Adrenal Cell Suspensions Research Articles

Histochemical targeting: Combining plasma immersion ion implantation and histochemical probes to target magnetic particles to intracellular cytological features

Paramagnetic microparticles are effective tools for cell purification, diagnostics, imaging and drug delivery and have been used for over 30 years to separate cell types from complex mixtures. The targeting of magnetic particles to cells and tissues is dependent upon the use of antibodies and adhesion molecules as targeting modalities. Here, we present a novel paradigm for the targeting of microparticles to cells according to intracellular cytological characteristics. Cell-permeable histochemical dyes, when covalently conjugated to magnetic microparticles, following plasma immersion ion implantation (PIII) imbue the particles with cell targeting properties in a process we term histochemical targeting. PIII treatment and conjugation to the lipophilic dyes Nile Red or Nile Blue led to changes in microparticle size, surface charge and lipophilicity. PIII-treated microparticles covalently conjugated with Nile Red, crossed the live cell membrane and homed to intracellular neutral lipid droplets. Nile Blue-conjugated particles crossed live cell membranes and remained in the cytosol where free fatty acids localize. Magnetic cell sorting could be performed on cultured adipocytes, as well as rat liver and adrenal cell suspensions using particles conjugated to Nile Red or Nile Blue. Taken together, our findings demonstrate that histochemical probes can be covalently conjugated to magnetic particles following PIII treatment resulting in intracellular targeting. With a vast armamentarium of cell-permeable probes available, histochemical targeting could be applied to many fields requiring magnetic particle delivery to specific sites or isolation of particular cell types.

Функциональная активность адренокортикоцитов крыс после инкубации в криозащитных средах на основе ДМСО, содержащих эмбриональную телячью сыворотку

Investigation of functional state of cells at various stages of cryopreservation is important for the development and improvement of freezing protocols of endocrine cells. This study assessed the effect of incubation in media with 5, 7, 10, and 15% dimethyl sulfoxide (DMSO), as well as the media with DMSO and 25% fetal bovine serum (FBS) on some indices of adrenal cortex cell suspension: viability (MTT-test), content of cells with active 3b-hydroxysteroid dehydrogenase, functionally active mitochondria (JC‑1 test), as well as the level of basal and stimulated aldosterone secretion. It was shown that presence of FBS in a cryoprotective medium reduced the toxic effect of DMSO on adrenocorticocytes during incubation, furthermore in some cases incubation in media with DMSO and FBS led to the increase in the number of cells with functionally active mitochondria and cells with active steroidogenesis if compared with the suspensions with cells not exposed to cryoprotectant solution. Probl Cryobiol Cryomed 2014; 24(1):47–56.

Localization, characterization, and second messenger coupling of pituitary adenylate cyclase-activating polypeptide receptors in the fetal human adrenal gland during the second trimester of gestation.

The distribution and pharmacological properties of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation. Autoradiographic studies, using [125I]PACAP27 as a radioligand, revealed that PACAP-binding sites are exclusively located on chromaffin cells of adrenals from fetuses 14-20 weeks old. Biochemical characterization of binding revealed the occurrence of a single class of PACAP-binding sites with a dissociation constant value of 0.32-0.74 nmol/L and a binding capacity of 0.30-0.81 pmol/mg wet tissue. PACAP27 and PACAP38 were equipotent in competing for [125I]PACAP27 binding (IC50 = 0.28-0.64 nmol/L and 0.15-0.81 nmol/L, respectively), and the Hill coefficients were close to 1. In contrast, vasoactive intestinal polypeptide was much less efficient in displacing the tracer (IC50 = 4-362 nmol/L), and the Hill coefficients were less than 0.6. PACAP38 induced a dose-dependent increase in cAMP production in fetal human adrenal cell suspension (ED50 = 0.07 +/- 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 +/- 1.8 nmol/L). In contrast, PACAP38 induced a modest increase in inositol phosphate formation. These data indicate that type I PACAP receptors are present in the early stages of the human medulla organization during the process of migration of chromaffin cells from the periphery to the central part of the gland. The present results suggest that PACAP could be involved in the regulation of the human adrenochromaffin cells during ontogenesis.

Xenogeneic adrenal medulla graft rejection rather than survival leads to increased rat striatal tyrosine hydroxylase immunoreactivity.

Adrenal medulla has often been used as a donor tissue for transplantation into damaged central nervous system, with functional effects ranging from very good to nonexistent. The grafts have often been associated with morphological evidence of stimulated recipient dopaminergic fiber plasticity. The interpretation of these results has been difficult due to variable but mostly poor graft survival. The present study combines two experiments which evaluated the effects of intrastriatal xenogeneic adrenal medullary cell suspension grafts on rat recipients. First, bovine adrenal medulla cell suspension grafts of various compositions were tested for their functional and morphologic effects on immunosuppressed hemiparkinsonian rats. In the second experiment, graft rejection was allowed to occur in half of the rats in order to determine a possible contribution of the inflammatory/immune response to increased dopaminergic fiber plasticity of the recipient. At 28 days, grafts of all cell types survived well in immunosuppressed rats, but none of the grafted cell types was associated with either an amelioration of amphetamine-induced rotation or an increase in striatal tyrosine hydroxylase immunoreactivity around the graft site. The latter phenomenon was observed only in the nonimmunosuppressed rats with rejected grafts. Our findings strongly support the role of inflammatory/immune response to grafting in stimulating dopaminergic fiber plasticity and in the appearance of sprouting.

Characterization of the adrenal cytochrome P450C17 in the hamster, a small animal model for the study of adrenal dehydroepiandrosterone biosynthesis.

The hamster, like the human produces cortisol as its major glucocorticoid, rather than corticosterone, typical of most enzyme rodents. It is not known, however, if the hamster cytochrome P450C17 (P450C17), a key enzyme for cortisol formation, also exhibits 17,20-lyase activity and if it catalyzes the formation of dehydroepiandrosterone (DHEA) at the adrenal level. To study this, we isolated the cDNA of P450C17 from a hamster adrenal library. This cDNA was sequenced and was found to have an open reading frame for a protein of 511 amino acids, as compared to the human P450C17, which contains 508 amino acids. The hamster P450C17 cDNA, in the coding region, is 76% homologous with the human P450C17 cDNA. The cDNA was then cloned in the expression vector pSV-SPORT 1, which was transiently transfected into COS 1 cells. The transfected cells were used for temporal studies on the transformation of radiolabeled C21-delta5- and C21-delta4-precursors. When transfected cells were incubated with [14C]pregnenolone, rapid formation of [14C]DHEA occurred. The intermediate 17alpha-hydroxypregnenolone accumulated initially with subsequent metabolism to DHEA. Likewise, when incubated with C21-delta4-steroids, [14C]progesterone and [3H]17alpha-hydroxyprogesterone, the 17,20-lyase product androstenedione was produced efficiently. In these studies, with respect to the delta5 pathway, the expressed hamster P450C17 gave similar results to bovine P450C17 cDNA inserted in the same expression vector. However, in contrast to the bovine enzyme, which converted low amounts of progesterone to androstenedione, the expressed hamster P450C17 enzyme showed an active metabolism via the delta4 pathway. Northern blot analysis, using the complete alpha-32P labeled hamster P450C17 cDNA as the probe, demonstrated a strong presence of P450C17 mRNA in hamster adrenals, a weaker presence in testes and ovaries, and no detectable species in brain, mesentery, and kidney. Immunoblotting analysis using an anti-rat P450C17 antibody demonstrated the presence of P450C17 protein in hamster adrenals, testes, and ovaries. Hamster adrenal cell suspensions and microsomal preparations were used to demonstrate the biosynthesis of [14C]17alpha-hydroxypregnenolone and [14C]DHEA from [14C]pregnenolone; both metabolites were formed during incubations. However, the ratio of [14C]DHEA/[14C]17alpha-hydroxypregnenolone was much lower in adrenal cells than in transfected COS 1 cells, indicating the presence of putative factors in hamster adrenal cells, favoring the 17alpha-hydroxylase activity rather than that of the 17,20-lyase. In conclusion, these studies demonstrate that the hamster adrenal is both a DHEA and a cortisol producer, and, therefore, this animal could be a suitable small animal model for the study of the role of DHEA in relation to human biochemistry and physiology.

Hormonal status and the neuroendocrine response to a novel heterotypic stressor involving subchronic noise exposure.

Despite a number of studies on noise-induced health effects, it is still unclear to what extent different neuroendocrine pathways are affected by noise exposure. Male Wistar rats were housed in sound-attenuated rooms isolated for noise from outside. Three groups of chronically cannulated rats were exposed to either background noise (+/-64 dB) only or irregular experimental white noise (90 dB, 2-22 kHz). Two protocols, with approximately the same total amount of noise but with different densities, were used: protocol N1 (180 min random noise per day for 18 days) or protocol N2 (540 min random noise per day for 8 days). Basal levels of circulating hormones (ACTH, corticosterone, prolactin and catecholamines) and plasma glucose were measured. In control animals, no significant changes in any of these parameters were observed over 18 days. Except for plasma prolactin, N1 did not induce a significant elevation in basal hormonal levels. N2 however induced significant elevation in basal prolactin, corticosterone and noradrenaline levels. At the end of the exposure period, all animals were subjected to a novel heterotypic stressor (restraint stress) to monitor differences in neuroendocrine activation (ACTH, corticosterone and prolactin). Compared to nonexposed control animals, N1 animals showed a normal ACTH and an enhanced corticosterone response, whereas N2 animals showed an increased ACTH but a normal corticosterone response. The prolactin response of both N1 and N2 animals was significantly decreased. Adrenal cell suspension experiments revealed that in noise-exposed rats both basal- and ACTH-stimulated corticosterone production were significantly increased as compared to control animals. These results indicate that chronic noise exposure at mild intensities induces subtle but significant changes in hormonal regulation.

A comparison of the responses of dispersed steroidogenic cells derived from embryonic adrenal tissue from the domestic chicken (Gallus domesticus), the domestic Pekin duck and the wild mallard duck (Anas platyrhynchos), and the domestic muscovy duck (Cairina moschata)

The steroidogenic responsiveness of adrenal cell suspensions prepared from domestic chicken adrenal tissue at the end of embryogenesis was compared to the responses of similar preparations derived from the wild and domesticated mallard duck (Anas platyrhynchos), and the domesticated muscovy duck (Cairina moschata). In all cases, the masses of corticosterone (B), aldosterone (Aldo), and deoxycorticosterone (DOC) released from cells incubated in medium containing 1–24 ACTH exceeded the estimated hormone content of the freshly dispersed cells; the induced rates of corticosteroid release were, therefore, presumed to reflect de novo hormone synthesis. When chicken cells were incubated in medium containing 1–24 ACTH, there were progressive, dose-dependent increases in B and DOC synthesis over a range of concentrations spanning two orders of magnitude; only small, nondose-related, albeit significant, increases in Aldo release were observed. The 1–24 ACTH-induced increases in B and Aldo synthesis by the mallard and Pekin duck cells exposed to the same range of concentrations were up to 40 and 60 times greater than the corresponding responses of the chicken cells. The rates of 1–24 ACTH-induced B and DOC release from muscovy duck cells were similar to those from the Pekin duck cells; compared with the mallard duck cells, however, the muscovy duck cells were less sensitive and the maximum inducible rate of B release was significantly lower. The pattern of 1–24 ACTH-induced Aldo release from the muscovy duck cells was indistinguishable from that of the mallard duck cells, although the maximum inducible increase occurred at a lower concentration. Angiotensin II (AII) induced very small, but significant increased in B, Aldo, and DOC release from the chicken cells but in a nondose-related fashion. In contrast, mallard, Pekin, and muscovy duck cells all responded in a dose-dependent manner when incubated in medium containing AII. In each instance the maximum rate of Aldo synthesis induced by AII was about one-tenth of the corresponding rate induced by 1–24 ACTH. The maximum rates of B synthesis induced by AII, however, were extremely low compared with the rates induced by 1–24 ACTH. Thus, when maximally stimulated with All the B:Aldo output ratio ranged from 1.0 to 1.9, whereas the corresponding ratio was between 9.0 and 17.0 when the cells were maximally stimulated with 1–24 ACTH.

The differential regulation of aldosterone output in hamster adrenal by angiotensin II and adrenocorticotropin

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K + concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K +. Angiotensin II (A II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10 −8 M A II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10 −8 M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A II and ACTH on adrenal cytochrome P-450 11 β involved in the last steps of aldosterone formation were evaluated by c combined in vivo and in vitro experiments. The P-450 11 β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A II differentially regulate P-450 11 β . It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.

Isolation and mode of action of rabbit corticostatic (antiadrenocorticotropin) peptides.

Corticostatic peptides are a family of arginine-rich cysteine-rich peptides that inhibit ACTH-stimulated corticosterone (B) production in rat adrenal cell suspensions. In this communication we describe a new method for the facile isolation and purification of these basic peptides from rabbit adult lung. We then describe the isolation and sequences of the four rabbit peptides, CSI, CSII, CSIII, and CSIV, and compare their biological activities in the ACTH (150 pg/ml) inhibition assay. CSI is by far the most potent of the four peptides. Using CSI as a model, we then studied its effects on the proximal and distal parts of the pathway leading to the generation of cAMP. CSI had no effect on (Bu)2cAMP action on forskolin or cholera toxin in their ability to mimic ACTH and increase B production in rat adrenal cells, nor did CSI have any effect on the stimulation of B production by pertussis toxin. Endogenous cAMP stimulated by ACTH decreased after the addition of CSI, which pointed to the inhibition of ACTH binding to explain the mode of action of this corticostatin. Displacement of the specific binding of labeled ACTH by CSI and the ACTH antagonist ACTH-(6-24) was determined, and indeed, CSI did displace ACTH from its binding site. The question of what portion of the ACTH molecule was involved in the action of CSI was answered by studying ACTH-(1-13) acetyl amide (alpha MSH) and ACTH-(1-18) amide. CSI had no effect on alpha MSH stimulation of B production, but did lower the production of B stimulated by ACTH-(1-18) amide. Therefore, CSI must act on ACTH-(14-18), which is part of the so-called address region of ACTH, which is -Gly14-Lys15-Lys16-Arg17-Arg18-, the very basic part of the molecule. These results indicate that CSI acts by competing with ACTH for its binding receptor on the adrenal cell and that this competition is confined to amino acids 14-18 of the molecule when it is bound to the receptor.

Atrial Natriuretic Peptide Inhibition of Calcium Ionophore A23187-Stimulated Aldosterone Secretion in Rat Adrenal Glomerulosa Cells*

The effect of atrial natriuretic peptide (ANP) on calcium ionophore A23187-stimulated aldosterone secretion was investigated using collagenase-dispersed rat adrenal glomerulosa cell suspensions. A23187 treatment induced a dose-dependent stimulation of aldosterone secretion, exhibiting an EC50 of approximately 75 nM. In agreement with the presumed action of A23187 as a Ca2+ ionophore, stimulation was dependent on the extracellular Ca2+ concentration, being completely inhibited in nominally Ca(2+)-free medium. In such Ca(2+)-free medium, stimulation of aldosterone secretion by bath applied 25-hydroxycholesterol was not inhibited, indicating that cells and biosynthetic pathway enzymes were not inhibited by low extracellular Ca2+ levels. A23187-induced aldosterone secretion was also inhibited by more than 90% when cells were simultaneously treated with ANP. Maximal ANP inhibition of A23187-stimulated aldosterone secretion was not overcome by concentrations of A23187 up to 10 microM or by increasing the extracellular Ca2+ concentration from 1.25 to 5 mM in the presence of A23187 and ANP. Addition of A23187 to ACTH-, angiotensin II-, or K(+)-stimulated glomerulosa cells did not overcome ANP-induced inhibition of aldosterone secretion stimulated by these secretagogues. In contrast to ANP inhibition of Ca(2+)-dependent A23187 stimulation of aldosterone secretion, ANP inhibition of dBcAMP-stimulated aldosterone secretion was readily overcome by increasing the dBcAMP concentration. These results indicated that ANP selectively and noncompetitively inhibited an intracellular step necessary for Ca(2+)-dependent stimulation of the early pathway of aldosterone biosynthesis in rat adrenal glomerulosa cells.

Effect of chronic administration of nicotine or cocaine on steroidogenesis in rat adrenocortical cells

Rats were implanted subcutaneously with osmotic mini-pumps containing either 0.9% NaCl, nicotine (1.5 or 4.5 mg kg-1 day-1), or cocaine (30 mg kg-1 day-1), for 14 days. Neither nicotine nor cocaine treatment significantly altered the maximal rate of steroidogenesis in adrenocortical cell preparations from the animals. However, pretreatment with cocaine increased the sensitivity of the preparation to stimulation by ACTH, the ED50 was 5 pM compared with 10 pM from control animals. Addition of nicotine or cocaine at concentrations up to 100 microM to adrenal cell suspensions from naive rats did not stimulate steroidogenesis or increase the sensitivity of cells to ACTH stimulation. These results suggest that the primary chronic effect of nicotine on steroidogenesis is exerted at the level of the hypothalamus and/or pituitary and not directly on adrenocortical cells. On the contrary, pretreatment with cocaine causes persistent changes in adrenocortical cells.

Isolation and Biological Activity of Corticostatic Peptides (Anti-Acth)

Corticostatins (CS's) are a family of low molecular weight peptides, rich in arginine and cysteine with the ability to inhibit ACTH stimulated adrenocortical steroidogenesis. They show a high degree of specificity in that they do not inhibit the action of angiotensin II in the adrenal cortex. Four corticostatins have been isolated from rabbit lung extracts and peritoneal neutrophil extracts and one from human neutrophils. Among them corticostatin I, CSI, is the most potent with an I.D.50 of 25 nM. Corticostatin activity is different from other published inhibitory factors such as TGF-B and ANF, which inhibit basal and angiotensin II stimulated steroidogenesis. Other factors such as macrophage secreted products, and septic shock plasma factors which have not been fully characterized also have suppressive activity on ACTH induced adrenocortical steroidogenesis in vitro. It is not yet clear whether there is any relationship between corticostatins and these macrophage products and shock plasma factor. Corticostatins do not inhibit dbcAMP induced steroidogenesis, however they do inhibit the accumulation of cAMP in response to ACTH in rat adrenal cell suspensions. Binding studies show that CSI is a competitive inhibitor of ACTH, probably acting by blocking, the address recognition site of the receptor. There is wide variation in the potency of the corticostatins ranging from an ID50 of 100 ng/ml for CSI to a completely inactive analog, HP1. In this paper we will describe the purification of the corticostatins and some recent results obtained on their mechanism of action.

Structure of a novel human granulocyte peptide with anti-ACTH activity

We report the purification, structure and biological properties of a peptide of novel sequence from human granulocytes that inhibits ACTH stimulated synthesis of corticosterone in rat adrenal cell suspensions. The peptide HP-4 is homologous to a previously described human granulocyte peptide HP-1 that has no anti-ACTH activity.

Adrenal medullary cells transmute into dopaminergic neurons in dopamine-depleted rat caudate and ameliorate motor disturbances

Adrenal medullary cell suspensions, derived from newborn rats (postnatal day 1–6), were implanted into the head of the caudate nucleus in 35 rats with unilateral 6-hydroxydopamine (6-OHDA) lesions in the nigrostriatal dopamine (DA) pathway. Behavioral recovery from Met-amphetamine induced circling, cell growth morphological features (tyrosine hydroxylase positive cells), and release of adrenaline (Ad), noradrenaline (NA), DA, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were investigated for 40 weeks after transplantation. Met-amphetamine induced circling decreased significantly in 43% (15/35) of the rats. The decrease was concurrent with transmutation of the tyrosine hydroxylase-like immunopositive (THLI) cells into mature neurons that had abundant elongated neurites with varicosities and synapses on neuronal elements in the host caudate. In the absence of behavioral recovery (57%, 20/35) THLI cells were very scant. DA, DOPAC and HVA were reduced more than 90% in perfusates collected by in vivo dialysis from the striata of the animals that were not improved by transplant. These levels recovered to 20–50% of controls in animals whose behavior recovered. Ad and NA were not detected in the perfusates of either recovered or non-recovered animals. The results suggest that some grafted adrenal medullary cells transform into dopaminergic neurons and the release of DA from these grafted cells functionally affects behavior improvement for at least 40 weeks.

ACTH and adrenal aerobic glycolysis. II: Effects of aminoterminal peptide fragments on lactic acid and steroid production by mouse adrenocortical cells.

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 alpha-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,Tyr2 from ACTH(1-18)-NH2 reduced 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of catecholamine on aldosterone release in isolated rat glomerulosa cell suspensions

Effect of adrenergic activity on the adrenal steroido-genesis and the modulation by catecholamines of aldosterone release were studied in isolated rat adrenal cell suspensions. Isoproterenol, norepinephrine and epinephrine, but not dopamine, caused statistically significant increase in aldosterone release. Both prazosin (α1 antagonist) and yohimbine (α2 antagonist) suppressed the norepinephrine-induced aldosterone release in a dose dependent manner, respectively. Both atenolol (β1 antagonist) and ICI 118–551 (β2 antagonist) also blocked (−)-isoproterenol-induced aldosterone release in a dose dependent manner, respectively. Neither (−)-isoproterenol nor (±)-norepinephrine at concentrations of 10 −6 M potentiated aldosterone release stimulated by angiotensin II or ACTH. These results suggest that catecholamines stimulate aldosteroidogenesis, but it appears unlikely that aldosterone release induced by ACTH or angiotensin-II is modulated by adrenergic stimulation.

The corticostatic (anti-ACTH) and cytotoxic activity of peptides isolated from fetal, adult and tumor-bearing lung

The possibility that corticosteroids influence lung maturation was suggested in 1968 by Buckingham et al. [1] and supported by studies mounted in humans and in rabbits by Liggins and Howie [2, 3] and by a collaborative study from the N.I.H. [4]. To our knowledge no reverse process, i.e. the regulation of adrenal function by the lung, has been postulated. In attempting to isolate ACTH from fetal lung we found peptides which depressed corticosterone production. Eventually, we isolated a group of peptides from rabbit fetal lung which showed potent inhibiton of corticosterone production in rat adrenal cell suspensions. Using high-resolution reverse-phase HPLC and high-sensitivity gas-phase sequencing techniques the primary structure of one of these peptides has been determined and it was called corticostatin. The adult rabbit lung contains a large quantity of a very similar peptide, which elutes close to corticostatin but whose amino acid composition differs by a single glycine and possibly a serine. This peptide is also potently corticostatic. We have isolated homologous human peptides but these show no apparent corticostatic activity in rat adrenal cell suspensions. They do, however, show interesting effects on in vitro growth of lung cell lines.

Chlorbutol, a new inhibitor of aldosterone biosynthesis identified during examination of heparin effect on aldosterone production.

This study was designed to examine the mechanism whereby routine heparin therapy inhibits adrenal aldosterone production. In bovine adrenal glomerulosa cell suspensions, pure heparin, in concentrations up to 500 U/ml, had no significant effect on basal or angiotensin II-stimulated aldosterone production. A therapeutic preparation of heparin for parenteral use containing the preservative chlorbutol (2.8 X 10(-2) M) inhibited aldosterone production [67 +/- 8.7% (+/- SE); P less than 0.005]. Chlorbutol alone, in a dose-dependent manner, inhibited basal aldosterone production from 1548 +/- 355 to 316 +/- 152 pg/ml (P less than 0.001) and inhibited angiotensin II-stimulated production from 4950 +/- 724 to 589 +/- 257 pg/ml (P less than 0.001). To elucidate the inhibitory mechanism of chlorbutol, we used trilostane, an inhibitor of the conversion of pregnenolone to progesterone, and aminoglutethimide, an inhibitor of the conversion of cholesterol to pregnenolone. Aldosterone production was completely suppressed by each inhibitor. Pregnenolone accumulation in trilostane-treated cells fell from 9.70 +/- 1.66 to 1.40 +/- 0.28 ng/ml (P less than 0.005) with the addition of chlorbutol. Aldosterone accumulation from corticosterone added to aminoglutethimide-treated cells fell from 715 +/- 96 to 348 +/- 59 pg/ml (P less than 0.02) in cells incubated with chlorbutol. Thus, chlorbutol is a potent inhibitor of aldosterone production, inhibiting both the early biosynthetic phase and, to a lesser extent, the late phase. Since chlorbutol is a widely used pharmaceutical preservative and has a slow metabolic clearance, these findings may be of toxicological significance and may account for the inhibition of aldosterone production previously attributed to heparin.

Effect of atrial natriuretic factor on the plasma aldosterone response to potassium infusion in rats - in vivo study

Previous in vitro studies have shown that atrial natriuretic factor inhibits the secretion of aldosterone stimulated by AII, ACTH, and potassium in adrenal cell suspensions. The present study investigated the effects of atriopeptin II on the plasma aldosterone response to a potassium infusion in conscious unrestrained rats in vivo. The infusion of potassium chloride solution increased plasma aldosterone level from 20.4 ± 3.7 to 168.4 ± 27.3 ng/dl. The simultaneous administration of atriopeptin II reduced the increase in plasma aldosterone level ((16.0 ± 2.1 to 63.3 ± 10.4 ng/dl). There was no significant difference in the plasma renin activity, corticosterone, or serum potassium levels between the two groups. These results suggest that atriopeptin II may be important in the regulation of aldosterone secretion.

Stimulation of pituitary-adrenocortical system by cepharanthine

We observed that cepharanthine might exert its anti-allergic action by stimulating the secretion of corticosterone. The present experiments were carried out to investigate stimulation of the pituitary-adrenocortical system by cepharanthine. Administration of cepharanthine to rats produced increases in plasma and adrenal corticosterone levels. Administration of cepharanthine to propranolol pretreated rats also produced increases in plasma and adrenal corticosterone levels and plasma ACTH level. The elevation of corticosterone level induced by cepharanthine was considered to be the specific effect of cepharanthine. Cepharanthine did not increase plasma corticosterone level in rats in the state of dexamethasone suppression of the pituitary-adrenocortical system, in which the level was lowered. Administration of cepharanthine to Bordetella pertussis vaccine induced beta-adrenergic blocked rats also produced increases in plasma and adrenal corticosterone levels. The production and release of corticosterone from an adrenal cell suspension were not influenced by cepharanthine in vitro. These results suggest that cepharanthine stimulates the pituitary-adrenotropic function.