Ultracytochemical localization of particulate guanylate cyclase after stimulation with natriuretic peptides in lamb olfactory mucosa

Summary The ultracytochemical localization of particulate guanylate cyclase has been studied in lamb olfactory mucosa after activation with rat atrial natriuretic factor (rANF), porcine brain natriuretic peptide (pBNP), porcine C-type natriuretic peptide (pCNP) or rat brain natriuretic peptide (rBNP). Particulate guanylate cyclase is the receptor for these peptides and recently two subtypes of the cyclase have been identified. These isoforms are stimulated differently by ANF, BNP and CNP. Under our experimental conditions, rANF, pCNP and pBNP were strong activators of particulate guanylate cyclase in lamb olfactory mucosa, as demonstrated by the presence of reaction product. Samples incubated in basal conditions without rANF, pCNP or pBNP, or samples incubated in presence of rBNP did not reveal any cyclase activity. The rANF-stimulated cyclase activity was localized in the apical portion of olfactory epithelium. pCNP-stimulated guanylate cyclase was detected to the lamina propria in association with secretory cells of Bowman's glands and with cells in close relation with Bowman's glands (elongated cells and myoepithelial cells). The cyclase activity stimulated by pBNP was limited to cells of Bowman's glands. The present data indicate that ANF and CNP are recognized by different receptors and that BNP and CNP bind to the same receptor.     

Ultrastructural demonstration of guanylate cyclase in rat lung after activation by ANF.

We studied the cytochemical localization of particulate guanylate cyclase (GC) activity after stimulation with atrial natriuretic factor (ANF) in rat lung, at the electron microscope level. Samples incubated in the absence of ANF did not reveal any GC reaction product. These results indicate that ANF is a strong activator of the enzyme in this organ. In intrapulmonary bronchi, the ANF-activated GC reaction product was localized on mucus secreting goblet cells. GC was seen in bronchioles, alveoli and capillaries. All of the GC reaction product was associated with plasma membranes of Clara cells, of great alveolar cells and of endothelial cells in alveolar capillaries. Our data suggest that, by activation of particulate GC, ANF acts directly on cells where Na+ reabsorption occurs.     

Porcine brain natriuretic peptide, another modulator of bovine adrenocortical steroidogenesis

Porcine brain natriuretic peptide (pBNP) significantly inhibited aldosterone production stimulated by an angiotensin II analog and ACTH-stimulated cortisol secretion, together with simultaneously increasing the formation of cGMP in dispersed bovine adrenocortical cells. Receptors for pBNP were identified in bovine adrenal gland using an in vitro receptor autoradiographic technique and studies of 125I-pBNP binding. In vitro receptor autoradiography demonstrated specific binding sites for 125I-pBNP in bovine adrenal cortex. Complete displacement of 125I-pBNP by unlabeled pBNP or human atrial natriuretic peptide (hANP) can take place at these sites. Analysis of 125I-pBNP binding to bovine adrenocortical membrane fractions showed that the adrenal cortex had high-affinity, low-capacity pBNP-binding sites, with a dissociation constant (Kd) of 2.32 +/- 0.33 x 10(-10) M (mean +/- SE) and a maximal binding capacity (Bmax) of 36.7 +/- 1.6 fmol/mg protein. Moreover, the specific binding sites for 125I-pBNP were completely displaced not only by unlabeled pBNP but also by unlabeled hANP. The hANP dose required for 50% inhibition of specific 125I-pBNP binding was almost identical to that for pBNP (IC50 values for hANP and pBNP: 8.5 x 10(-10) and 6.5 x 10(-10) M, respectively). These results suggest that pBNP exerts a suppressive effect on bovine adrenocortical steroidogenesis via a receptor which may be shared with ANP.     

Porcine brain natriuretic peptide receptor in bovine adrenal cortex

The action of porcine brain natriuretic peptide (pBNP) on the steroidogenesis was investigated in cultured bovine adrenocortical cells. Porcine BNP induced a significant dose-dependent inhibition of both ACTH- and A II-stimulated aldosterone secretion. 10(-8) M and 10(-7) M pBNP also significantly inhibited ACTH-stimulated cortisol and dehydroepiandrosterone (DHEA) secretions. Binding studies of [125I]-pBNP to bovine adrenocortical membrane fractions showed that adrenal cortex had high-affinity and low-capacity pBNP binding sites, with a dissociation constant (Kd) of 1.70 x 10(-10) M and a maximal binding capacity (Bmax) of 19.9 fmol/mg protein. Finally, the 135 Kd radioactive band was specially visualized in the affinity labeling of bovine adrenal cortex with disuccinimidyl suberate (DSS). These results suggest that pBNP may have receptor-mediated suppressive actions on bovine adrenal steroidogenesis, similar to that in atrial natriuretic peptide (ANP).     

Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-beta (1-24) ACTH or anti-alpha (17-39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold. The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.     

Brain natriuretic peptide interacts with atrial natriuretic peptide receptor in cultured rat vascular smooth muscle cells

The effect of synthetic porcine brain natriuretic peptide (pBNP), a novel brain peptide with sequence homology to alpha-human atrial natriuretic peptide (hANP), on receptor binding and cGMP generation, was studied in cultured rat vascular smooth muscle cells (VSMC) and compared with that of alpha-hANP. 125I-pBNP bound to the cells in a time-dependent manner similar to that of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for pBNP with affinity and capacity identical to those of alpha-hANP. pBNP and alpha-hANP were almost equipotent in inhibiting the binding of either radioligand and stimulating intracellular cGMP generation. These data indicate that BNP and ANP interact with the same receptor sites to activate guanylate cyclase in rat VSMC.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

Autoradiographic localization of binding sites for atrial natriuretic factor

The distribution of atrial natriuretic factor (ANF) binding sites in Wistar rat tissues, as well as tissues from other species was studied. Using autoradiography of slide mounted tissue sections incubated with 125I-labelled ANF, high densities of binding sites were found in the renal glomeruli and papilla, aortic smooth muscle, iliac vein, choroid plexus, anterior pituitary, lung, and adrenal zona glomerulosa. Results from renal, aortic, adrenal, and lung tissues from spontaneously hypertensive rats did not differ from those of Wistar rats. Binding sites were also observed in guinea pig glomeruli, renal medulla, and aorta as well as in the rabbit aorta.     

Identification of the atrial natriuretic factor-R1C receptor subtype (B-clone) in cultured rat aortic smooth muscle cells.

The present report demonstrates the presence in cultured rat aortic smooth muscle cells of a natriuretic factor receptor subtype with a specificity typical of the ANF-R1C (B-clone) receptor subtype. To prove the existence of this receptor subtype in this cell line we show that pCNP-(82-103) is the most potent activator of the intrinsic guanylate cyclase activity, and that [125I]pCNP-(82-103) binds to a specific receptor subtype which is insensitive to the ANF-R2 specific ligand, C-ANF. The investigation of its binding characteristics show the rank potency order of the natriuretic factors in competing for pCNP binding to be pCNP greater than pBNP greater than rANF. Furthermore it was possible to covalently photolabel this receptor subtype with underivatized]125I]pCNP and show that it is composed of a single subunit of 130 kDa with very high specificity for pCNP.     

Atrial natriuretic factor: radioimmunoassay and effects on adrenal and pituitary glands

A simple and sensitive radioimmunoassay was developed for measurement of immunoreactive atrial natriuretic factor (IR-ANF) in rat and human plasma and in rat atria. The two atria contain about 20 micrograms ANF per rat. The right atrium contained 2.5 times more ANF than did the left. Ether anesthesia and morphine markedly increased IR-ANF in rat plasma. The concentration of IR-ANF in plasma of clinically normal human subjects was 65.3 +/- 2.5 pg/ml. Paroxysmal tachycardia and rapid atrial pacing significantly increased IR-ANF in human plasma. Two- to seven-fold higher concentrations were found in coronary sinus blood than in the peripheral circulation. In the plasma of rats and humans, circulating ANF is probably a small-molecular-weight peptide. ANF acts on the adrenal and the pituitary. ANF inhibits aldosterone secretion from rat zona glomerulosa and steroid secretion by bovine adrenal zona glomerulosa and fasciculata. ANF stimulated the basal secretion of arginine vasopressin (AVP) in vitro and inhibited KCl-stimulated release of AVP.     

Receptors for insulin and insulin-like growth factor-I in the human adrenal gland

Human adrenal glands contain high-affinity receptors for insulin and insulin-like growth factor I (IGF-I). Comparative studies with rat, hamster and human adrenal membranes confirmed that IGF-I receptors are most abundant in rat and hamster adrenals, whereas insulin and IGF-I receptors are present in equivalent numbers in human adrenal glands. Covalent crosslinking studies revealed that the human adrenal gland IGF-I receptor binding subunit migrated on dodecyl sulfate polyacrylamide gels with Mr = 135,000, which is identical to the migration of IGF-I receptor binding subunits isolated from other tissues. Autoradiography of frozen human adrenal slices incubated with [125I]insulin showed prominent, displaceable binding of this radioligand to the zona reticularis, zona glomerulosa, vasculature and medulla; in contrast, [125I]IGF-I binding to human adrenal tissue was most prominent in the zona reticularis and negligible in the medullary region.     

Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

Summary The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-(1–24) ACTH or anti (17–39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold.The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.T. Garcia-Caballero has a Postdoctoral Fellowship from Xunta de Galicia.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

In vitro studies of nuclear volume of rat adrenocortical cells: lace of ACTH effect

The effect of ACTH on nuclear volume of adrenocortical cells in the zona fasciculata of rat adrenal cortex was examined in vitro. Sections of adrenal gland were incubated for 60 or 90 min in Krebs-Ringer's solution with 1% glucose in the presence of ACTH, actinomycin D, cycloheximide and aminoglutethimide. ACTH, despite its clear effect in stimulating steroidogenesis, did not exert a direct effect on the nuclear volume of cells studied. This phenomenon is not dependent upon the stimulation of steroidogenesis, since aminoglutethimide does not influence the nuclear volume of adrenocortical cells studied; rather, ACTH in the presence of aminoglutethimide leads to a decrease in their volume. Actinomycin D does not influence nuclear volume while after incubation with cycloheximide nuclei were larger than the control. The presence of ACTH did not alter this effect. These results indicate no relationship between the degree of corticosterone output and nuclear volume in rat adrenocortical cells of the zona fasciculata in vitro.     

Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex

An assumption that the aldosterone-synthesizing enzyme exists only in zona glomerulosa cells apparently contradicts our recent findings that a purified bovine adrenocortical cytochrome P-45011 beta catalyzes the aldosterone formation and the enzyme exists in both zones of the adrenal cortex. To gain more insight into the zone specificity of aldosterone production, the aldosterone-synthesizing activity of mitochondria prepared from the isolated zones of adrenal cortex of various animal species was investigated. The intact mitochondria from the bovine or porcine zonae fasciculata-reticularis could not produce aldosterone whereas those from the zona glomerulosa produced it at a significant rate. When the mitochondria from the zonae fasciculata-reticularis were solubilized by the addition of cholate, they produced aldosterone from corticosterone at a rate comparable to that of those from the zona glomerulosa. The presence of specific factor(s) in the zonae fasciculata-reticularis mitochondria inhibiting expression of the aldosterone synthetic activity is discussed. The mitochondria of the rat zonae fasciculata-reticularis could hardly catalyze aldosterone synthesis under the detergent-solubilized conditions, whereas those of the zona glomerulosa could. Immunoblot analysis revealed that the mitochondria of the zonae fasciculata-reticularis contained a protein of Mr 51,000 which was immunocrossreactive with a monoclonal antibody directed against P-45011 beta, whereas those of the zona glomerulosa contained two immunocrossreactive proteins of Mr 51,000 and 49,000. These results suggest that in the case of rat adrenal cortex, a specific aldosterone-synthesizing enzyme exists in the zona glomerulosa.     

Particulate guanylate cyclase and adenylate cyclase activities after activation with various agents in rabbit platelets. An ultracytochemical study

Summary The cytochemical localization of particulate guanylate cyclase and adenylate cyclase activities in rabbit platelets were studied after stimulation with various agents, at the electron microscope level. In the presence of platelet aggregating agents such as thrombin and ADP, the particulate reaction product of guanylate cyclase activity was detectable on plasma membrane and on membranes of the open canalicular system. In contrast, samples incubated with platelet-activating factor showed no activation of the cyclase activity. Atrial natriuretic factor stimulated the particulate guanylate cyclase. The ultracytochemical localization of this activated cyclase was the same as that of thrombin-or ADP-stimulated guanylate cyclase. Adenylate cyclase activity was studied in platelets incubated with prostaglandin E₁ plus or minus insulin. The enzyme reaction product was found at the same sites where guanylate cyclase was detected. Therefore guanylate and adenylate cyclase activities do not seem to be preferentially localised in platelet membranes.     

Cyclic nucleotide phosphodiesterase activity in rat adrenal gland zones

The distribution of cyclic 3′, 5′ -nucleotide phosphodiesterase activity in the rat adrenal gland has been studied. Phosphodiesterase activity was 10-fold higher in the zona glomerulosa than in the zona fasciculata-reticularis. Kinetic studies carried out at low substrate concentrations suggest the possible presence of multiple forms of phosphodiesterase activity in both zones of the adrenal; however, these forms appear to have similar apparent Km's for cAMP. Thus, the well known differences in the steroidogenic response of the two zones to ACTH stimulation may be partially explained by large differences in total activities of the various forms of phosphodiesterase.     

Rat atrial natriuretic polypeptide stimulation of adrenal zona glomerulosa cell growth

Rat atrial natriuretic polypeptide (rANP) was found to stimulate [3H] thymidine incorporation into the DNA of bovine adrenal glomerulosa cells in a primary culture in a dose-dependent manner. The minimum effective dose was a very low concentration (10(-12) M of ANP), suggesting that ANP had a physiological effect. These findings are the first indication that ANP possesses growth-stimulating activity with regard to adrenal zona glomerulosa cells.     

Affinity cross-linking of atrial natriuretic factor to its receptor in bovine adrenal zona glomerulosa

125I-Labeled atrial natriuretic factor (ANF) was covalently cross-linked to its binding sites in bovine adrenal zona glomerulosa membranes using the hydrophilic cross-linker bis(sulfosuccinimidyl) suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol revealed that two protein bands with apparent Mr 68,000 and 114,000 were specifically labeled. The labeling of the two bands was prevented in a dose-dependent fashion by unlabeled ANF with a significant inhibition observed at 10(-10) M. High concentrations of angiotensin II and adrenocorticotropic hormone did not compete with 125I-ANF for binding and cross-linking. The dose-response curve for inhibition of covalent cross-linking of 125I-ANF by unlabeled ANF coincided with the dose-response curve for inhibition of binding to the receptor. No radioactive bands were observed in liver membranes. Experiments in which adrenal membranes were prepared and incubated in the presence of protease inhibitors showed no difference in the labeling pattern. Electrophoresis in the absence of reductant showed that the affinity-labeled species are not derived from larger disulfide-linked components. The apparent molecular weight of the two labeled species was not affected by a 100-fold variation in cross-linker concentration, and the labeling of both species increased in parallel. Possible relationships between the two labeled species are discussed.     

Sex differences in adrenocortical structure and function. V. The effects of postpubertal gonadectomy and gonadal hormone replacement on nuclear-cytoplasmic ratio, morphology and histochemistry of rat adrenal cortex.

Studies were carried out on adult rats of Wistar strain. Six weeks after postpubertal gonadectomy some of the orchiectomized rats were injected with a single dose of testosterone cypionate and ovari-ctomized rats with estradiol cypionate (17 beta-cyclopentyloproprionate esters of testosterone or estradiol). The control rats were sham operated. Investigations were carried out 8 weeks after surgery. Absolute and relative adrenal weight is lower in the male than in the female rat. Orchiectomy increases these weights while testosterone restores them to their normal level. Ovariectomy has no effect on the weight of adrenal, estradiol, however, increases the relative weight of the gland. The adrenal cortex of the adult female is wider than in the adult male rat and in female gland sudanophobic zone is lacking. Orchiectomy leads to the broadening of the adrenal cortex and testosterone replacement has an opposite effect. Ovariectomy has no effect on the structure of adrenal cortex and estradiol replacement resulted in the narrowing of zona glomerulosa. There were no differences in the nuclear-cytoplasmic ratio of zona glomerulosa cells in male and Female rats and neither gonadectomy nor gonadal hormone replacement has no effect on this parameter. The nuclear-cytoplasmic ratio of zona fasciculata cells in the male is markedly higher than in female. Orchiectomy lowers this ratio and testosterone restores it to the normal level. Neither ovariectomy nor estradiol replacement has effect on the nuclear-cytoplasmic ratio of zona fasciculata cells. Similar changes as those in the zona fasciculata were observed in zona reticularis cells. Among the oxidoreductases studied, the most marked sex differences were found in alpha-glycerophosphate dehydrogenase activity. In control female rats an intense reaction for this enzyme is observed in broad band of cells of the zona fasciculata interna, while in male rats only individual cells showed this reaction. In orchiectomized rats the reaction for this enzyme is similar as in control female rats and testosterone restores it to normal. Ovariectomy has no effect on localisation of reaction while after estradiol replacement reaction was more intense. Regarding the remaining oxidoreductases studied, in the adrenal cortex of rats of both sexes no marked differences were observed in localization, however, intensity of reaction depended upon applied experimental conditions. More distinct sex differences were observed in topochemistry of some hydrolases and intensity of reaction depended upon the applied experimental conditions.