are directly injected into an HPLC-EC system following addition of a small aliquot of solubilized ascorbate-oxidase. DA and DOPAC are both separated and quantitated in 3-5 min. Twelve to 15 samples can be analyzed each hour with a nominal detection limit of 1.0 pg per injection for each compound or 10-20 pg/ml of superfusate. The present method was used to study changes in DA and DOPAC outflow and overflow in superfusates of single striatal slices following electrical field stimulation, both in the absence and presence of the catecholamine uptake inhibitor nomifensine. Studies of 1 min superfusate collections clearly showed that electrical field stimulation produced a latent increase in DOPAC as compared to DA. The routine sensitivity and sample throughout of the method allows for studies of both outflow and overflow of DA and DOPAC, as well as studies involving time-dependent overflow of these compounds.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1989

Timolol-induced up-regulation of polymorphonuclear leukocyte beta 2-adrenergic receptors in the elderly.

Drug-induced up-regulation of beta-adrenergic receptors is impaired in the brains of aged rats but not in myocardia. To investigate age-related changes in receptor regulation in human beings, young (24 to 35 years of age) and elderly (62 to 78 years of age) healthy volunteers were treated with the beta-adrenergic receptor blocking agent timolol maleate (10 mg b.i.d.) for 8 days. Baseline densities of beta 2-adrenergic receptors on polymorphonuclear leukocyte (PMNL) membranes and heart rates were the same in the two age groups. However, systolic blood pressures were higher in the elderly subjects. Administration of timolol produced similar plasma levels in the two groups. In response to timolol, the density of PMNL beta-adrenergic receptors increased at a similar rate and to the same extent (threefold) in both age groups. Likewise, hemodynamic changes were not related to age. These results suggest that up-regulation of peripheral beta 2-adrenergic receptors in human beings is not impaired with aging.

Adult↗

PubMed1988

A new densitometric procedure to measure protein levels in tissue slices used in quantitative autoradiography.

A new quantitative staining technique for the assay of protein in tissue sections using the dye Coomassie brilliant blue G 250 is discussed. This densitometric procedure uses commercially available hardware and software employed in the quantitation of receptor autoradiographs. Rather than assuming a homogeneous distribution of protein in the tissue section, regional levels of protein are measured in the same tissue slice used to produce the autoradiograph. Additionally, the process of staining tissue with Coomassie blue for protein is reversible; the tissue can be destained after the measurement of protein is complete and then restained with a standard histological stain such as Cresyl violet. This technique is a more reliable method for normalizing receptor densities by tissue protein levels and allows for a more accurate comparison between QAR and membrane binding techniques.

Animals↗

PubMed1988

Biphasic modulation of evoked [3H]D-aspartate release by D-2 dopamine receptors in rat striatal slices.

It has been hypothesized that dopamine (DA) inhibits glutamate release from corticostriatal fibers via presynaptically located D-2 DA receptors although the evidence presented in the literature has not been conclusive. In the present experiments, the effect of D-2 receptor ligands on K+-stimulated tritium release from rat striatal slices preloaded with the nonmetabolizable glutamate analog [3H]D-aspartate ([3H]ASP was measured. The D-2 receptor antagonist S-sulpiride increased stimulated [3H]ASP release by 75% (EC50 value = 240 nM) and the biologically less-active isomer R-sulpiride, although equally effective, was tenfold less potent. The D-2 receptor agonists pergolide and (+)-4-propyl-9-hydroxynapthoxazine (+PHNO) inhibited [3H]ASP release at nM concentrations; however, this effect was small (20%). This low efficacy of the exogenous agonists was apparently due to competition by high concentrations of endogenous DA since the effect of pergolide was increased in rats whose striatal DA levels were decreased by 97%. These data support the hypothesis that D-2 DA receptors modulate [3H]ASP release in an inhibitory fashion. However, when the agonists were tested at lower concentrations, [3H]ASP release was increased significantly by 20% in control rats and 60% in DA-depleted rats. Both the facilitory and inhibitory effects of pergolide were blocked by 10 microM S-sulpiride, suggesting D-2 receptor mediation. In addition, the facilitory effect of pergolide was blocked by tetrodotoxin (TTX) and by the GABAA antagonist bicuculline, implying mediation of this D-2 effect by an inhibitory GABAergic interneuron. The inhibitory effect of pergolide was decreased by the muscarinic antagonist atropine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Repeated cocaine administration results in supersensitivity of striatal D-2 dopamine autoreceptors to pergolide.

Groups of rats administered cocaine-HC1 (10 mg/kg, i.p.) or saline either acutely or once daily for 8 or 14 days were killed 24 hrs after the last dose. In striatal slices prelabelled with [3H]DA, modulation of [3H]-overflow by pergolide was used to measure D-2 autoreceptor activity. Compared to the contemporaneous control group pergolide produced a greater inhibition only in striatal slices from rats treated repeatedly with cocaine. In radioligand binding studies using striatal membranes from control rats, pergolide had a 500-fold greater affinity for the D-2, as opposed to the D-1, dopamine (DA) receptor subtype. These results indicate that repeated treatment with cocaine produces supersensitive striatal D-2 release-modulating autoreceptors consistent with a compensatory change to diminish the effect of elevated synaptic concentrations of DA produced by cocaine. In contrast, supersensitivity of D-2 receptors was not detected in [3H]spiperone binding assays.

Animals↗

PubMed1988

An improved calibration procedure for computer-based quantitative autoradiography utilizing a mathematical model for the non-linear response of camera and film.

A mathematical model which accounts for the non-linear response of both the camera and film in computer-aided quantitative autoradiography is derived. This model and the algorithm to fit the data for radioactive standards by a non-linear least-squares procedure to this model are described. The usefulness of this technique is demonstrated by employing it to analyze a set of 14C-labeled brain paste standards which were exposed to Ultrofilm in such a way that the full response range of the film was used. This technique can be readily implemented on most commercially available image analysis systems and is compatible with most types of video cameras.

Algorithms↗

PubMed1988

Comparison between the effects of aging on antagonist and agonist interactions with beta-adrenergic receptors on human mononuclear and polymorphonuclear leukocyte membranes.

Age-related differences in the binding properties of beta-adrenergic receptors on lymphocyte membranes isolated from healthy individuals have been reported. The purpose of the present studies was to determine whether or not beta receptors on polymorphonuclear leukocyte (PMN) membranes showed similar age-related changes. Plasma Percoll gradients were used to isolate PMN and mononuclear leukocyte (MN) cells from blood drawn in the supine position from young (25-34 years) and elderly (60-76 years) healthy volunteers. Both blood pressures and plasma norepinephrine levels were significantly elevated in the elderly subjects. Saturation analysis with [125I]-(-) iodopindolol (IPIN) showed a 2.5-fold higher density of beta 2 receptors on the MN, but not PMN, membranes from the elderly. Neither the affinities of the receptor for IPIN or the agonist isoproterenol, nor the GTP-induced shift in agonist affinity, differed with subject age in either cell type. These results suggest that subpopulations of MN cells on which beta-adrenergic receptors are localized may increase in the elderly and that it is important to measure receptor properties on a more homogeneous leukocyte population such as PMN cells or subpopulations of MN cells as a function of aging.

Adrenergic beta-Agonists↗

PubMed1988

Propafenone interacts stereoselectively with beta 1- and beta 2-adrenergic receptors.

In order to determine whether the new antiarrhythmic agent propafenone interacts stereoselectively with beta-adrenergic receptors, the potencies of both the (-) and (+) isomers were determined using in vitro binding assays. (-)-Propafenone was the more potent isomer and competed with 125I-pindolol in a simple manner in both rat cerebral cortical and cerebellar membranes with Ki values of 32 +/- 1.7 and 77 +/- 5.8 nM, respectively. In contrast, competition curves for (+)-propafenone in the same tissues were more complex and revealed two binding sites with affinities 10- to 75-fold less potent than those for (-)-propafenone. Moreover, the (+)-propafenone was found by high performance liquid chromatography (HPLC) analysis to be contaminated with 3% of the more potent (-)-isomer; this contamination accounted for most of the apparent activity of the (+)-propafenone. These data suggest that interactions of propafenone with both beta 1- and beta 2-receptors are markedly stereoselective for the (-) isomer. It is possible that beta-adrenergic receptor blockade by the (-) isomer may be responsible for some of the adverse clinical effects that have been reported with propafenone therapy.

Animals↗

PubMed1988

Sensitization to cocaine in the nigrostriatal dopamine system.

Behavioral sensitization involving the nigrostriatal dopaminergic tract is manifested after treatment with only a single dose of cocaine and is augmented following repeated treatment. One neurochemical change observed that is consistent with behavioral sensitization is the increase in amphetamine-induced 3H-DA release from striatal slices seen after one injection of cocaine. One day after repeated administration of cocaine, however, the increase is no longer evident. It is possible that transient compensatory changes, such as increased D-2 autoreceptor inhibition, may obscure this effect when it is measured at relatively short times after the repeated administration has been terminated. One day after cessation of repeated cocaine administration, D-2 autoreceptors in both striatum and substantia nigra compacta were upregulated consistent with a compensatory mechanism and the development of behavioral tolerance rather than sensitization. In contrast, DA content, neuronal DA uptake, and postsynaptic D-2 DA receptors in striatum were not regulated by this treatment. Likewise, D-1 DA receptors in striatum and substantia nigra were unaffected. In the mesolimbic system, both the pre- and postsynaptic receptor changes are consistent with sensitization. Amphetamine-stimulated release from nucleus accumbens has not yet been measured in cocaine-sensitized animals. It is possible that changes similar to those seen in striatum may occur in this area. It is interesting that, in general, presynaptic parameters associated with the DA neuron, with the notable exception of the uptake pump, appear to be more sensitive to regulation by cocaine administration than do postsynaptic parameters. The long-lasting effects of a single moderate dose of cocaine are also surprising. It will be important to determine the molecular mechanisms underlying this regulation and whether or not similar changes are induced in mesolimbic dopaminergic systems by single and repeated administration of cocaine.

Animals↗

PubMed1987

The use of 14C-labeled tissue paste standards for the calibration of 125I-labeled ligands in quantitative autoradiography.

The relationship between the exposure of Ultrofilm to 5-30-microns slices of 14C-labeled or 125I-labeled brain paste standards was characterized using quantitative autoradiography. After exposing these slices to Ultrofilm for 24, 48 or 72 h, the autoradiograms from the 14C-labeled brain pastes were used to generate standard curves with computerized densitometry. These autoradiographic standard curves were then used to estimate the level of radioactivity present in brain paste slices containing known amounts of 125I, which also had been exposed to the film for the same length of time. The relationship between the calculated concentration of 125I and the radioactivity of 125I as determined by direct gamma counting of slices scraped from slides was a direct linear one. The use of 14C-labeled, as opposed to 125I-labeled, standards obviates the major disadvantage of using 125I-radioligands for quantitative autoradiographic studies.

Animals↗

PubMed1987

Modulation of gamma-[3H]aminobutyric acid release from rat cortical slices by alpha 2-adrenoceptors.

Modulation of gamma-aminobutyric acid (GABA) release by alpha 2-adrenoceptor agonists has not been consistently demonstrated. This could be due to high levels of norepinephrine (NE) concomitantly evoked by stimulation parameters needed for GABA release. In the present experiments, NE release was preferentially decreased by omission of calcium (Ca2+) and alpha 2-modulation of [3H]GABA release from cortical slices was measured. The antagonist rauwolscine increased only Ca2+-dependent [3H]GABA release, while the agonists guanabenz and clonidine inhibited only Ca2+-independent GABA release. These results suggest that release of endogenous NE diminished the effect of alpha 2-agonists but reveals the effect of antagonists and support the hypothesis that endogenous NE inhibits GABA release in cortex.

Animals↗

PubMed1987

Effects of cell isolation procedures and radioligand selection on the characterization of human leukocyte beta-adrenergic receptors.

Radioligand binding techniques are commonly used in the characterization of beta-adrenergic receptors on human peripheral leukocytes. Accurate interpretation of receptor binding parameters necessitates appropriate radioligand selection. In addition, cell isolation techniques should have minimal effect on the binding parameters of receptors. Our observation of curvilinear Scatchard plots with (-)-[125I]iodocyanopindolol (ICYP) resulted in a re-evaluation of this radioligand and the influence of cell isolation techniques on leukocyte beta-adrenergic receptor binding parameters. Membranes from mononuclear (MN) and polymorphonuclear (PMN) cells isolated by a standard procedure (Ficoll-Hypaque) resulted in biphasic Scatchard plots with ICYP in three of four subjects. In contrast, linear Scatchard plots were observed for ICYP binding to membranes from MN and PMN cells isolated from the same four subjects with an alternative procedure utilizing plasma Percoll. Competition and saturation binding assays with ICYP identified a high degree of nonspecific binding. Decreased stereoselectivity with (-)- and (+)-propranolol was observed with membranes from Ficoll-Hypaque cells as compared to plasma Percoll cells. Kinetic analysis with ICYP demonstrated apparent irreversible binding whether displacement was initiated with a beta-adrenergic receptor antagonist or agonist. These problems with ICYP prompted evaluation of an alternative radioligand, (-)-[125I]iodopindolol (IPIN); this radioligand demonstrated rapid and completely reversible binding, improved stereoselectivity, and low nonspecific binding. Using IPIN, Scatchard plots from three additional subjects were linear for both cell isolation procedures. Based on these observations, the preferred method of human leukocyte beta-adrenergic receptor analysis incorporates the plasma Percoll cell isolation technique and the radioligand IPIN.

Binding, Competitive↗

PubMed1987

One injection of cocaine produces a long-lasting increase in [3H]-dopamine release.

A single cocaine exposure has been reported to sensitize animals to the behavioral effect of subsequent cocaine administration for up to one week. We now report that a single injection of cocaine results in an augmentation in amphetamine-induced release of tritium from rat striatal slices preloaded with [3H]-dopamine. The augmentation appears within 24 hr and persists for at least 2 weeks after injection. This increase in release may result in increased synaptic concentrations of dopamine possibly caused by a change in the membrane transporter for dopamine. Increased dopaminergic synaptic transmission could explain behavioral sensitization.

Animals↗

PubMed1986

The acute effects of 6-hydroxydopamine treatment on noradrenergic function in the rat hippocampus in vitro.

The electrophysiological consequences of in vitro treatment with 6-hydroxydopamine (6-OHDA) were examined in the CA1 region of the rat hippocampal slice. In control slices, norepinephrine (NE) increased the amplitude of the population spike response elicited by synaptic stimulation of hippocampal pyramidal neurons with a threshold of approximately 5 microM. When hippocampal slices were pretreated with 500 microM 6-OHDA for 10 min, perfusion with a subthreshold concentration of NE (0.5 microM) produced responses similar to those observed with a 10-fold higher concentration of NE in untreated slices. Baseline electrophysiological responses were unchanged following the 6-OHDA exposure. The potentiation of the response to NE by in vitro pretreatment with 6-OHDA was accompanied by a greater than 40% decrease in NE content and greater than 90% decrease in [3H]NE accumulation. In vivo treatment with 6-OHDA or N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP4) also potentiated the electrophysiological response to NE in a manner similar to that observed with acute in vitro 6-OHDA pretreatment. This action does not appear to be due to development of beta-adrenergic receptor supersensitivity, because the apparent potency of isoproterenol in increasing the population spike amplitude was unaffected. These data suggest that the increase in the potency of NE in slices pretreated with 6-OHDA is due to the rapid disruption of the high-affinity NE uptake mechanism characteristic of noradrenergic nerve terminals.

Adrenergic Fibers↗

PubMed1986

DSP4-induced noradrenergic lesions increase beta-adrenergic receptors and hippocampal electrophysiological responsiveness.

Following profound (greater than 90%) depletions of norepinephrine (NE) by the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4), the numbers of beta-adrenergic receptors were significantly increased (20-25%) in rat hippocampal and somatosensory cortical membranes; however, the numbers of alpha 1-adrenergic receptors and the affinities of both types of receptors were unaffected. This selective up-regulation of beta-adrenergic receptors was evident 1 week after DSP4 administration and was maintained for at least 2 more weeks. In electrophysiological experiments in the hippocampal slice preparation, responses to threshold as well as maximal concentrations of isoproterenol were enhanced 150% and 33%, respectively, in the DSP4-lesioned animals. The results demonstrate that nearly complete depletion of brain NE produced by administration of DSP4, like that produced by 6-hydroxydopamine, results in increased numbers of beta- but not alpha-adrenergic receptors, and suggest that the density of the former are regulated by afferent noradrenergic fibers. Furthermore, the functional significance of the increased number of hippocampal beta-adrenergic receptors is directly manifested in a greater electrophysiological responsiveness to an exogenously administered beta-adrenergic receptor agonist.

Action Potentials↗

PubMed1986

Dopamine, acting through D-2 receptors, inhibits rat striatal adenylate cyclase by a GTP-dependent process.

This report demonstrates that the D-2 dopamine receptors that are present in rat striatum can directly inhibit the activity of adenylate cyclase in a GTP-dependent manner. N-n-propylnorapomorphine evoked a more pronounced inhibition than did dopamine. However, in the presence of the D-1-selective antagonist, SCH 23390, dopamine was also observed to inhibit the enzyme. Forskolin facilitated the detection of D-2 receptor-mediated inhibition by markedly stimulating striatal adenylate cyclase activity. The inhibition was antagonized in a dose-dependent manner by the D-2 receptor antagonist spiperone (Ki value = 70 pM) and was absolutely dependent on the presence of both GTP and sodium ions. Inhibition produced via D-2 receptors was additive with that produced via opiate or adenosine A1 receptors. The nonhydrolyzable GTP analogue, 5'-guanylylimidodiphosphate [Gpp(NH)p], did not substitute for GTP in promoting the D-2 receptor-mediated inhibition. It thus appears that D-2 receptors mediate adenylate cyclase inhibition by processes that have been observed for other neurotransmitters in the striatum and elsewhere. In addition, Gpp(NH)p displayed a Ca2+/calmodulin dependency for its inhibitory effects that was not shared by receptor-mediated, GTP-dependent inhibition.

Adenylyl Cyclase Inhibitors↗

PubMed1986

Robust modulation of [3H]dopamine release from rat striatal slices by D-2 dopamine receptors.

Conflicting results have been reported for D-2 dopamine (DA) receptor modulation of DA release from rat striatal slices. After systematic examination of the assay conditions used to evoke release of [3H]DA, we report robust modulation of calcium-dependent stimulation-evoked 3H-overflow by D-2 receptors in rat striatal slices preloaded with [3H]DA. In the presence of the DA uptake inhibitor, nomifensine, the amount of 3H-overflow evoked by low numbers (15-30) of pulses was not dependent on the frequency of stimulation (0.25-3.0 Hz). When larger numbers (60-300) of pulses were applied, 3H-overflow was related inversely to the frequency of stimulation. In contrast, 3H-overflow increased linearly as the total number of pulses applied was increased when examined at any of these frequencies. Nomifensine (10 microM) augmented 3H-overflow at each level of stimulation while only slightly increasing spontaneous release. Modulation of evoked 3H-overflow by S-sulpiride, a selective D-2 DA receptor antagonist, was dependent on the number of pulses delivered, rather than on the frequency of stimulation. In contrast, modulation by pergolide, a selective D-2 DA receptor agonist, was dependent on stimulation frequency. S-sulpiride produced greater augmentation of 3H-overflow after delivery of low numbers of pulses in the presence of nomifensine. However, pergolide produced greater inhibition of 3H-overflow at low frequencies of stimulation in the absence of nomifensine. These results indicate that D-2 receptors in rat striatum modulate DA release and are most effective when a low concentration of endogenous DA is present in the synaptic cleft.

Animals↗

PubMed1985

Behavioural sensitivity to PIA in selectively bred mice is related to a number of A1 adenosine receptors but not to cyclic AMP accumulation in brain slices.

In a dose of 0.1 mg/kg, PIA had marked behavioural effects in long-sleep mice (which show a high sensitivity to ethanol, while no significant effect was observed in short sleep mice (low sensitivity to ethanol). The number of [3H]PIA binding sites in cortex and subcortical brain regions was significantly higher in long-sleep than in short-sleep mice. The KD value was higher in cortex and cerebellum in the short-sleep mice, but there were no differences in the number of hippocampal beta-adrenoceptors or in the adenosine analogue-induced increase in cyclic AMP accumulation in slices of mouse hippocampus.

Adenosine↗