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The effects of nucleotides and nucleosides on steroidogenesis and aerobic lactic-acid production were examined in unsectioned mouse adrenal glands preincubated for 1 h and then incubated for 2 h in Krebs–Ringer bicarbonate and 0·01 m-glucose medium equilibrated with 95% O2:5% CO2. Of all the compounds tested, at a concentration of 10 mmol/l (cyclic AMP, cyclic GMP, AMP, ADP, ATP, GMP, IMP, adenosine, guanosine and inosine), only cyclic AMP was capable of stimulating steroidogenesis and induced a nine- to 12-fold increase in corticosterone production. Cyclic GMP inhibited corticosterone production by 40–55%. The nucleotides and nucleosides, except for ATP, all increased lactic-acid production. Cyclic AMP caused a three- to fivefold stimulation, cyclic GMP an increase of only 20–30%, and GMP, AMP and ADP increases of 80–100%. Cyclic GMP, protected from hydrolysis, may thus inhibit lactic-acid as well as steroid production in the mouse adrenal gland. By contrast, cyclic GMP was nearly as effective as cyclic AMP in stimulating glycolysis and steroidogenesis of rat adrenal glands. The proportion of corticosterone to 18-hydroxydeoxycorticosterone (18-OH-DOC) obtained with cyclic GMP was, however, always lower than that obtained with cyclic AMP. Cyclic AMP, as opposed to cyclic GMP, increased the formation of corticosterone and lactic acid in the presence of exogenous deoxycorticosterone (DOC) beyond that expected from an additive response. Lactic-acid production was inhibited by 18-OH-DOC, a major secretory product of the rat but not the mouse adrenal. This steroid, furthermore, greatly reduced the stimulation of glycolysis evoked by added DOC, 11β-hydroxyprogesterone and corticosterone, facts that could account for the greater glycolytic activity of mouse compared with rat adrenals. The yield of corticosterone in the presence of added 11β-hydroxyprogesterone, but not of DOC, was also reduced by 18-OH-DOC, denoting a selective inhibition of 21-hydroxylation. A structural analogue of 18-OH-DOC, 18,20-cyclo-20,21-dihydroxypregn-4-en-3-one, added by itself stimulated lactic-acid production. Added in combination with other steroids, it specifically counteracted the inhibitory effect of 18-OH-DOC, a steroid of potentially adverse biological properties. Our results are compatible with the concept that adrenal aerobic glycolysis is to a significant extent, but not exclusively, steroid-mediated. The glycolytically active but steroidogenically inert nucleotides and nucleosides offer examples of a dissociation between the two events.

The effects of the dibutyryl derivatives of cyclic GMP and cyclic AMP on lactic acid and steroid production were compared in intact mouse adrenal glands at concentrations of 0·5–1 mmol/l and in mouse adrenal cell suspensions at concentrations of 0·01–1 mmol/l. The dibutyryl derivative of cyclic GMP had little or no effect on lactic acid production in either tissue preparation. It caused a slight stimulation of corticosteroid output in intact glands at a concentration of 1 mmol/l, amounting to one-tenth of the response observed with 1 mm-dibutyryl cyclic AMP. Dose-dependent increases in lactic acid and steroid production were obtained with dibutyryl cyclic AMP in cell suspensions. AMP and GMP increased lactic acid but not steroid production.All the substrates tested (glucose, glucose-6-phosphate, fructose, fructose-6-phosphate, fructose-1,6-diphosphate, 10 mmol/l; pyruvate and glycerol, 20 mmol/l) stimulated basal glycolysis in intact glands and cell suspensions and none affected basal steroid production significantly. By far the greatest increase in lactic acid production was noted with fructose-1,6-diphosphate. However, only glucose and, in unsectioned glands, pyruvate exerted a potentiating effect on the glycolytic response to ACTH. Glucose potentiated the steroidogenic response to ACTH also, but only in intact glands.The relative ineffectiveness of dibutyryl cyclic GMP is in accord with the species-dependent differing responses to the free form of the cyclic nucleotides noted in mouse and rat adrenal glands. The substrate requirements are in keeping with a rate-limiting role of phosphofructokinase and an action of ACTH at some site between the entry of glucose into the cell and the formation of fructose-1,6-diphosphate.J. Endocr.(1985) 104, 105–111

The effects of shortening the ACTH molecule from either end of the peptide chain on adrenal glycolysis and steroidogenesis were examined in mouse adrenal cell suspensions. Shortening the (1–24) sequence to (1–17), (1–16) and (1–14), thereby interfering with the basic tetrapeptide (15–18) assigned to the address message, progressively reduced both glycolytic and steroidogenic potencies by four, six and ten orders of magnitude respectively, without impairing the capacity for maximal excitation. The glycolytic potency of the (1–18) sequence, which was amidated at the C-terminal, equalled that of ACTH(1–24), but the steroidogenic potency was reduced by an order of magnitude. The (1–13) sequence of α-MSH, which contains substitutions at both terminals, had glycolytic and steroidogenic potencies intermediate between those of ACTH(1–16) and ACTH(1–17). Deletion of Ser1,Tyr2from ACTH(1–18)-NH2reduced both potencies by an order of magnitude. ACTH(11–24) and (7–38) were inactive or inhibitory.The capacity for excitation was further examined by comparing responses to peptide fragments (1–4), (1–10), (1–13), (4–10), (4–11), (5–10), (5–14), (7–13) and (11–24) at a concentration of 1 mmol/l. All fragments, excepting (1–4), (5–10) and (11–24) were active. The activities of fragments (5–14) and (7–13), as opposed to (5–10), suggest that the requirements for methionine in position 4 may be replaced by the (11–13) tripeptide. The relative glycolytic responses of fragments containing the (11–13) sequence exceeded the steroidogenic responses, suggesting that the (11– 13) sequence may be specifically implicated in a receptor involved in glycolysis. With this exception, the functional domains within the ACTH molecule responsible for excitation, potentiation and affinity appear to be in similar locations for both evocation of glycolysis and steroidogenesis, judging from the parallel responses to reduction in chain length.J. Endocr.(1987) 115,71–76

The structural requirements in the ACTH molecule for evocation of the glycolytic response in suspensions of mouse adrenal cells were investigated by examining the effects of analogues containing modifications at positions 8,9 and 10 and of peptides containing homologies with the amino-terminal segment of ACTH. Introduction of a nitrophenylsulphenyl (NPS) group into the tryptophan moiety at position 9 of ACTH(1–24) greatly reduced both the potency and the capacity for maximal glycolytic response. It also virtually abolished cyclic AMP formation. In contrast, the capacity for a maximal steroidogenic response remained unimpaired in the NPS derivative, although steroidogenic potency was reduced to 0·4% of that of ACTH(1–24).Replacement of the tryptophan moiety with phenylalanine had intermediate inhibitory effects on glycolysis and steroid output; replacement with alanine virtually abolished both these responses. Replacement of arginine in position 8 with lysine in the Phe9analogue caused a fifty-fold increase in glycolytic potency, but rendered it steroidogenically inactive. Cyclic AMP production was abolished in the Ala9analogue and greatly impaired in the Phe9and Lys8,Phe9analogues. Replacement of the glycine moiety in position 10 with l-alanine, d-alanine, β-alanine or α-aminoisobutyric acid had little or no effect on steroidogenic or glycolytic capacity, although potency was reduced with all substitutions excepting l-alanine.Vasoactive intestinal peptide, which contains homologies with positions 3, 7, 15 and 16 of ACTH, proved completely inactive in dispersed mouse adrenal cells under our experimental conditions, in contrast to human parathyroid hormone(1–34) (hPTH (1–34)) which contains homologies with positions 3, 4, 5, 9 and 11 and was steroidogenic at the lowest concentration tested (0·1 nmol/l), eliciting an eleven-fold increase in steroid production, a response which might be physiologically relevant. It induced near maximal steroidogenesis at a concentration of 10 nmol/l, without affecting cyclic AMP production, and stimulated glycolysis at concentrations above 10 nmol/l, accompanied by a slight rise in cyclic AMP levels.The examples of dissociation between glycolysis and steroidogenesis suggest that different receptors may mediate the two responses. The examples of increased steroidogenesis unaccompanied by a rise in cyclic AMP accord with the concept that cyclic AMP is not an obligatory second messenger for the steroidogenic response.J. Endocr.(1987) 115,61–69

A study was made of the serum peptidase activity of 37 normal control subjects, of 80 subjects suffering from various pathological conditions, of eight normal subjects following administration of ACTH, and of one case of normal pregnancy. The substrate used was l-leucylglycylglycine and in some cases glycylglycylglycine. The highest values for peptide hydrolysis were observed in cases of lymphatic leukemia. Normal, or close to normal, values were found in the serum of patients with aleukemic lymphatic leukemia and with myelo genous leukemia. In a case of Hodgkin's disease and in four out of six cancer patients the peptidase activity was elevated depending on the severity of the disease. Normal activity was observed in a case of multiple myeloma and nonmalignant tumor. In five out of 11 patients suffering from tuberculosis the peptidase activity of the serum was high. In most cases of acute infection the peptidase activity was elevated, in cases of mild respiratory infections the activity was within or close to the normal range. There was no clear-cut rela tionship between the cellular composition of the blood and the serum peptidase level. Injection of ACTH usually had little or no effect on the serum peptidase level of normal subjects. No change in serum peptidase activity was observed in a subject during the course of a normal pregnancy. The implication of these findings is discussed.