1.1. Goldfish gill and kidney ATPases are sensitive to ionic strength.2.2. The optimal conditions for goldfish gill Na+ + K+-ATPase activity at 37°C are pH 7.5, 2 mM magnesium, 3 mM ATP, 18 mM potassium, 12 mM sodium.3.3. The optimal conditions for goldfish kidney Na+ + K+-ATPase activity at 37°C are pH 7.0, 2 mM magnesium, 2 mM ATP, 12 mM potassium, and 48 mM sodium.4.4. Under optimal conditions, the Na+ + K+-ATPase activity in the gill is 2.82 μmol Pi/mg protein/hr and in the kidney 15.95 μmol Pi/mg protein/hr. Under these conditions, the Mg2+-ATPase activity is 17.92 μmol Pi/mg protein/hr in the gill and 13.29 μmol Pi/mg protein/hr in the kidney.5.5. The described methodology provides ouabain sensitive active goldfish enzyme without use of detergents or sodium iodide.

In BALB/c female mice with melanoma transplants, the incidence of "takes" is decreased and survival is increased by hydroquinone, a melanocytolytic agent. The mechanism of drug action is suggested by via DNA. The significant and high degree of positive response to hydroquinone treatment in vivo is encouraging for the clinical management of melanoma with melanocytolytic agents.

The acute in vitro actions of two potent melanocytolytic agents, hydroquinone (HQ) and beta-mercaptoethanolamine (MEA), were determined in the B-16, Cloudman S-91 and Harding-Passey (HP) murine melanomas grown in vivo. Drug treated melanoma dice (5--480 min) were analyzed for tyrosinase activity and cyclic nucleotide levels (cAMP, cGMP). HQ and MEA effects on tyrosinase activity are complex and vary with tumor type, duration of treatment and agent tested. MEA or HQ inhibited B-16 tyrosinase activity. With combined drug therapy, low concentrations of MEA plus HQ stimulate B-16 tyrosinase activity while high concentrations of the drugs have little effect on enzymatic activity. MEA depresses tyrosinase activity while HQ elevates enzymatic activity in the S-19 melanoma. Both high and low concentrations of the combined drugs (MEA plus HQ) elicit the same response, stimulation at 10 min followed by continued depression of tyrosinase activity for the remainder of the 4 h study period. MEA initially stimulates HP tyrosinase activity followed by depression of enzymic activity. In contrast, HQ initially depresses HP tyrosinase activity followed by stimulation of enzyme activity. In combination the drugs inhibit HP tyrosinase activity. The effects of MEA and/or HQ on murine melanoma cyclic nucleotide levels are equally complex. MEA or HQ elevate cAMP and cGMP levels in all three tumors with the exception of S-91 cGMP levels which are not altered. In combination the drugs increase cyclic nucleotide levels in each of the three tumor types but at different times. No correlation is present between cyclic nucleotide levels and tyrosinase activity. Thus, the action of increased cyclic nucleotide levels in melanogenesis can not be separated from the direct actions of MEA and HQ upon melanogenesis. The divergent effects of MEA and/or HQ on tyrosinase activity and cyclic nucleotide levels in these melanomas are not correlated with the known in vivo melanocytolytic activity of these drugs. Thus, these parameters appear to be inadequate indicators of melanoma cell viability in chemotherapeutic screening of drugs effective in destroying malignant melanoma.

A technique is described for the subcutaneous injection of small fish using the goldfish (Carassius auratus L.) as a model. Using radioactive tracers, (109Cd, 125I), maximum levels occur in the plasma at 4 and 16 min, with statistical plateaus evident by 2 and 4 min, respectively. The plateau level of injected material occurring in the plasma shortly after the injection exposes the tissues to a constant dose of the injected material for the duration of the plateau. In the case of 109Cd, this period is in excess of 4 h. Subcutaneous injection by this method provides low variability in delivery, rapid plasma – delivery site equilibration of the injected material, and a constant plasma level of the injected material for a significant time interval.

The acute in vitro action of ACTH and corticosterone individually and in combination were determined in the B-16 melanoma grown in vivo. ACTH elevated levels 10-fold while tyrosinase activity generally was depressed. Corticosterone depressed cAMP levels and tyrosinase activity. ACTH in the presence of corticosterone produced a coincident peak in tyrosinase activity and cAMP levels followed by a depression of enzymatic activity. The results demonstrate that cAMP is not the sole modulator of tyrosinase activity and that ACTH, corticosterone and cAMP interact in the regulation of B-16 melanoma tyrosinase activity.

Compared to normal humans, lung carcinoma patients show increased tyrosinase activity. 7 serum enzymic fractions or carriers were present in the diseased state. Further, serum tyrosinase inhibitory factors generally were decreased in lung carcinoma patients compared to normal individuals.

The activity of dominant human melanoma tyrosinase isozyme was greatly decreased by certain reducing agents. The number and geometry of phenolic groups as well as free-SH group appear important for enzymic inhibition.

The in vitro effects of theophylline and aminogluthetimide upon basal and ACTH stimulated cAMP, cortisol and aldosterone responses of normal human adrenocortical tissue were evaluated. Theophylline increased basal cAMP levels and cortisol output, however, basal aldosterone output was depressed. Theophylline in concert with ACTH depressed cortisol and aldosterone output. Aminogluthetimide alone did not affect basal cAMP levels, however, the normal cAMP response to ACTH was delayed in aminogluthetimide pre-treated adrenals. Aminogluthetimide also depressed basal and ACTH stimulated cortisol and aldosterone output with the latter being more sensitive. The findings indicate that both theophylline and aminogluthetimide produce effects upon the adrenal in addition to inhibition of phosphodiesterase and cholesterol side-chain cleavage, respectively. Further, theophylline depression of ACTH stimulated steroid output may be helpful in understanding the interplay between a number of factors in the control of adrenal steroid biosynthesis and release.

1. The direct actions of cAMP and 5′-AMP upon goldfish integumental and mushroom tyrosine activity were examined.

The evolutionary origin of the ACTH-cyclic nucleotide-adrenocorticoid relationship was investigated in vitro. The adrenocortical production of cAMP and cGMP as well as corticosterone output in response to ACTH were examined in the blue (Prionace glauca) and the mako (Isurus oxyrinchus) sharks. Basal (control) cAMP levels remained relatively constant in both species, but were higher in the blue shark adrenal. Initially undetectable basal cGMP levels were increased with incubation time. A basal level of corticosterone output was present in both species. Porcine ACTH significantly increased shark adrenocortical cAMP and cGMP levels. Increased cGMP levels preceded cAMP elevation. Elevation of both cyclic nucleotides preceded increased steroid output. The mako shark adrenal corticosterone response to a given ACTH dose was 100% greater than that of the blue shark. The steroid response was inversely related to the total amount of cAMP generated and the temporal duration of the cAMP response. Both cyclic nucleotides may participate in the events preceding and ultimately determining the duration of the steroid response. The results indicate that the ACTH-cyclic nucleotide-steroid output relationship is established early in the evolution of adrenocortical tissue.

ACTH and corticosterone altered tyrosinase activity in the B-16, S-91 and Harding-Passey melanomas but did not evoke any changes in peroxidase activity. Tyrosinase activity, but not peroxidase activity, was correlated with the degree of melanin pigmentation present in the melanomas. Thus, tyrosinase is the enzyme of physiological importance in the regulation of melanin pigmentation.

The in vitro modulating effects of the E and F series prostaglandins upon the cAMP and cortisol output of normal human adrenal dice were evaluated with time and compared to the effects of ACTH. PGE1 and PGE2 significantly increased human adrenal cAMP levels; cortisol output increased in a dose related manner. Although the cortisol levels produced by E prostaglandins and ACTH were quantitatively similar, on a molar basis ACTH was greater than 106 fold more effective. PGE1α, PGF2α, PGF1β and PGF2β depressed adrenal cAMP, except PGF2α and PGF2β at 100 μg/ml. PGF1α and PGF2α depressed cortisol levels at all doses. Similarly, PGF1β and PGF2β also depressed cortisol output, except PGF2β at 100 μg/ml which was stimulatory. In both series of prostaglandins the temporal responses were dose related in regard to the cyclic nucleotide and steroid alterations. The findings demonstrate the E and F series prostaglandins act antagonistically in respect to cAMP and cortisol output. In addition, as the cAMP level and cortisol output are not always correlated, it appears that these prostaglandin mediated events are separable. The relationship between adrenal prostaglandins and cyclic nucleotides, therefore, invites a more sophisticated second messenger concept in terms of adrenocortical function, than currently proposed.

The mechanism of ACTH action in the adrenal of the frog (Rana berlandieri forreri) was evaluated in vitro. Basal cAMP levels remained constant, but the total output of corticosterone and aldosterone increased with time. The rate of basal steroid output, however, decreased with time. ACTH, frog pituitary homogenate, and theophylline increased cAMP levels rapidly and temporally preceding increased outputs of corticosterone and aldosterone. The responses were rapid and dose-dependent. The role of cAMP in the frog adrenal steroid output is similar to that described in mammals and contrasts with that present in the crocodile. The mechanism of ACTH action in the frog adrenal involves cAMP in the stimulation of corticosterone and aldosterone output.

The acute in vitro action of adrenocorticotropin (ACTH) and corticosterone alone and in combination were determined in the Cloudman S-91 melanoma grown in vivo. Hormone-treated melanoma dice (5-240 min) were analyzed for tyrosinase activity (EC 1.14.18.1), cyclic AMP (cAMP) and cyclic GMP (cGMP). ACTH elevated cAMP levels in the S-91 melanoma. However, these increases in cAMP were not accompanied by increased tyrosinase activity. Corticosterone depressed cAMP levels while stimulating tyrosinase activity. ACTH plus corticosterone produced an early cAMP peak followed by depression. ACTH plus corticosterone stimulated tyrosine activity coincident with the early cAMP peak followed by a drop in tyrosinase activity which was subsequently elevated. cGMP levels were not altered by any hormone treatment. The results indicate that cAMP is not the sole modulator of tyrosinase activity and suggest the interaction of ACTH, corticosterone and cAMP in the regulation of melanoma tyrosinase activity.

The acute in vitro action of adrenocorticotropin (ACTH) and corticosterone alone and in combination were determined in the Harding-Passey (HP) melanoma grown in vivo. Hormone treated melanoma dice (5–240 min) were analyzed for tyrosinase activity, cyclic AMP (cAMP) and cyclic GMP (cGMP). ACTH elevated cAMP and cGMP levels 20-and 13-fold, respectively, in the HP melanoma. However, these large increases in cyclic nucleotide levels were accompanied by only a 49% increase in tyrosinase activity. Corticosterone elicited a similar response. ACTH plus corticosterone produced an early cAMP and cGMP peak followed by depression. ACTH plus corticosterone stimulated tyrosinase activity coincident with the early cyclic nucleotide peak followed by a drop in tyrosinase activity which was subsequently elevated. The results indicate that neither cAMP nor cGMP are the sole modulators of tyrosinase activity and suggest the interaction of ACTH, corticosterone and cyclic nucleotides in the regulation of melanoma tyrosinase activity.

1.1. A tyrosinase-positive form of oculocutaneous albinism in the goldfish is described.2.2. Both tyrosinase and peroxidase activity are present in the integuments of xanthic and albino goldfish.3.3. The lack of integumental melanin pigmentation in both xanthic and albino goldfish is due, in part, to the presence of tyrosinase inhibitors.4.4. The tyrosinase inhibitors present in the integument of xanthic and albino goldfish have different properties.5.5. Triton X-100 treatment solubilizes xanthic and albino goldfish integumental tyrosinase.

Indomethacin or 7-oxa-13-prostynoic acid does not alter basal adrenal cortisol output. Preincubation in either, depresses subsequent ACTH-stimulated cortisol output below ACTH alone and controls. Either inhibitor following ACTH preincubation blunts normal ACTH-stimulated cortisol production.