A comparison of trihydroxyindole and HPLC/electrochemical methods for catecholamine measurement in adrenal chromaffin cells.

Three commonly used methods for the determination of epinephrine and norepinephrine levels in adrenal medullary tissue were compared. Two variations of the trihydroxyindole procedure, which utilized oxidation at room temperature or 0 degrees C, underestimated levels of total catecholamines in certain standard solutions and were unable to determine correctly their norepinephrine:epinephrine ratios. However, both the variability and the underestimation of the trihydroxyindole procedure carried out at room temperature were more pronounced than that of the trihydroxyindole assay at 0 degrees C. In addition, we tested an isocratic HPLC method utilizing electrochemical detection which separates epinephrine from norepinephrine. The ability of this method to measure correctly total and individual catecholamine levels was superior to either trihydroxyindole procedure, as was its variability. When the three assay methods were used to measure total and individual catecholamine levels in cultured adrenal bovine chromaffin cells, both the trihydroxyindole (0 degrees C) method and the HPLC method yielded values in agreement with those in the literature. However, the HPLC method produced data with lower error estimates. The trihydroxyindole (room temperature) assay was unable to reliably measure levels of epinephrine and norepinephrine in chromaffin cells. These comparisons of catecholamine assays demonstrated that there are circumstances under which the use of each is appropriate. In experiments where the epinephrine:norephinephrine ratio may be changing, the more accurate and precise HPLC assay may be essential, since the trihydroxyindole assays underestimate total catecholamines to varying degrees depending on this ratio. However, the HPLC method suffers from a requirement for technical sophistication for routine use. Therefore, in some laboratories and for repetitive measurement of many samples, the trihydroxyindole assay has a distinct advantage due to its easy utilization and ubiquitous materials. However, the superior results obtained with the trihydroxyindole (0 degrees C) assay over the trihydroxyindole (room temperature) assay emphasizes the need to evaluate the trihydroxyindole procedure for the required purpose, especially if differential oxidation is used for estimation of individual catecholamine levels.

Glucocorticoid receptors and regulation of phenylethanolamine-N-methyltransferase activity in cultured chromaffin cells.

Glucocorticoids are known to regulate the enzyme phenylethanolamine-N-methyltransferase (PNMT) in the adrenal medulla of the rat and are thereby thought to control the synthesis of epinephrine. We have examined the details of this relationship in a simplified system, chromaffin cell primary cultures derived from bovine adrenal medulla. Cultured chromaffin cells were found to have a cytosolic, high affinity, saturable glucocorticoid-binding protein with the steroid specificity of a classical glucocorticoid receptor and a Kd of approximately 1 nM. Treatment of cultured cells with dexamethasone or hydrocortisone at any time up to 21 days in culture increased PNMT activity in the soluble fraction of the cell. The concentration of hormone required to produce a half-maximal response was 10 nM dexamethasone when cells were cultured in the presence of 5% fetal calf serum, or 1 nM in a defined serum-free medium. These dose-response relationships are consistent with mediation of this effect by the glucocorticoid receptor. Unexpectedly, however, the glucocorticoid-induced increment in PNMT activity was not inhibited by cycloheximide at concentrations up to 50 microM, and an acceleration of protein synthesis by insulin treatment did not augment the glucocorticoid effect on PNMT. Treatment of the cells with dexamethasone (100 microM) prevented the decline in the epinephrine-to-norepinephrine ratio seen over time in culture, an effect consistent with increased PNMT activity. However, there was no effect of dexamethasone on the ability of the cells to secrete catecholamines in response to stimulation with high KCl or 30 microM nicotine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity of estrogen target tissues: implications for estrogen regulation of serum luteinizing hormone.

In the rat, it is generally accepted that the primary site of estrogen's stimulatory (positive) effects on serum LH is the preoptic area-anterior hypothalamus (POA/AH). In contrast, the primary site of estrogen's inhibitory (negative) effects on serum LH levels has not been conclusively identified. There is evidence to suggest both a medial basal hypothalamic (MBH) and an anterior pituitary site of action. The present studies utilized a unique characteristic of these estrogen effects to investigate their putative loci. Extensive dose-response curves of estrogen's induction of positive and negative feedback indicated that the negative feedback response was activated at a lower concentration of serum estradiol than the positive feedback response. The differential sensitivities of these two responses suggested that the tissues mediating them might also be differentially sensitive to estradiol. In a previous paper, we showed that receptor translocation is an index of estrogen sensitivity. We measured receptor translocation in response to a series of estradiol doses in the POA/AH, the MBH, and the pituitary. Dose-response curves for estrogen's effect on receptor translocation showed that the pituitary receptor translocation mechanism is activated at significantly lower levels of serum estradiol than that of either the POA/AH or the MBH. These results are consistent with the POA/AH as a site of estrogen's positive feedback effects. In addition, they suggest that negative feedback in the rat may be mediated via estrogen's action at the anterior pituitary. Estrogen's negative feedback effect on serum LH occurs at a serum level of estrogen at which no receptor is translocated in the MBH. Therefore, the pituitary, which does possess nuclear receptors at these estradiol dose levels, is more likely to be the primary mediator of estrogen's negative feedback effects. In another experiment, pituitary, but not hypothalamic, receptor was translocated to the nuclear fraction with an injection of 100 micrograms clomiphere (Clomid). Under these conditions serum LH is depressed, thus strengthening the hypothesis that, in the rat, estrogen action on the pituitary can cause suppression of serum LH independently of the hormone's action in the hypothalamus.

Resolution of estrogen binding species in hypothalamus and pituitary.

In addition to the classic Type I estrogen receptor, a second estrogen binding species has been reported in rat uterine nuclear material. We have examined nuclear material from hypothalamus and pituitary for the presence of Type II estrogen receptor by the sucrose pad/exchange assay. Extensive (0.05-40.0 nM) saturation analyses were performed on crude chromatin isolated from hypothalami and pituitaries of hyperestrogenized ovariectomized rats. Analysis of data by an adaptation of the graphical method of Rosenthal suggests that there is only a single class of estrogen binding sites in these tissues with a Kd (0.2 nM) close to that reported for Type I receptors in other systems. However, a definitive resolution of the binding component(s) was not possible due to noise in the upper regions of the saturation plot. Therefore, we pooled 7 hypothalamic and 4 pituitary saturation experiments and analyzed the data with LIGAND, a nonlinear curve fitting program. Computer generated curves indicate that the data from both target tissues are approximated most closely by a model describing a single high affinity binding species (Type I) (Kd = 0.17 - 0.38 nM) and a linear or very low affinity nonspecific binding component. Thus, we conclude that there is no evidence for the presence of a second or lower affinity estrogen binding component in hypothalamus or pituitary. The absence of this binding species in these two estrogen target tissues is consistent with the concept that Type II sites may be involved in estrogen's control of tissue hypertrophy and hyperplasia--estrogen responses not found in hypothalamus or pituitary.

Purification of synexin by pH step elution from chromatofocusing media in the absence of ampholytes.

Synexin can be purified to virtual homogeneity by a modification of the conventional chromatofocusing technique. Ampholytes are omitted from the procedure altogether and the protein is eluted by a specific pH step chosen on the basis of the pI of the protein. This modification of the conventional method eliminates the separation of ampholytes from the purified protein, an insurmountable problem in our case, and reduces the cost of purification profoundly.

Atrial gland cells synthesize a family of peptides that can induce egg laying in Aplysia.

Endogenous peptides induce egg laying in the marine mollusc Aplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying. Atrial glands were incubated in a mixture of 3H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE. Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparent Mr 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparent Mr 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm. Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule. Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pI's in peak D.(ABSTRACT TRUNCATED AT 400 WORDS)

Restricted diffusion of tyrosine hydroxylase and phenylethanolamine N-methyltransferase from digitonin-permeabilized adrenal chromaffin cells.

Tyrosine hydroxylase [TyrOHase; tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] and phenylethanolamine N-methyltransferase, EC 2.1.1.28) are involved in catecholamine biosynthesis and are considered soluble proteins. However, they may actually be localized on the surface of the chromaffin granule. We have used the detergent digitonin to permeabilize the plasma membrane of cultured adrenal chromaffin cells to investigate the subcellular localization of TyrOHase and PMTase. A digitonin titration of the release of proteins and catecholamines revealed the existence of at least three subcellular compartments that are distinguished by their digitonin sensitivity: (i) soluble proteins, which were released upon treatment of the cells with low digitonin concentrations (5 microM), (ii) a "digitonin-sensitive" cytoplasmic protein pool, which required higher concentrations of digitonin for release (10 microM) and included TyrOHase and PMTase, and (iii) the chromaffin granule, which was insensitive to digitonin. Analysis of the rates of release of all of these proteins revealed that the rate of TyrOHase and PMTase release was slower at 10 microM than at 40 microM digitonin, while the rates of release of the other proteins were similar at both concentrations and varied in proportion to their respective sizes. Treatment with cytoskeletal disrupting agents had no effect on TyrOHase or PMTase efflux. These data suggest that TyrOHase and PMTase are in a detergent-labile association in the cell. This is consistent with the concept that TyrOHase and PMTase may be localized on the surface of the chromaffin granule.