Immunoassay reagents for psychoactive drugs. Part 4. Quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay.

Methods for the quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay (FPIA) is described. One immunoassay allows for the accurate quantification of amitriptyline in the presence of nortriptyline while the second immunoassay allows for the accurate quantification of nortriptyline in the presence of amitriptyline.

Immunoassay reagents for psychoactive drugs. Part 3. Removal of phenothiazine interferences in the quantification of tricyclic antidepressants.

Phenothiazines and their metabolites are known to interfere in the quantification of tricyclic antidepressants (TCAs). A method for selective chemical modification of phenothiazines by chloramine-T in the presence of TCAs is described. This method allows for accurate quantification of the TCA analyte in a serum sample without interference from the modified phenothiazine.

Immunoassay reagents for psychoactive drugs. I. The method for the development of antibodies specific to amitriptyline and nortriptyline.

Amitriptyline and nortriptyline were structurally modified by the attachment of spacer arms to the aromatic ring which were subsequently attached to bovine serum albumin (BSA). Rabbits inoculated with these conjugates yielded polyclonal antisera with high selectivity and good titers. This approach required novel spacer arms and new conjugation methods. The antisera produced were characterized with respect to their cross-reactivity with amitriptyline, nortriptyline and their hydroxy metabolites as well as selected structurally related compounds.

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain.

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.

High-affinity uptake of 67Cu into a veratridine-releasable pool in brain tissue.

We have previously characterized two saturable, ligand-dependent processes for 67Cu uptake by hypothalamic slices: a high- and low-affinity process (22). In this study, we wished to ascertain if veratridine, a secretagogue that mimics a physiological release process, stimulates the release of newly taken up 67Cu and whether uptake of 67Cu into the releasable pool of copper is dependent on the process of 67Cu uptake. Hypothalamic or caudate slices from male rats were loaded for 30 min with 67Cu complexed to histidine (His) under conditions favoring high- or low-affinity uptake. First, we assessed the stability of the newly taken up 67Cu and found that, regardless of the mode of 67Cu entry into the tissue, greater than or equal to 85% of the 67Cu is retained in tissues incubated for 3 h in 67Cu-free buffer. Moreover, the 67Cu taken up by the high-affinity process was not displaced by 15-fold molar excess of nonradiolabeled Cu2+, histidine, albumin, or Zn2+, and only 20-30% of the 67Cu taken up by the low-affinity process was displaced by 10-fold excess Cu2+ or albumin. Next, we assessed veratridine stimulation of 67Cu release and found that 67Cu release occurred only from tissues loaded with the high- but not with the low-affinity process. This effect of veratridine was calcium dependent and was blocked by Tetrodotoxin, a specific blocker of the voltage-sensitive Na+ channel. In addition, we confirmed our earlier observation that a depolarizing concentration of K+ stimulates 67Cu release.(ABSTRACT TRUNCATED AT 250 WORDS)

A correlation between the ligand specificity for 67copper uptake and for copper-prostaglandin E2 stimulation of the release of gonadotropin-releasing hormone from median eminence explants.

We have previously shown that copper (Cu) leads to a 3- to 4-fold amplification of prostaglandin E2 (PGE2) stimulation of LHRH release from explants of the median eminence area (MEA), that this amplification is a saturable function of [Cu], and that complexed Cu but not ionic Cu is the active form of the metal. This implicates a ligand-specific site in Cu action. In this study we address the following questions. Is there a ligand specificity for Cu amplification of PGE2 stimulation of LHRH release, and if so, does it correlate with the ligand specificity for Cu uptake? MEAs of 1-month-old female rats were incubated for 5 min with 150 microM Cu solution and then for 15 min with 10 microM PGE2 (Cu/PGE2); LHRH released into the medium was evaluated by RIA. To assess Cu uptake, MEAs were incubated with 100 microM67 Cu solution for 15 min, and 67Cu accumulation by the MEA was evaluated. The Cu was complexed to one of the following ligands: histamine, His, Cys, Thr, Gly, glutathione, Gly-His-Lys, or albumin. There was a high degree of correlation (r = 0.943) between the ligand specificity for Cu/PGE2 stimulation of LHRH release (Cu action) and 67Cu uptake. Complexation of ionic Cu with His facilitated Cu action and 67Cu uptake 3-fold each, and this was completely prevented by the inclusion of His in a 100-fold excess over the concentration of the Cu/His complex. Histamine, the amino acids, and the peptides facilitated Cu action and 67Cu uptake, whereas albumin did not do so. Of these facilitatory ligands, histamine and His were the most effective and Gly-His-Lys was the least effective. In summary, both 67Cu uptake and Cu action are ligand-dependent and ligand-specific; the Cu interactive sites have a common recognition for the Cu-ligand complex and for the ligand itself; and the ligand specificity for 67Cu uptake and for Cu action are highly correlated. These results are consistent with the ligand specificity for Cu uptake being the primary determinant of the ligand specificity for Cu/PGE2 stimulation of LHRH release.

Evidence for synergism between copper and prostaglandin E2 in stimulating the release of gonadotropin-releasing hormone from median eminence explants: Na+/Cl- requirements.

Copper (Cu) and PGE2 are known to stimulate LHRH release from explants of the median eminence area (MEA) by two mechanisms distinguishable by their Ca2+ dependence. Moreover, exposure to Cu and PGE2 results in an amplified release of LHRH which is partially Ca2+ dependent, thus, resembling the release process stimulated by PGE2 alone. We have shown that LHRH release stimulated by Cu alone is Na+/Cl- dependent. By defining the Na+/Cl- dependence of PGE2- and Cu/PGE2-stimulated release of LHRH, we wished to ascertain if there is synergism between Cu and PGE2 actions. MEA of adult male rats were incubated for 5 min with 150 microM Cu and then for 15 min with 10 microM PGE2 (Cu/PGE2). Controls were incubated with Cu or PGE2. LHRH release into the medium was evaluated by RIA. Substituting Cl- in the incubation buffer with the non-permeant anion, isethionate, did not alter PGE2 stimulation of LHRH release, but it drastically inhibited Cu/PGE2 stimulation of LHRH release, indicating that this process requires a permeant monovalent anion. PGE2 and Cu/PGE2 stimulation of LHRH release were both inhibited when Na+ was substituted with Li+, or when 0.5 mM ouabain was included in the Na+-containing buffer; neither 10 microM tetrodotoxin (TTX) nor 100 microM amiloride were inhibitory. To ascertain if Na+ is required for Cu uptake, we evaluated the uptake of 67Cu by MEA explants and found that neither ouabain nor Li+ inhibited uptake, indicating that the extracellular Na+ and the activity of Na+/K+ ATPase are required for the process of LHRH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for release of copper in the brain: depolarization-induced release of newly taken-up 67copper.

The potential importance of copper (Cu) in neurosecretion can be inferred from the demonstration that extracellular Cu modulates the secretory function of peptidergic neurons (in vitro studies) and from the presence of high Cu concentrations in nerve terminals and secretory vesicles, primarily within the soluble matrix of the latter. We have previously hypothesized that vesicular Cu is released from neurons undergoing exocytosis and that such extracellular Cu plays an important modulatory role in the central nervous system. To test this Cu release hypothesis, rat hypothalami were incubated under in vitro conditions for 1 or 2 hr with 20 nM radiolabeled Cu (67Cu), and then 67Cu release was stimulated by a depolarizing concentration (60 mM) of K+. K+ markedly (P less than 0.001) stimulated 67Cu release in a Ca2+-dependent manner (stimulated release was 95 fmol/10 min/mg protein after 1 hr 67Cu loading and 160 after 2 hr). These amounts of released 67Cu account for about 10% of the total 67Cu taken up by the tissue. These results indicate that part of the 67Cu taken up by hypothalamic explants is directed into an intracellular compartment from where it can be released by a Ca2+-dependent mechanism, thus providing strong support to our hypothesis that release of copper is operative in situ in the brain.

Brain tissue accumulates 67copper by two ligand-dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process.

We characterized the mechanism of copper accumulation by the brain, using rat hypothalamic tissue slices incubated with 67Cu as a model system. Two ligand-dependent saturable processes were discerned: a high affinity, low capacity process and a low affinity, high capacity process. Vo versus [S] for the high affinity process was a hyperbolic function having an apparent Km and Vmax of 6 microM copper and 23 pmol/min/mg protein, respectively. Vo versus [S] for the low affinity process was a sigmoidal function having an "apparent Km" (So5) and maximal velocity at saturating [S] of 40 microM copper and 425 pmol/min/mg protein, respectively. The two processes were similar in that each exhibited: (a) a requirement for complexing of copper for optimal 67Cu accumulation; (b) a broad ligand specificity with respect to amino acids (histidine, cysteine, threonine, glycine) and peptides (Gly-His-Lys, glutathione) and ineffectiveness of albumin in serving as a facilitatory ligand; (c) a requirement for thermic but not metabolic energy. In spite of these similarities, a 50- or 1000-fold molar excess of ligand (histidine) inhibited 67Cu accumulation by the low affinity process by 60 and 85%, respectively, whereas excess histidine facilitated 67Cu accumulation by the high affinity process by 1.6-4-fold. These results are consistent with 1) a carrier-mediated facilitated diffusion, analogous to that of neutral amino acids, as a means of transporting complexed copper into brain tissue, and 2) the existence of two distinct carrier sites interacting in a positive cooperative manner: a high and a low affinity site.

Distribution and calcium-sequestering ability of smooth endoplasmic reticulum in olfactory axon terminals of frog brain.

In the present study, the structural and functional role of smooth endoplasmic reticulum was investigated in bullfrog olfactory axon terminals. Structural evidence obtained from this study indicated that this vesiculotubular organelle becomes a more elaborate network of anastomosing tubules near the nerve terminal, located in the olfactory lobe of frog brain. Further structural evidence suggested that membranes of the smooth endoplasmic reticulum pinch off to give rise to some electron-lucent vesicles of approximately 50 nm diameter (microvesicles). Ultrastructural cytochemistry was employed in the present study to demonstrate that olfactory axon terminal smooth endoplasmic reticulum actively sequesters Ca2+. However, a variable amount of electron-dense product (calcium oxalate) was associated with microvesicles located at a distance from the synapse, in contrast to those clustered near the synapse which usually did not contain this reaction product. Results from Ca2+-Mg2+-adenosine-5'-triphosphatase (ATPase) cytochemistry showed a similar pattern of distribution, with smooth endoplasmic reticulum being densely labeled with ATPase reaction product (lead phosphate), but aggregated microvesicles in the nerve terminal generally lacking this electron-dense product. Therefore, it is concluded that olfactory axonal smooth endoplasmic reticulum plays a role in the regulation of intraneuronal Ca2+ levels, and that the Ca2+-sequestering activity of this membranous organelle is dependent upon enzymatic hydrolysis of ATP. Conversely, the microvesicles, particularly those accumulated near the synapse, lack this Ca2+-pumping capacity. Thus, if some of the microvesicles originate from smooth endoplasmic reticulum membranes which are capable of pumping Ca2+, but these vesicles themselves lack this capacity, one can postulate that the Ca2+ pumps are either removed from the newly formed microvesicle membranes or are somehow incapacitated in situ in the membrane.

Responsiveness of immature versus adult male rat hypothalami to dibutyryl cyclic AMP- and forskolin-induced LHRH release in vitro.

In the present study, we have investigated the effects of intermittent dibutyryl cyclic AMP (dbcAMP, 5 X 10(-8) M), butyrate (5 X 10(-8) M) and forskolin (10(-4) M) on immunoreactive luteinizing hormone-releasing hormone (LHRH) release from superfused hypothalamic fragments from intact male rats of age 25, 30, 45, or 60-75 day (adult). The results indicate that at 25 days of age, male rat hypothalami were most responsive to cyclic AMP (162% of preinfusion basal LHRH release); by 30 days of age, dbcAMP also elicited increased LHRH release (120% of basal). By 45 days of age, the dbcAMP effect on in vitro LHRH release was slightly inhibitory (78% of basal); however, by adulthood, the effect of this cyclic nucleotide on LHRH release was minimal (92% of basal). Butyrate also induced age-dependent modifications in in vitro LHRH release from male rat hypothalami, with slight increases following butyrate delivery at 25 days of age (115%), slight decreases at 30 (82%) and 45 days of age (68%), and little change in adulthood (94%). This latter finding emphasizes the importance of using butyrate as a control for butyryl derivatives of cyclic AMP, which are known to liberate butyric acid as a product of hydrolysis of parent compounds. To address the possibility that the lack of effectiveness of dbcAMP in the older animal preparations was solely due to an increasing sensitivity of male rat medio-basal hypothalamus fragments to butyrate, we examined the effect of forskolin (10(-4) M), an adenylate cyclase stimulator, on LHRH release.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors altering the secretion of LHRH from superfused fragments of rat hypothalamus.

This paper deals with the effect of temperature (t degrees), ions, prostaglandin (PGE2) and testosterone on the rate of release of LHRH from the superfused medial basal hypothalamus (MBH) of normal or castrated male rats in an effort to further validate the biological properties of this preparation. LHRH in the perfusate is nondetectable when the MBH is at 0 C, warming to 37 C produces a sharp rise in LHRH and then stabilization at a level at least threefold the one calculated for the 0 C experiment. Basal release rate of LHRH is t degrees and calcium dependent, as is the stimulatory action of potassium. Infusion of high K+ induces approximately a fivefold increase in the release rate of LHRH and stays at this high level for as long as the infusion lasts (50 min). LHRH stimulation by PGE2 (pulse) proceeds in the absence of external calcium, and the effect lasts longer than the potassium (pulse) action. Surprisingly, MBHs from long-ter castrated males (60-90 days) release less LHRH per minute than intact male MBHs during the steady state phase of LHRH secretion; testosterone propionate (TP, 200 microgram/100 g BW x 3 days) increases significantly the rate of release in castrate rats and is not related to the amount of LHRH assayed in the MBH. MBHs of untreated castrated rats or castrated rats treated with TP, show a reduced LHRH secretion in response to PGE2. It is concluded that this preparation can be used at advantage to study the rate of LHRH release in a variety of experimental paradigms.