Effects of protease inhibitors on adenylate cyclase activation and aldosterone production in rat adrenal zona glomerulosa cells

It has been shown that serine proteases are involved in aldosterone and 18-hydroxycorticosterone production by the rat adrenal zona glomerulosa in response to a variety of stimulants. From evidence presented for various tissues, including the rat adrenal cortex, the observation that adenylate cyclase can be activated by proteolytic enzymes and inhibited by protease inhibitors has led to the suggestion that serine proteases may also be involved in the hormonal stimulation of adenylate cyclase. In studies designed to test this hypothesis using protease inhibitors, only high concentrations (greater than 10(-4) M) of TAME (p-tosyl-L-arginine methyl ester) inhibited ACTH stimulated steroid and cAMP production in rat adrenal glomerulosa cells. TPCK (tosyl-L-phenylalanine chloromethylketone) and TLCK (tosyl-L-lysine chloromethylketone) were found to have a similar effect at very high concentrations (10(-2) M) but had no effect at the serine protease inhibitory concentration of 5 X 10(-6) M. Other protease inhibitors tested had no effect on ACTH-stimulated cAMP but the inhibitory effect of high concentrations of protease inhibitors on ACTH-stimulated adenylate cyclase was duplicated by the polyanion dextran sulphate. The results suggest that the inhibitors act through non-specific membrane effects and that proteases are not involved in the activation of zona glomerulosa adenylate cyclase by ACTH. In view of these findings it is concluded that a more rigorous approach should be applied to the use of protease inhibitors in whole cell systems, and that the concept of hormonal activation of adenylate cyclase via proteolytic events, which is based on studies with such inhibitors, should be reconsidered.     

The effects of furosemide on adenosine 3':5'-cyclic monophosphate, guanosine 3':5'-cyclic monophosphate and corticosterone production stimulated by adrenocorticotropin in monolayer cultured rat adrenal cells

Furosemide has been reported to have a suppressive effect on ADH-, PTH- and adrenaline-stimulated adenosine 3':5'-cyclic monophosphate (cAMP) production, but the effect on adrenocorticotropin (ACTH) action has not yet been elucidated. In the present study, therefore, the effects of furosemide on cAMP and also on guanosine 3':5'-cyclic monophosphate (cGMP) and corticosterone, stimulated by ACTH in monolayer cultured rat adrenal cells, were investigated. The intra- and extracellular cAMP stimulated by ACTH was dose-dependently suppressed by furosemide within the concentration range of 10(-3) M to 3 X 10(-3) M, and the suppressive effect of the drug was accompanied with decreased corticosterone production. However, non-stimulated basal corticosterone production was not influenced by the drug even at 3 X 10(-3) M. A similar suppressive effect of dibutyryl cAMP-stimulated corticosterone production by 3 X 10(-3) M furosemide was observed. The intracellular cAMP bound to its binding protein in sonicated adrenal cell extract was also suppressed in a very similar dose-dependent manner to total cAMP. However, though the effect on corticosterone production was also observed when the calcium concentration in the loading medium was changed, the magnitude of the effectiveness (percent of control) was relatively constant at each calcium concentration, suggesting that furosemide may not affect the site(s) at which calcium acts. Intracellular cGMP, on the other hand, was increased by 10(-3) M to 3 X 10(-3) M of furosemide, suggesting an intensifying effect of furosemide on guanylate cyclase activity. Dibutyryl cGMP-stimulated corticosterone production was also increased at the same concentration range. These results indicated that furosemide may act not only on adenylate cyclase but also on the additional step(s) to suppress the resultant corticosterone production. In contrast to the effects of furosemide on such cAMP-mediated processes, this drug treatment appeared to enhance cGMP-mediated corticosterone production.     

ACTH-induced refractoriness in cultured adrenal cell line (Y1).

Treatment of cultured mouse adrenal cells Y₁ with ACTH induced cell refractoriness to further hormonal stimulation. When ACTH was added to the cells every 2 hours the first addition increased the levels of 20αOH-progesterone and cAMP secreted into the medium. Upon the second and third additions of ACTH the levels of 20αOH-progesterone and cAMP secreted were greatly diminished and upon the fourth addition of ACTH were absent. Prolonged incubation (14 hours) with different concentrations of ACTH (5 × 10^?11 M to 10^?6 M) induced a dose-related steroidogenic refractoriness to further ACTH stimulation, 10^?8 M ACTH inducing complete refractoriness. The number of ACTH binding sites of cell particles prepared from desensitized cells was similar to that of the control but ACTH failed to stimulate the adenylate cyclase of desensitized cells, whereas the enzyme responded fully to NaF and Gpp(NH)p. The cAMP phosphodiesterase activity was similar in both desensitized and control cells. In addition the steroidogenic response to dibutyryl cAMP of desensitized cells was abolished. Thus, ACTH-induced adrenal cell desensitization seems to be related to at least two phenomena : a defect in the “coupling” between the hormone-receptor sites and the adenylate cyclase and an alteration of certain steps beyond cAMP formation.     

Effect of forskolin on collagen production in clonal osteoblastic MC3T3-E1 cells

The effect of forskolin on collagen production in osteoblasts was investigated by using clonal osteoblastic MC3T3-E1 cells cultured in a-minimum essential medium containing 0.1% bovine serum albumin. Forskolin increased the adenylate cyclase activity in membranes pelleted from homogenates of the cell line in a dose-dependent manner. The drug caused a 13-fold stimulation at 10(-4) M, indicating that the compound directly acts on adenylate cyclase, leading to an increase in the intracellular cAMP content of the cells. Collagen accumulation in the cultures was elevated by one-day treatment with 5 X 10(-5) M forskolin to about twice that in the controls. The stimulation was mainly due to an elevation in collagen synthesis but not to an inhibition of intracellular collagen degradation because forskolin dose-dependently increased collagen synthesis; it also significantly increased the amount of low-molecular-weight hydroxyproline found in the cultures. Cells treated with forskolin produced mainly type I collagen, as found in bone matrix in situ, with only small amounts of other types of collagen. Furthermore, forskolin time-dependently inhibited DNA synthesis in the cells, indicating that the increase in type I collagen synthesis by forskolin was not due to stimulated cell proliferation. These results suggest that cAMP is closely linked to the differentiation of osteoblasts in vitro.     

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

A cAMP independent inhibitory action of high doses of forskolin in rat Leydig cells

In addition to well known direct stimulatory and potentiatory actions of forskolin, we have previously reported that low doses of this diterpene (10(-9), 10(-12) M) markedly inhibit the production of cAMP and testosterone in rat Leydig cells through a pertussis toxin sensitive G-protein (A. Khanum and M. L. Dufau, J. Biol. Chem. 261, 1986). A different type of inhibitory effect of forskolin is described in this study. Forskolin (10(-5) M) markedly stimulates basal adenylate cyclase activity (about 200%) in rat Leydig cell membranes and potentiates the stimulatory effect of gonadotropin (10(-9), 10(-7) M) on adenylate cyclase in presence or in absence of GTP (10(-5) M). Similarly a time-dependent stimulation of forskolin (10(-5) M) alone is noted on all cAMP pools and testosterone production. Using a supramaximal steroidogenic dose of hCG (0.26 nM) or choleragen (0.1 microM), forskolin potentiates the gonadotrophin and toxin-induced responses of all cAMP pools significantly while inhibiting testosterone production. Moreover, forskolin also inhibits 8-Bromo-cAMP stimulated steroidogenesis. In contrast, pregnenolone synthesis was not altered by the diterpene. We have demonstrated in this study that the inhibitory effect of high doses of forskolin on steroidogenesis is distal to cAMP generation, and resulted from a steroidogenic block residing beyond pregnenolone synthesis.     

Inhibition of basal and hormone-stimulated adenylate cyclase in adipocyte ghosts by the prostaglandin endoperoxide prostaglandin H2.

The prostaglandin endoperoxide PGH2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid), at a concentration of 2.8 x 10(-5) M inhibited basal adenylate cyclase activity 11% and epinephrine-stimulated activity 30 to 35%. PGH2 inhibited epinephrine-stimulated enzyme activity in the presence of 10 mM theophylline, 2.5 mM adenosine 3':5'-monophosphate (cAMP), or in the absence of inhibitors or substrates of the cAMP phosphodiesterase. When the cAMP phosphodiesterase was assayed directly using 62 nM and 1.1 muM cAMP, PGH2 did not affect the 100,000 x g particulate cAMP phosphodiesterase from fat cells. The inhibition of adenylate cyclase by PGH2 was readily reversible. A 6-min preincubation of ghost membranes with PGH2, followed by washing, did not alter subsequent epinephrine-stimulated adenylate cyclase activity. During epinephrine stimulation, the PGH2 inhibition was apparent on initial rates of cAMP synthesis, and the addition of PGH2 to the enzyme system at any point during an assay markedly reduced the rate of cAMP synthesis. Between 2.8 x 10(-7) M and 2.8 x 10(-5) M, PGH2 inhibited epinephrine-stimulated enzyme activity in a concentration-dependent manner. The stimulation of adenylate cyclase by thyroid-stimulating hormone, glucagon, and adrenocorticotropic hormone as well as by epinephrine was antagonized by PGH2, suggesting that PGH2 may be an endogenous feedback regulator of hormone-stimulated lipolysis in adipose tissue.     

Adenosine 3':5'-cyclic monophosphate (cAMP) is not the mediator of kappa opiate effect on human placental lactogen release.

We previously reported that kappa opiates stimulated the release of human placental lactogen (hPL) from human placental cells. In this study, we investigated the role of adenylate cyclase as a potential cellular mediator of such an effect. Incubations with ethylketocyclazocine (EKC) led to a time- and dose-dependent inhibition of adenylate cyclase activity. The maximal inhibition was 45 +/- 5% of control value after 15 min exposure to 10(-7)M EKC. This inhibition was reversed by opiate antagonist naloxone and was specific to kappa opiate type. Preincubation of human trophoblastic cells with 0.1 microgram/ml Islet-Activating-Protein (IAP; also called pertussis toxin) did not modify basal adenylate cyclase activity but abolished the inhibition of adenylate cyclase activity by EKC, indicating that the effect of opiates on cAMP production was mediated by an IAP-sensitive GTP binding protein. Also, IAP stimulated basal hPL release; the control levels were 22.4 ng/ml and 46.5 ng/ml without and with IAP respectively. However, the EKC-stimulated hPL levels were unchanged by preincubation with IAP. This difference in cAMP and hPL response in IAP-treated cells suggested that the opiate receptors are not directly coupled to adenylate cyclase. This hypothesis was confirmed by 1) experiments on placental membranes showing that in absence of the cytoplasmic elements (membranes only), EKC had no effect on membrane adenylate cyclase and 2) experiments on placental cells showing that dibutyryl-cAMP (dbcAMP) stimulated hPL release.     

Adenosine 3', 5'-monophosphorothioate (Rp-isomer) induces down-regulation of surface cyclic AMP receptors without receptor activation in Dictyostelium discoideum

cAMP induces the activation and subsequent desensitization of adenylate cyclase in Dictyostelium discoideum. cAMP also induces down-regulation of surface cAMP receptors. Desensitization of adenylate cyclase is composed of a rapidly reversible component (adaptation) and a slowly reversible component related to down-regulation of surface cAMP receptors (Van Haastert, P.J.M. (1987) J. Biol. Chem. 262, 7700-7704). The agonistic and antagonistic activities of the cAMP derivative adenosine 3',5'-monophosphorothioate ((Rp)-cAMPS) for these responses were investigated. (Rp)-cAMPS competes with cAMP for binding to different receptor forms with an apparent Ki = 5 microM. (Rp)-cAMPS does not activate adenylate cyclase and antagonizes the cAMP-induced activation with an apparent Ki = 5 microM. (Rp)-cAMPS induces down-regulation of surface cAMP receptors with EC50 = 5 microM. (Rp)-cAMPS induces desensitization of adenylate cyclase, which is not rapidly reversible. These results indicate that desensitization of adenylate cyclase by (Rp)-cAMPS is due to down-regulation of surface cAMP receptors and not to adaptation. We conclude that down-regulation of surface cAMP receptors does not require their activation or modification involved in adaptation.     

Mechanisms of insulin inhibition of ACTH-stimulated steroid secretion by cultured bovine adrenocortical cells.

Results of previous studies indicated that insulin at levels comparable to those in humans during hyperinsulinemia decreased ACTH-stimulated cortisol and androstenedione secretion by bovine adrenal fasciculata-reticularis cells in primary culture. In the present studies this inhibitory action was examined further by comparing the effects of insulin on ACTH-stimulated corticosteroid secretion with its effects on 8-(4-chlorophenylthio)-cAMP (cpt-cAMP), forskolin- and [5val]angiotensin II (Ang II)-stimulated corticosteroid secretion. Effects on corticosteroid secretion were correlated with effects on cAMP accumulation and rates of cAMP production. Monolayers were incubated for 24 h in the absence or presence of each agonist alone or in combination with insulin. Insulin (1.7 x 10(-9) or 17.5 x 10(-9) M) caused about a 50% decrease in cortisol and androstenedione secretion in response to ACTH (10(-11) or 10(-8) M). Insulin also decreased ACTH-stimulated aldosterone secretion by cultured glomerulosa cells. Cpt-cAMP (10(-4) or 10(-3) M)-stimulated increases in cortisol and androstenedione secretion were inhibited by insulin, but to a lesser extent than those in response to ACTH. The inhibition of cpt-cAMP-stimulated steroid secretion was not related to increased degradation of the cyclic nucleotide. Increases in cortisol and androstenedione secretion caused by a submaximal concentration (10(-6) M) of forskolin were decreased 50-70% by insulin. In contrast, insulin failed to significantly affect cortisol or androstenedione secretion caused by a maximal concentration (10(-5) M) of forskolin. The secretory responses to Ang II (10(-8) M) were also unaffected by insulin. The effect of insulin to inhibit ACTH-stimulated steroid secretion was accompanied by a reduction in cAMP accumulation as well as an apparent inhibition of adenylate cyclase activation. These data indicate that the effect of insulin to attenuate ACTH-stimulated corticosteroid secretion results from both an inhibition of ACTH-stimulated adenylate cyclase activity and an antagonism of the intracellular actions of cAMP.     

Adrenal adenylate cyclase and steroidogenic activities of 63 day old ovine fetuses: in vitro effects of ACTH1-24 and forskolin

This study examines the activity of the adenylate cyclase system and that of some enzymes of the steroidogenic pathway of adrenal cells from 62-63 day old ovine fetuses. Synthetic corticotropin (ACTH1-24), cholera toxin and forskolin stimulated both cAMP and corticoid productions by freshly isolated adrenal cells. The cAMP response to ACTH1-24 was lower than that to forskolin. However, forskolin-induced steroidogenesis was significantly lower than the ACTH1-24-induced steroid output. Freshly isolated cells metabolized quickly [14C]-labeled pregnenolone mainly through the 17-deoxy pathway. The amounts of cortisol and of corticosterone formed, in the presence of exogenous pregnenolone, were roughly 15-fold higher than under maximal stimulation by ACTH1-24. When the cells were cultured for 6 days in the absence or presence of ACTH1-24 (10(-8) M) or forskolin (10(-5) M), a small development of the cAMP response to these factors was observed in the course of the experiment. However, the mechanism of this development appeared different, according to the conditions of culture. The amounts of corticosterone secreted on day 6 by ACTH1-24- or forskolin-treated cells were 2- to 4-fold higher than on day 1, whereas cortisol outputs were much lower on day 6 than on day 1. The response to ACTH1-24 of cells maintained in ACTH-free media decreased dramatically during the culture in terms of both cortisol and of corticosterone. On day 6 of the experiment, the metabolism of [14C]pregnenolone was lower than on day 1 under all 3 conditions of culture. Only the 3 beta-hydroxysteroid dehydrogenase/isomerase activity could be maintained by continuous treatment with forskolin. However, both ACTH1-24 and forskolin enhanced the production of pregnenolone from an endogenous substrate. In conclusion, these results present evidence that: 1) the adenylate cyclase system is not a bottleneck in the steroidogenic response to ACTH1-24 of freshly isolated adrenal cells from 62-63 day old ovine fetuses; 2) the main rate-limiting step for steroidogenesis by these cells is the availability of pregnenolone; 3) neither ACTH1-24 nor forskolin is able to maintain the activity of most enzymes involved in the metabolization of pregnenolone by cultured cells while increasing pregnenolone availability; 4) some inhibiting factors are involved in the loss of adrenal cells responsiveness to ACTH between days 50 and 100 of gestation, and they probably act mainly on the adenylate cyclase system.     

The modulation of transcobalamin II (TC-II) production by cyclic adenosine 3',5'-monophosphate in the murine macrophage cell line J774: relationship to growth behavior

We undertook a study to define the role of cyclic AMP [cAMP] in modulating the secretion of transcobalamin II (TC-II) in the mouse macrophage like cell line J774. J774 was observed to secrete large amounts of TC-II, particularly in the presence of 8-bromo cAMP or cholera toxin or when grown in medium supplemented with low concentrations of horse serum (1% or 5%) or in serum-free medium. Variant cell lines derived from J774 and deficient either in adenylate cyclase (ac -) or cAMP-dependent protein kinase (pk -) activity showed very low and intermediate levels of basal secretory activity of TC-II, respectively, compared to J774. Maximum secretory activity of TC-II was observed in J774 under conditions in which growth was poorest (in the presence of 8-bromo-cAMP or 1% or 5% horse serum-supplemented medium or in serum-free medium). Cells grown in serum-free medium were found to have elevated basal adenylate cyclase activity and cAMP levels compared to those grown in medium supplemented with 20% horse serum. The data from this study demonstrate a negative correlation between growth activity and TC-II secretion in the J774 cell line. The stimulatory effect of exogenous cAMP on TC-II secretion by J774, the reduced secretory activity of the variant lines ac- and pk- and the observed increase in cell cAMP levels under conditions of serum starvation in which TC-II secretion is considerably enhanced, suggest that cell cAMP is an important modulator of TC-II secretion and growth behavior in the J774 cell line.     

Dibutyryl cyclic AMP modulation of gap junctions in SW-13 human adrenal cortical tumor cells

Cultured human adrenal cortical adenocarcinoma cells (SW-13) form a confluent monolayer of epithelial-like cells when seeded into culture flasks. Following a 24-48 hr non-mitotic period, cells begin to divide and become confluent within a week after seeding at 5 X 10(4) cells/cm2. The SW-13 cells were exposed to dibutyryl cyclic AMP (DbcAMP), cyclic AMP (cAMP), sodium butyrate, and adrenocorticotropin (ACTH). The rate of SW-13 cell proliferation was measured with a DNA microfluorometric assay, as well as by procedures measuring the incorporation of 3H-thymidine. In addition, following administration of ACTH and DbcAMP, the fractional area of membrane covered by gap junctions was quantitated with freeze-fracture electron microscopic techniques. Dibutyryl cyclic AMP at a concentration of 1 X 10(-3) M decreased the growth rate of the cell population. There was a corresponding increase in the fractional area of gap junctions found on the cell membrane in 96-hr DbcAMP-treated cultures. ACTH (40 mU/ml) exposure failed to produce an increase in the fractional area of gap junctions or to alter the rate of cell proliferation. From these data it can be suggested that elevations in cAMP levels within the cell can be related to both the proliferation of gap junctions and the decrease in cell proliferation in the SW-13 tumor cell.     

ACTH receptor-mediated induction of leukocyte cyclic AMP

Studies were conducted to determine whether lymphocyte ACTH receptors behave as their structurally similar adrenal cell counterparts, in terms of adenylate cyclase activation and cyclic AMP (cAMP) production in the presence of ACTH. Treatment of mouse mononuclear splenocytes with ACTH (10(-5) to 10(-10) M) induced a consistent rise in cAMP. ACTH treatment of more homogenous cell populations, represented by Molt 4 T lymphoblast and S49A T cell lymphoma lines, yielded a dramatic, dose-related increase in cAMP levels for S49A cells but not for Molt 4 cells. Immunofluorescence assays, employing an antiserum to the adrenal cell ACTH receptor, indicated that 45% of splenocytes, 69% of S49A cells, and less than 1% of Molt 4 cells possess ACTH receptors. Radioligand binding studies confirmed that Molt 4 cells possess many fewer receptors than S49A cells, and probably fail to respond to ACTH because they lack the appropriate receptor. This is the first report of ACTH induction of leukocyte cAMP, evidence important to understanding the mechanisms by which this neuroendocrine hormone influences immune responses.     

GRF is a highly potent activator of adenylate cyclase in the normal human, bovine and rat pituitary: interaction with somatostatin

The effect of GRF adenylate cyclase activation was studied in normal human, bovine and rat pituitary tissues. Human GRF (hGRF) activates adenylate cyclase in normal human pituitary membrane preparations in a concentration dependent manner (ED5 0 = 10(-11) M). In bovine pituitary cells hGRF stimulates GH secretion into the medium (ED5 0 = 7 X 10(-12) M) and activates adenylate cyclase (ED5 0 = 10(-11) M). In normal rat pituitary cells in monolayer culture, rat GRF (rGRF) stimulates adenylate cyclase (ED5 0 = 3 X 10(-11) M). In normal human pituitary membrane preparations and in normal rat pituitary cells in culture, somatostatin inhibits GRF-stimulated adenylate cyclase in a non-competitive manner, while it does not affect basal (i.e. non-stimulated) adenylate cyclase levels. VIP, a peptide which is structurally homologous to hGRF and rGRF is a weak GRF-agonist and activates adenylate cyclase in human and rat pituitary preparations at concentrations greater than 10 nM.     

The effect of L-glutamate and related agents on adenylate cyclase in the cestode Hymenolepis diminuta.

The effect of the putative amino acid transmitter, L-glutamate, on adenylate cyclase in crude membrane preparations of the rat tapeworm Hymenolepis diminuta was investigated to determine if glutamate effects the generation of the second messenger cAMP. Addition of glutamate at 10(-3) and 5.5 x 10(-9) M resulted in significant elevations in basal activity of adenylate cyclase, while concentrations in the 10(-5)-10(-7) M range caused significant depressions below basal activity. Assays with glutamate agonists and other acidic compounds showed glutamate to be the only amino acid, dicarboxylic acid, or acidic compound capable of this pattern of stimulation and inhibition. While the response of adenylate cyclase to glutamate agonists suggested that an N-methyl-D-aspartic acid (NMDA) type receptor may be present, glutamate agents acting as NMDA antagonists in vertebrate systems were agonists. Metabolic end products of glycolysis stimulated adenylate cyclase, suggesting that these, along with metabolic glutamate may regulate glycolytic enzymes. Only 10(-3) M L-glutamate significantly stimulated adenylate cyclase activity in tissue slices, and this response was restricted to those slices rich in nervous tissues. L-Glutamate eliminated the 5-hydroxytryptamine (5-HT) stimulated adenylate cyclase response suggesting that glutamate can modulate the 5-HT stimulated elevations in adenylate cyclase activity. The data support the hypothesis that L-glutamate is a neurotransmitter-modulator in the cestode.     

PTH and PTH-related peptide enhance steroid secretion from human adrenocortical cells

Parathyroid hormone (PTH) and PTH-related peptide (PTH-RP) are two hypercalcemic hormones that share a common receptor subtype, the PTH/PTH-RP receptor. PTH and PTH-RP concentration dependently enhanced basal aldosterone and cortisol secretion from dispersed human adrenocortical cells, with a maximal effective concentration (approximately 2-fold increase) of 10(-8) M. The secretagogue effect of 10(-8) M PTH or PTH-RP was abolished by the PTH/PTH-RP receptor antagonist [Leu11,D-Trp12]-PTH-RP-(7-34)-amide (10(-6) M). PTH and PTH-RP (10(-8) M) raised cAMP and inositol-triphosphate release by dispersed adrenocortical cells, and these effects were blocked by the adenylate cyclase inhibitor SQ-22536 (10(-4) M) and the phospholipase C (PLC) inhibitor U-73122 (10(-5) M), respectively. SQ-22536 (10(-4) M) and U-73122 (10(-5) M) partially inhibited aldosterone and cortisol response to 10(-8) M PTH and PTH-RP; when added together, they abolished it. Similar results were obtained by using the protein kinase (PK)A and PKC inhibitors H-89 and calphostin C (10(-5) M). It is concluded that PTH and PTH-RP exert a sizeable secretagogue action on the human adrenal cortex, probably acting through the PTH/PTH-RP receptor coupled with both adenylate cyclase/PKA- and PLC/PKC-dependent signaling cascades.     

In situ analysis of adenylate cyclase activity in permeabilized rat adipocytes: sensitivity to GTP, isoproterenol, and N6-(phenylisopropyl)adenosine

Adenylate cyclase activity in rat adipocyte suspensions was assayed in situ using a digitonin permeabilization technique. Recovery of activity was dependent on digitonin concentration, reaching a maximum at 20 micrograms/ml digitonin and paralleling the effect on cell permeability. Maximum adenylate cyclase activity recovered in permeabilized cells was 75% of that in comparable homogenates. Isoproterenol, a beta-adrenergic agonist, activated adenylate cyclase by 1.4, 2.2 and 4.5 fold at 10(-6), 10(-5) and 10(-3) M, respectively, despite perturbation of the plasma membrane. Exogenous GTP was not required for expression of beta-adrenergic activation, but 10(-5) M GTP maximally increased both basal and isoproterenol-dependent activity. The response to 10(-6) M isoproterenol was increased 2.1 fold by 10(-5) M GTP. N6-(Phenylisopropyl)adenosine at 10(-6) M inhibited both basal and isoproterenol-dependent adenylate cyclase activity by approximately 30%, demonstrating that the adenosine-dependent inhibitory pathway (Ni) remained functional in the digitonin-permeabilized cells. In situ analysis of adenylate cyclase is not only simple and rapid, but provides a unique approach to studying regulation of this key enzyme.     

Exogenous steroids alter steroidogenesis in cultured Y-1 adrenal tumor cells by actions preceding cyclic AMP

Using cultured Y-1 mouse adrenal tumor cells which produce 20 alpha-hydroxy-4-pregnen-3-one (20-DHP), it was found that 0.01 mM corticosterone and deoxycorticosterone increased basal and inhibited ACTH-induced 20-DHP production during consecutive 30 and 120 min incubations. Steroid effects were concentration-dependent and reversible. Six other steroids tested did not stimulate 20-DHP production and varied in ability to inhibit ACTH-stimulated steroidogenesis. Experiments demonstrated that 20-DHP production following treatment with cholera toxin, N,0'-dibutyryl cyclic AMP (dbcAMP), or pregnenolone was not inhibited by exogenous steroids. Corticosterone (0.01 mM) increased basal and inhibited ACTH-induced intracellular cyclic AMP (cAMP) production. Cytochalasin D, a microfilament perturbing agent, inhibited steroid-stimulated 20-DHP production, suggesting that ACTH and steroid stimulation mechanisms were similar. These findings taken together suggest that exogenous steroids can alter steroidogenesis by modifying plasma membrane adenylate cyclase activity.