ACTH stimulation of purine nucleotide biosynthesis in the adrenal cortex. ACTH stimulation of purine biosynthesis

In the presence of azaserine an inhibitor of phosphoribosylformylglycineamidine synthetase (EC 6.3.5.3) the incorporation of [2-¹⁴C]glycine into 5′-phosphoribosylglycineamide and its formyl derivative was measured in 105,000g supernatant fraction prepared from a homogenate of adrenal cortex. Corticotropin at a level of 1-0.001 nm markedly stimulated in 10 min these early steps of purine biosynthesis. The stimulus was in addition to that achieved with added glucose-6-phosphate and NADP. Increased synthesis of precursors of purine nucleotides is due to ACTH activation of adrenal glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and thus the pentose cycle with an increase in 5′-phosphoribosylpyrophosphate. The generation of this latter compound is presumed to be a rate-limiting factor to 5′-phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) the first enzyme of de novo purine biosynthesis.     

Interaction of ACTH synthetic fragments with rat adrenal cortex membranes.

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.     

3-D structure of mitochondrial cristae in rat adrenal cortex varies after acute stimulation with ACTH and CRH

We attempted to determine whether acute treatment with adrenocorticotropin hormone (ACTH) and corticotropin releasing hormone (CRH) affects mitochondrial morphology, as evaluated by the HRSEM and osmium maceration methods. We quantified CRH and ACTH effects on HRSEM images in rat glomerulosa and fasciculata. After ACTH or CRH treatment, mitochondrial cristae increased the number of globular expansions, whereas mitochondrial volume decreased in glomerulosa. As the morphological variations reported may be linked to increased hormonal production, further studies using parallel measurements of circulating and tissue hormones are now in progress, and may aid in clarifying their functional significance.     

ACTH stimulation of adrenal epinephrine and norepinephrine release

Epinephrine (E) and norepinephrine (NE) levels were measured simultaneously in the adrenal veins of 6 patients before and after stimulation with 0.25 mg beta 1-24 ACTH. In 1 patient with Cushing's syndrome, E and NE were also measured before and 30 min after dexamethasone. There was a significant increase in NE and E secretion (p less than 0.002) from both adrenal glands after ACTH stimulation. In the patient with Cushing's syndrome, there was also a slight increase in plasma E levels after dexamethasone. It is postulated that ACTH stimulated NE and E secretion by augmenting blood flow through the adrenals and by induction of tyrosine hydroxylase and dopamine beta-hydroxylase, although a direct effect of ACTH on NE and E secretion cannot be excluded. It is also possible that the increase in adrenal catecholamine secretion after ACTH may be due to ACTH augmentation of catecholamine secretion by endogenous opioids such as beta-endorphin.     

Inhibitory effects of a pineal extract on adrenal cortex. Lack of competition with ACTH

The purpose of the present investigation was to study the mode of action of a crude aqueous pineal extract (CAPE) on corticosterone (B) production from ACTH-mediated isolated adrenal cortex cells. Corticosterone production from a heterogenous adrenal cortex cell population, isolated from 8 male Sprague-Dawley rats, was measured fluorimetrically. CAPE (25 microliters) was tested in this system using ACTH (0--5,000 pg/ml) and dibutyryl-c-AMP (0--100 nM/ml) as stimuli for a period of 1 h. In a separate experiment, CAPE (25 microliters) was administered to ACTH (50 pg/ml) stimulated adrenal cortex cells for 15, 30, 60, and 120 min incubation periods. CAPE significantly decreased B produced by adrenal cortex cells at all doses of ACTH administered. CAPE also decreased the B produced by adrenal cortex cells when dibutyryl-c-AMP was used as a stimulus. The inhibitory effect of CAPE was manifest at some point in time between 30 and 60 min. It was significant at 60 min and highly significant at 120 min. It is evident from these data that CAPE and ACTH are not competing for the same receptor site.     

Affinity labeling of ACTH receptors in bovine adrenal cortex membranes

Adrenocorticotropin labeled with 125I at Tyr23 [(125I-Tyr23]ACTH) was prepared by radioiodination of ACTH1-39 followed by reverse phase HPLC purification. When incubated with bovine adrenal cortical membranes, the radioligand bound specifically to a 40-kDa membrane protein as revealed by affinity labeling. This result indicates that the bovine adrenal ACTH receptor, whose identification and characterization have proved difficult, has an Mr of about 40,000.