Guinea Pig Adrenals Research Articles

Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at -60 mV. This inward current was partially suppressed by quinine with an IC(50) of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at -78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K(+) (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of -5 mV and a negative slope below -40 mV. External application of La(3+) had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve.

P419 FENOFIBRATE ATTENUATES NICOTINE-INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION IN THE RAT

Chromaffin cells isolated from guinea-pig adrenal glands secrete catecholamines in response to acetylcholine, nicotine, pilocarpine, veratridine, and high [K+]. Both substance P and somatostatin inhibit acetylcholine-induced catecholamine secretion. The maximal inhibition of acetylcholine-induced catecholamine secretion produced by substance P and by somatostatin is approximately 60%: the concentrations of the peptides required for half-maximal inhibition of secretion are approximately 0.8 and 2 μM. respectively. The maximal inhibition of catecholamine secretion produced by somatostatin and that caused by substance P are not additive. The effects of the peptides on secretion are readily reversible. Somatostatin and substance P also inhibit nicotine-induced catecholamine secretion, but they do not inhibit catecholamine secretion stimulated by pilocarpine, veratridine, or high [K+]. Thus, these peptides specifically inhibit catecholamine secretion linked to stimulation of nicotinic receptors. The inhibition of acetylcholine-induced catecholamine secretion by somatostatin is noncompetitive with respect to acetylcholine, Na+ or Ca2+.Immunoreactive somatostatin and substance P are present in guinea-pig adrenal glands. It is suggested that these peptides may play a role in the regulation of catecholamine secretion from the adrenal medulla.

Glucocorticoids potentiate effect of ACTH on cortisol secretion in isolated perfused guinea pig adrenal glands.

To clearly understand the autoregulation of glucocorticoid secretion by the glucocorticoid level, we studied the effects of cortisol and dexamethasone on the cortisol secretion in the perfused guinea pig adrenal glands. The basal cortisol secretion was suppressed by cortisol infusion at 0.1 microgram/ml, but not affected by dexamethasone infusion at 1 microgram/ml. The cortisol response to ACTH after pre-perfusion for 35 min was much quicker and larger in the presence of 0.1 microgram/ml cortisol, and was quicker in the presence of 1 microgram/ml dexamethasone than in the absence of glucocorticoid. It is considered that glucocorticoids play a permissive role in the action of ACTH on the adrenal gland.

Cross-regulation of cortisol secretion by PAF, ACTH, and calcium ionophore A23187 in perfused guinea pig adrenals

Cross-regulation of cortisol secretion by PAF, ACTH, and calcium ionophore A23187 in perfused guinea pig adrenals

Species differences in 3-methylsulphonyl-DDE bioactivation by adrenocortical tissue

The CYP11B1-activated adrenocortical toxicant 3-methylsulphonyl-DDE (3-MeSO2-DDE) is proposed as a lead compound for an improved chemotherapy for adrenocortical carcinoma. We compared the binding of 3-MeSO2-[14C]DDE in the adrenal cortex of four rodent species; hamster, guinea pig, mouse and rat, using a precision-cut adrenal slice culture system ex vivo. Localization and quantification of the bound radioactivity were carried out using light microscopy autoradiography and radioluminography. The results revealed major species differences since 3-MeSO2-[14C]DDE was extensively bound to the hamster adrenal tissue while the guinea pig adrenals were devoid of binding. A high binding in mouse adrenal cortex was confirmed while binding in rat adrenal cortex was very weak. The results support previous observations that metabolic activation of 3-MeSO2-DDE is highly species dependent. Since CYP11B1 could be expressed in tissues other than the adrenal cortex, final toxicological characterization should be carried out in a species that can bioactivate this compound.

Cross-regulation of cortisol secretion by adrenocorticotropin and platelet-activating factor in perfused guinea pig adrenals

We examined the cross-regulation of signaling between ACTH-and platelet-activating factor (PAF)-mediated steroidogenesis in the perfused guinea pig adrenal gland. Our method of in situ perfusion using an artificial medium can evaluate whether cortisol secretion in response to ACTH and PAF is interactive. Treating adrenal glands with 100 pg/ml ACTH diminished the subsequent cortisol response to 10 nM PAF. By contrast, PAF resulted in subsequent potentiation of ACTH-induced cortisol secretion. A mixture of 50 microM L-alpha-1-oleoyl-2-acetyl-sn-glycerol (OAG), an activator of protein kinase C (PKC), and 3.3 microM calcium ionophore (A23187), or 10 microM forskolin (FRK) diminished the cortisol response to PAF, whereas that to ACTH was unaffected. Each of PAF, ACTH, or FRK eliminated the cortisol response to OAG plus A23187, whereas that to FRK was unaffected. These data show that the protein kinase A (PKA)-dependent processes activated by ACTH or FRK can interfere with PAF-induced signal transduction at receptor and post-receptor levels. In contrast, PKC-dependent processes activated by PAF promoted ACTH-signaling at receptor and post-receptor level. Cross-regulation between processes activated by PAF receptor-PKC and by ACTH receptor-PKA might function in the multifactorial regulation of adrenocortical steroidogenesis.

Neuropeptide Y and glucocorticoid secretion from guinea pig adrenal gland: An in vivo and in vitro study

The effects of neuropeptide Y (NPY) on adrenal glucocorticoid secretion are controversial, and we have investigated this issue in guinea pigs, where, like in humans and cows, the main glucocorticoid hormone is cortisol. In vivo experiments showed that prolonged NPY administration markedly lowered cortisol plasma concentration not only in normal guinea pigs, but also in animals whose hypothalamic-pituitary-adrenal axis and renin-angiotensin system had been pharmacologically interrupted by the simultaneous administration of dexamethasone and captopril. In vitro experiments ruled out the possibility that in vivo glucocorticoid anti-secretagogue action of NPY can ensue from a direct effect on the adrenal gland. In fact, NPY did not affect cortisol secretion from dispersed guinea pig inner adrenocortical cells. In contrast, NPY raised cortisol production from adrenal slices containing medullary tissue, and this effect was blocked by the beta-adrenoceptor antagonist l-alprenolol. This finding, coupled with the demonstration that NPY enhanced catecholamine release from guinea pigadrenomedullary tissue, strongly suggests that NPY may stimulate glucocorticoid secretion in this species through an indirect mechanism involving catecholamines, that in a paracrine manner promote the secretion of inner adrenocortical cells. In light of these observations, the conclusion is drawn that the in vivo effects of NPY are mediated by mechanism(s) independent of either the suppression of the main adrenal agonists ACTH and angiotensin-II or the direct inhibition of adrenal secretion. The possibility merits an investigation into whether NPY enhances the production of peptides, which, like leptin, inhibit adrenal glucocorticoid secretion acting as circulating hormones.

IGF-I enhances cortisol secretion from guinea-pig adrenal gland: in vivo and in vitro study

Insulin-like growth factor (IGF)-I is a ubiquitously synthesized peptide that, along with IGF-II, acts via the IGF-R type I receptor. IGF-I and its receptor are expressed in the adrenal gland of humans and bovines, the secretion of which they seem to stimulate. As in humans and cows, the main glucocorticoid hormone secreted by guinea-pig adrenals is cortisol, and hence we have studied the adrenocortical effects of IGF-I in this species. In vivo experiments showed that prolonged IGF-I administration raised the plasma concentration of cortisol in both normal and dexamethasone/captopril-treated guinea pigs, thereby ruling out the possibility that IGF-I may act by activating the hypothalamic-pituitary-adrenal axis and the renin-angiotensin system. In vitro experiments demonstrated that IGF-I enhanced basal, but not maximally agonist [ACTH and angiotensin-II (Ang-II)]-stimulated, cortisol secretion from freshly dispersed guinea-pig inner adrenocortical cells. The IGF-I immuno-neutralization suppressed the IGF-I secretagogue effect, without altering the cortisol response to both ACTH and Ang-II. IGF-I raised cyclic-AMP and inositol triphosphate release from dispersed guinea-pig cells, and the effect was reversed by the adenylate cyclase inhibitor SQ-22536 and the phospholipase-C (PLC) inhibitor U-73122. SQ-22536, U-73122, the protein kinase (PK) A inhibitor H-89 and the PKC inhibitor calphostin-C decreased by approximately 50% the cortisol response of dispersed cells to IGF-I, and the combined exposure to SQ-22536 and U-73122 abolished it. We conclude that IGF-I stimulates glucocorticoid secretion from guinea-pig adrenocortical cells, acting via selective receptors coupled to both the adenylate cyclase/PKA- and PLC/PKC-dependent signaling cascades.

Effect of PAF on ACTH- and cholera toxin-induced cortisol secretions by perfused guinea pig adrenals

Effect of PAF on ACTH- and cholera toxin-induced cortisol secretions by perfused guinea pig adrenals

Atrial natriuretic peptide enhances cortisol secretion from guinea-pig adrenal gland: Evidence for an indirect paracrine mechanism probably involving the local release of medullary catecholamines

Atrial natriuretic peptide (ANP) is a regulatory hormone widely expressed, along with its receptors, in organs and body tissues. ANP is well known to inhibit aldosterone secretion from mammalian adrenals, but its effect on glucocorticoid-hormone production is controversial. In vivo experiments showed that prolonged ANP administration raised the plasma concentration of cortisol in both normal and dexamethasone/captopril-treated guinea pigs (i.e. in animals with pharmacologically interrupted hypothalamic-pituitary-adrenal axis and renin-angiotensin system). ANP did not affect cortisol secretion from dispersed guinea pig zona fasciculata-reticularis cells, but enhanced catecholamine release from adrenomedullary cells. ANP stimulated cortisol output from guinea pig adrenal slices containing medullary chromaffin tissue, and the beta-adrenoceptor antagonist l-alprenolol blocked this effect. The conclusion is drawn that ANP, when the structural integrity of the adrenal gland is preserved, is able to enhance glucocorticoid secretion in guinea pigs, through an indirect mechanism involving the rise in the catecholamine release, which in turn, acting in a paracrine manner, stimulate secretion of inner adrenocortical cells.

Cross-talk between ACTH- and PAF-induced cortisol and aldosterone secretion by perfused guinea pig adrenals

Cross-talk between ACTH- and PAF-induced cortisol and aldosterone secretion by perfused guinea pig adrenals

Characterization of the guinea pig 3β-hydroxysteroid dehydrogenase/Δ 5–Δ 4-isomerase expressed in the adrenal gland and gonads

The guinea pig adrenal gland, analogous to the human, possesses the capacity to synthesize C 19 steroids. In order to further understand the control of guinea pig adrenal steroidogenesis we undertook the characterization of the guinea pig 3β-hydroxysteroid dehydrogenase/Δ 5–Δ 4-isomerase (3β-HSD) expressed in the adrenal gland. A cDNA clone encoding guinea pig 3β-HSD isolated from a guinea pig adrenal library is predicted to encode a protein of 373 amino acid residues and 41,475 Da. Ribonuclease protection assay suggests that this cDNA corresponds to the predominant, if not the sole, mRNA species detectable in total RNA from the guinea pig adrenal gland, ovary and testis. The guinea pig 3β-HSD shows a similar affinity for both pregnenolone and dehydroepiandrosterone, and in addition, a 17β-HSD type II-like activity was also observed. A phylogenetical analysis of the 3β-HSD gene family demonstrates that the guinea pig is in a parallel branch to the myomorpha group supporting the hypothesis that the guinea pig lineage has branched off after the divergence among primates, artiodactyls and rodents, suggesting the paraphyly of the order rodentia.

Platelet-activating factor acts on cortisol secretion by perfused guinea-pig adrenals via calcium-/phospholipid-dependent mechanisms

Bilateral adrenals of the guinea pig were perfused in situ with an artificial medium equilibrated with 95% O2/5% CO2. Platelet-activating factor (PAF) induced biphasic cortisol responses, which reached a maximum at 10 nM PAF and declined at 100 nM. The effect of the PAF receptor antagonists CV-3988 and CV-6209 on PAF-stimulated cortisol secretion was examined. Prior exposure of adrenal glands to 10 microM CV-3988 or a simultaneous incubation with 10 microM CV-6209 abolished the cortisol response to 10 nM PAF. Lyso-PAF (a PAF precursor and breakdown product) did not affect cortisol secretion. Concentrations of 5-12.5 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C (PKC) inhibitor, abolished subsequent cortisol secretion in response to 10 nM PAF. N-[2-(Methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-8), a protein kinase A inhibitor, was less effective. A calcium ionophore (A23187) at 3.3 and 10 microM increased cortisol secretion, but the activator of PKC, l-alpha-1-oleoyl-2-acetyl-sn-3-glycerol (OAG), at 50 microM had no effect. When infused simultaneously, OAG (50 microM) and A23187 (3.3 microM) stimulated cortisol secretion synergistically. The secretory response of cortisol to repeated infusions of adrenocorticotrophin (100 pg/ml) or forskolin (10 microM) was essentially reproducible. By contrast, cortisol secretion in response to repeated infusions of PAF (10 nM) or OAG plus A23187 was not reproducible and the second response was diminished compared with the first. Our findings suggest that PAF plays a role in the regulation of steroidogenesis via a mechanism mediated by the PAF receptor and PKC.

Comparative Enzymology of 11β-Hydroxysteroid Dehydrogenase Type 1 from Glucocorticoid Resistant (Guinea Pig) Versus Sensitive (Human) Species

Type 1 11 beta-hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo. We compared kinetic characteristics of the human and guinea pig 11 beta-hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity. Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli. Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar K(m) values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels. Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme. Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with K(m) values of 0.8 microm (human) and 0.6 microm (guinea pig), close to the values obtained from intact cells. Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme. The kinetic data obtained argue against the involvement of 11 beta-hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs. Instead, the expression of 11 beta-hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species.

Inhibitory effects of cortical steroids and adrenocorticotropic hormone on catecholamine secretion in guinea-pig perfused adrenal glands.

1 We investigated the effects of exogenously applied steroids and endogenously released cortisol on catecholamine (CA) secretion induced by cholinergic agonists in perfused guinea-pig adrenal glands. 2 Acetylcholine (ACh) and electrical stimulation induced CA secretion, which was reversibly inhibited by cortisol. Adrenocorticotropic hormone (ACTH) increased the concentration of cortisol in the perfusion effluent and partly inhibited the secretory response to ACh. 3 Cortisol or aldosterone dose-dependently inhibited secretory responses to nicotine and muscarine. These inhibitory effects were not antagonized by mifepristone and spironolactone, respective cortisol and aldosterone receptor blockers. 4 Dexamethasone, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, prednisolone and cholesterol inhibited nicotine-evoked CA secretion. The secretory response to muscarine was inhibited by these compounds except for dexamethasone and prednisolone. 5 Dexamethasone, cortisol and aldosterone had no effect on CA secretion induced by high KCl. 6 These results suggest that steroids affect nicotinic and muscarinic ACh receptor-mediated responses through distinct mechanisms, and that cortisol released from the adrenal cortex inhibits CA secretion from the adrenal medulla.

Testosterone sulfotransferase: Evidence in the guinea pig that this reaction is carried out by 3α-hydroxysteroid sulfotransferase

During the course of isolating, characterizing, and cloning estrogen and 3-hydroxysteroid sulfotransferases from the guinea pig adrenal gland, it was noted that cytosolic preparations from this tissue would also sulfonate testosterone. Therefore, we set out to isolate and clone the enzyme that performs this reaction. Testosterone sulfotransferase (TST) was isolated from the guinea pig adrenal by using the standard procedures of ion exchange, affinity, and high-performance liquid chromatography. When purified, TST was examined by liquid-phase nondenaturing isoelectric focusing, it was found that the TST activity profile completely overlapped with the activity profile of the 3α-hydroxysteroid sulfotransferase (3αHST) isoform, but not the 3β-hydroxysteroid sulfotransferase (3βHST) isoform. This finding was further investigated by overexpressing the cDNAs for 3αHST and 3βHST in Escherichia coli and examining the expressed proteins for TST activity. This experiment confirmed that 3αHST does indeed function as a TST. In addition, 3αHST was also found to sulfonate estradiol but not estrone, a finding that further suggested that 3αHST may function as a general 17β-hydroxysteroid sulfotransferase.

Light and electron microscopic immunohistochemistry of the localization of adrenal steroidogenic enzymes.

Recent immunohistochemical studies have revealed the precise localization of the enzymes involved in adrenal steroidogenesis. Light microscopical investigations showed that cytochromes P450 of cholesterol side-chain cleavage enzyme (P450scc) and of 11 beta-hydroxylase (P45011 beta), 3 beta-hydroxysteroid dehydrogenase/ delta 5-4 isomerase (3 beta HSD), and 21-hydroxylase (P450C21) are localized in all the adrenocortical cells, especially in those of the zona fasciculata-reticularis. 17 alpha-Hydroxylase/C17-C20 lyase (P45017 alpha,lyase) is present in the zona fasciculata-reticularis cells of human, bovine, pig, and guinea-pig adrenals, but absent in the adrenals of some rodents such as rat, hamster, and mouse. Aldosterone synthase (P450aldo) is contained only in the zona glomerulosa cells. In the rat adrenal, P45011 beta, which catalyzes the conversion of deoxycorticosterone to corticosterone, is localized in the zona fasciculata-reticularis cells. Electron microscopic investigations demonstrated that P450scc and P45011 beta are colocalized in the matrix side of inner mitochondrial membrane including cristae, while 3 beta HSD, P450C21, and P45017 alpha, lyase are present in the membranes of smooth endoplasmic reticulum (SER). These results clearly indicate that aldosterone, the most potent mineralocorticoid, is synthesized in the zona glomerulosa cells, and glucocorticoids, such as corticosterone and cortisol, are produced in the zona fasciculata-reticularis cells. The conversion of cholesterol to pregnenolone and the final steps of corticosteroid synthesis occur in the mitochondria, while the intermediate steps, leading to the synthesis of deoxycorticosterone or deoxycortisol from pregnenolone, take place in the SER membranes.

P-10 - Effects of P2-purinoceptor antagoniste on the release of catecholamines from the perfused guinea-pig adrenal gland : the role of endogenous ATP

P-10 - Effects of P2-purinoceptor antagoniste on the release of catecholamines from the perfused guinea-pig adrenal gland : the role of endogenous ATP

Molecular Cloning and Functional Expression of the Cytochrome P450 11B-Hydroxylase of the Guinea Pig

A cDNA library has been constructed from adrenal glands of guinea pigs receiving a low sodium/high potassium diet. From this library a complementary DNA encoding an 11β-hydroxylase (EC 1.14.15.4) was isolated. Northern blot analysis revealed a major transcript of 2.2 kb size. The cDNA was cloned into an eucaryotic expression vector and the activity of the enzyme towards various substrates was investigated after expression in COS-1 cells. The enzyme was able to perform 11β-hydroxylations of 11-deoxycorticosterone, 11-deoxycortisol and androstendione as well as 18-hydroxylations and 19-hydroxylations to a small extent. Phylogenetic analyses with thirteen protein sequences of theCYP11Bsubfamily showed an early divergence of the guinea pig, but could not establish a definite branching order.

Biochemical alterations in guinea pig adrenal cortex following administration of PD 132301-2, an inhibitor of acyl-CoA:cholesterol acyltransferase.

To assess whether previously reported ultrastructural alterations of adrenocortical mitochondria induced by the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor PD 132301-2 are accompanied by functional deficits in tissue energy stores, phosphorylated adenine nucleotide levels in guinea pig adrenal cortex were quantitated. Adrenals of male guinea pigs were obtained at 1, 2, 6, or 24 hours after oral administration of 100 mg/kg PD 132301-2 or 0.5% methylcellulose vehicle. In treated animals, ATP levels and ATP/ADP ratios were decreased approximately 50% with concurrent increases in AMP. Calculated energy charge was also decreased; these decreases were maximal by 6 hours. Cholesterol esterification in adrenal cortex was inhibited, resulting in progressive accumulation of free cholesterol up to 3-fold over control by 24 hours, consistent with the ACAT inhibitory activity of the drug. These data suggest that PD 132301-2 distributes to the adrenal cortex where early alterations of tissue bioenergetics occur in a time frame consistent with ultrastructural alterations of mitochondria.