Garland A. Johnson

Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine

Modification of the original single isotope radioenzymatic assay of Passon and Peuler (1) permits the direct and simultaneous analysis of norepinephrine, epinephrine and dopamine in plasma samples of 50 μl or less. Plasma or cerebrospinal fluid without prior extraction of catecholamines or deproteinization is added directly into a mixture of 100 μl. This catechol-O-methyl-transferase-catalyzed assay is sensitive to 1 pg (20 pg/ml of plasma) for norepinephrine and epinephrine and 6 pg (120 pg/ml) for dopamine. A rapid thin layer chromatographic separation of the three 3H-methylcatecholamines contributes to the excellent specificity of the differential assay of the three catecholamines. The differential analysis of 15–20 plasma samples can be completed easily within one day. A total assay which omits the chromatographic step and, thus, measures norepinephrine plus epinephrine at the same sensitivity can be completed in 20 samples in one-half a working day.

Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma

Abstract The addition of a commercially available sulfatase to the incubation mixture for the single isotope radioenzymatic assay of the catecholamines permits the simultaneous assay of the catecholamine sulfates in plasma. The sulfatase is compatible with the catechol-0-methyl transferase of the radioenzymatic assay and cleaves the sulfate to form the free catechol. With the multiple enzyme action it is possible to assay total (sulfated plus free) catecholamines and DOPA. A second series of assays performed in the absence of the sulfatase provides results for each of the free catecholamines and DOPA. In a study with ten normotensive male subjects supine free NE was 19% (range of 10–24%) of the total, E was 12% (range 6–16%), DA was 1% (range 0.5–2%) and DOPA was 45% (range 14–71%) of the total. Upon standing, free NE increased an average of 71% from 196±53 pg/ml (supine) to 336±69 pg/ml. Free NE (30%) and free E (18%) comprised a larger proportion of the total plasma NE and E in standing subjects than in the plasma of the supine subjects. Changes in the free levels and in the ratio of free to total levels of DA and DOPA were less than those noted for NE and E. This assay methodology may be useful in demonstrating individual variations in the ability to sulfate endogenous catechols.

Sulfation of minoxidil by liver sulfotransferase

The 100,000 g supernatant fraction of rat liver homogenate contains a sulfotransferase activity which catalyzes the sulfation of minoxidil. Synthetic minoxidil N-O sulfate and the enzyme synthesized product had identical chromatographic characteristics on high pressure liquid chromatography. Minoxidil sulfate, which yields minoxidil when treated with sulfatase, was slowly hydrolyzed in water. Several N-oxides of other heterocycles, including several other pyrimidines, triazines and imidazoles, were also substrates for this sulfotransferase.

Sulfation of minoxidil by human platelet sulfotransferase

In an attempt to determine whether (1) sulfotransferase activity in human platelets would convert minoxidil to minoxidil sulfate and (2) inter-subject variations in this sulfotransferase activity could be noted, platelet homogenates were incubated with minoxidil and 35S-PAPS in HEPES buffer at 37 degrees C for 30 min. Radioactivity which was extracted into ethyl acetate and shown by HPLC to elute with authentic minoxidil sulfate was counted by scintillation counting. Aliquots of the platelet homogenates were also preincubated at 43 degrees C for 15 min to determine the thermal stability of the sulfotransferase activity. Sulfotransferase activity in platelets from 48 adult males ranged from 0.9-13.2 pmol minoxidil sulfate produced/10(7) platelets per 30 min (mean 4.91 +/- 2.84 pmol/10(7) platelets per 30 min +/- SD). Thermal stable sulfotransferase activity ranged from 0.2-7.6 pmol minoxidil produced/10(7) platelets per 30 min and varied from 15 to 57% of the total sulfotransferase activity. Thus, the results indicate that human platelets can effect the sulfation of minoxidil and that sulfotransferase activity does show inter-subject variation.

Minoxidil sulfation in the hair follicle.

The in vivo model which may be the most accurate for the ability to predict hair growth in humans, and which was utilized in the preclinical development of minoxidil, is the adult stumptailed macaque. Previous reports have suggested that the enzyme activity which accounts for the activation of minoxidil, i.e., minoxidil sulfotransferase, is present in skin. We have demonstrated that scalp skin from the stumptailed macaque contains minoxidil sulfotransferase activity, and further with dissection of that scalp skin into epidermis, dermis and hair follicle, most of sulfotransferase activity was present in the follicle. Sulfotransferase activity in the hair follicle in freeze-dried scalp skin sections from 9 stumptailed macaques ranged from 47 to 84% of the total (mean 61 +/- 12%). Much less minoxidil sulfotransferase activity was measured in the epidermis (mean 18 +/- 11%, with a range of 2-37%) and the dermis (mean 21 +/- 8%, with a range of 4-35%) of these scalp sections. These results indicate that the scalp skin from the stumptailed macaque contains minoxidil sulfotransferase activity and this activity is largely localized in the hair follicle which may account for its ability to stimulate hair growth in this animal model.

Plasma levels of catecholamines and dihydroxyphenylglycol during antidepressant drug treatment

Plasma norepinephrine, epinephrine, and 3,4-dihydroxyphenylglycol levels were measured in depressed outpatients treated in a double-blind controlled clinical trial with 150 mg/day of amitriptyline or 60 mg/day of phenelzine for 6 weeks. Both antidepressant drug treatments were associated with a significant decline in plasma dihydrox-yphenylglycol concentrations, which was more pronounced with phenelzine. Plasma norepinephrine levels also declined during phenelzine but not amitriptyline treatment, and the posttreatment values correlated with clinical improvement with the monoamine oxidase inhibiting drug. Reductions in norepinephrine and dihydroxyphenylglycol correlated highly with the degree of platelet monoamine oxidase inhibition. Mechanisms of these antidepressant drug effects on amine metabolism and their implications are discussed.

CYCLIC AMP AND PHOSPHODIESTERASE IN SYNAPTIC VESICLES FROM MOUSE BRAIN

The isolation of synaptic vesicles from mouse brain by a modification of previously reported methodology is described. Homogeneity of the preparations was ascertained by electron microscopy. Vesicles thus isolated contained cyclic AMP (365 ± 44 pmol/mg of protein; mean ± S.E.M.) and adenosine 3′,5′‐monophosphate phosphodiesterase activity (500 pmol/mg of protein per 30 min). The number of vesicles in three of the vesicle preparations was determined by a visual count of grid fields under the electron microscope. The content of cyclic AMP in the vesicles was calculated at 5000‐7500 molecules/vesiclc. The presence of cyclic AMP in the synaptic vesicles from presynaptic nerve endings would support a role for this nucleotide in nerve transmission.

Mechanism of Norepinephrine Depletion by 5-Hydroxytryptophan

Summary The 5-HTP (600 mg/kg, i.p.) reduced rat brain NE to 70% of control levels after 2 hours. The decrease in brain NE after large doses of 5-HTP may be due to inhibition of the biosynthesis of this biogenic amine. In support of this suggestion 5-HTP reduced both incorporation of tyrosine-14C into rat brain NE, and specific activity of the isolated NE. Data are also presented to show that the decrease in brain NE is not due to release such as that produced by reserpine or d-amphetamine.

Radioenzymatic assay of dihydroxyphenylglycol (DOPEG) and dihydroxyphenylethanol (DOPET) in plasma and cerebrospinal fluid

Abstract Modification of the radioenzymatic assay methodology for the assay of catecholamines now permits the simultaneous assay of dihydroxyphenylglycol (DOPEG) and dihydroxyphenylethanol (DOPET) in plasma and cerebrospinal fluid. Conditions of the incubation reaction are essentially those employed for the catecholamine assay, but extraction and isolation of the resulting 3 H-O-methyl derivatives contrasts with those utilized for the cathecholamines. A rapid thin layer chromatographic separation of the 3 H-3-O-methyl derivative lends specificity to the assay which is linear over the range of 30 to 10,000 pg of each substrate. Initial results indicate the presence of DOPEG in plasma and cerebrospinal fluid. DOPET levels in both fluids are usually below the sensitivity of the assay methodology.

Stimulation of cyclic GMP formation in smooth muscle cells by atriopeptin II.

Addition of synthesized atriopeptin II (AP-2), a 23 amino acid peptide of rat atria, to rat thoracic aorta smooth muscle cells results in the stimulation of cyclic GMP production by the cells. The EC50 for the effect is 81 nM and a 7 fold increase occurs at 10 microM AP-2. Cyclic GMP levels increased within 15 seconds after the addition of AP-2 and were maximal at 5 minutes. Cyclic GMP levels in primary rabbit kidney cells were increased 15 fold by 10 microM AP-2. However, no increase in cyclic GMP was detected in WI-38 fibroblast cells after the addition of 10 microM AP-2. Cyclic AMP levels were not affected by AP-2 in any of these cell systems. The effect upon cyclic GMP accumulation was specific for AP-2; none of the other compounds or peptides tested affected cyclic GMP levels.