Aldosterone secretion: effect of phorbol ester and A23187

The effects of the divalent ionophore, A23187, the phorbol ester, and/or 12-0-tetradecanoyl-phorbol-13-acetate on aldosterone secretion from adrenal glomerulosa cells were compared to those of angiotensin II (AII). AII causes a prompt and sustained increase in secretion. A23187 causes an initial increase followed by a gradual decline to values less than 25 percent of those seen with AII. TPA causes no initial increase but a slowly progressive rise in secretion rate to a less than maximal value. When TPA and A23187 act together, there is a prompt and sustained increase in aldosterone production rate similar to that seen after AII addition. The effect of TPA is dependent on the free Ca2+ concentration of the cell cytosol. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system operate to control respectively the initial and sustained phases of the secretory response. The first phase occurs as a consequence of amplitude modulation of the calmodulin branch of the system by a rise in [Ca2+]c, and the second phase as a consequence of the sensitivity modulation of the C-kinase branch by diacylglycerol.     

Insulin secretion: Combined effects of phorbol ester and A23187

The effect of the ionophore, A23187, and/or the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on insulin secretion were compared with those of glucose. Glucose induces a biphasic pattern of insulin secretion; A23187 a comparable initial spike but no second phase; and TPA a slowly progressive increase. Combined A23187 and TPA evoke a pattern similar to that induced by glucose. Forskolin enhances both phases of glucose-induced and of TPA-A23187-induced insulin secretion. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system, the calmodulin branch and the C-kinase branch, control, respectively, the initial and sustained phases of insulin secretion.     

Combined effect of phorbol ester and, A23187 or dibutyryl cyclic AMP on pepsinogen secretion from isolated gastric glands

In isolated guinea pig gastric glands, pepsinogen secretion was stimulated by the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in a dose dependent manner. Calcium-deprivation from the medium resulted in the decrease in TPA-induced pepsinogen secretion. The combination of 0.4 microM Ca2+ionophore A23187 and TPA stimulated pepsinogen secretion slightly higher than the calculated additive value for each agent. This synergistic effect of the agents supports a role of calcium-activated, phospholipid-dependent protein Kinase (protein Kinase C) in gastric pepsinogen secretion. Furthermore, pepsinogen secretion was also stimulated by dibutyryl cyclic AMP (dbc AMP) and dbc AMP slightly enhanced TPA-induced pepsinogen secretion. Results suggest that gastric chief cells possess at least two different secretory pathways for pepsinogen which are probably dependent on protein kinase C and cyclic AMP, respectively.     

Synergism between phorbol ester and A23187 in superoxide production by neutrophils

Concentrations of phorbol myristate acetate and the calcium ionophore, A23187, which by themselves are minimally effective in stimulating superoxide generation in human neutrophils show marked mutual potentiation when given together. This supports the hypothesis that synergism between cytosolic calcium and protein kinase C is involved in the stimulus/activation coupling of the respiratory burst in the neutrophil.     

Synergistic action of A23187 and phorbol ester on human B cell activation

We have investigated the existence of a synergy occurring between the calcium ionophore A23187 and phorbol myristic acetate (PMA) with respect to human B cell proliferation and differentiation. The combination of A23187 (250 to 500 nM) with nonmitogenic concentrations of PMA (1 to 3 ng/ml) resulted in a strong proliferative response in human tonsillar, spleen, and peripheral blood B cells. This proliferation could not be blocked by anti-Tac antibody at concentrations that effectively inhibited T cell proliferation under similar culture conditions, suggesting that IL 2 and its receptor are not involved in B cell proliferation in this system. During a 3-day culture period, A23187 (500 nM) did not activate B cells in terms of changes in cell size or in the expression of transferrin receptor, HLA-DR, and Tac antigen. PMA at a nonmitogenic concentration (3 ng/ml) enhanced the expression of the first two markers. Combination of the ionophore with PMA induced the occurrence of Tac and further increased the expression of transferrin receptor and HLA-DR. A23187 similarly enhanced the PMA-mediated increase in cell size. PMA and A23187 did not induce differentiation to lg production. However, when cells were prestimulated with a combination of the two agents and were recultured in the presence of a preparation containing B cell differentiation factor, a strong increase in IgM, IgG, and IgA production was found. We conclude that PMA and A23187 synergistically trigger intracellular events in human B cells, leading to proliferation and to responsiveness to differentiation factors.     

Synergistic stimulation of prolactin release by phorbol ester, A23187 and forskolin

The effects of 12-0-tetradecanoyl-phorbol-13-acetate (TPA), A23187, forskolin and thyrotropin-releasing hormone (TRH) on prolactin release from GH4C1 cells were compared. TPA caused a 2-fold release, maximum after 6 or more min, that was sustained for 30 min or more. A23187 caused only a small and variable response that peaked within 4 to 6 min. Combination of TPA and A23187 caused a rapid 3- to 5-fold increase in release that declined slowly. TRH increased prolactin release 3- to 5-fold, reaching a maximum within 4 min, followed by sustained release at lower rates. Forskolin had little effect by itself, but potentiated release caused either by combined TPA and A23187, or by TRH. These data are consistent with a model in which two branches of the Ca2+ messenger system participate in the action of TRH, a calmodulin branch and a C-kinase branch that interact to cause large amounts of sustained release. Forskolin, by regulating the cyclic AMP content of the cell determines the set point around which the Ca2+ messenger system operates.     

Synergistic activation of rat hepatocyte glycogen phosphorylase by A23187 and phorbol ester

The combination of 1.6 microM 4 beta phorbol, 12 beta myristate, 13 alpha acetate (PMA) and 1 microM A23187 produced a five-fold greater stimulation of rat hepatocyte glycogen phosphorylase activity than was seen with PMA alone. Vasopressin activation of glycogen phosphorylase was comparable to that seen with PMA plus A23187. Glycogen phosphorylase activity due to PMA plus A23187 was increased significantly after 30 sec, maximal at 120 and sustained at elevated levels for 240 sec. In contrast, activation due to vasopressin was maximal at 30 sec followed by a decrease. The addition of PMA 5 min prior to the A23187 abolished the synergism between these two agents. These data are compatible with the hypothesis that diacylglycerol and Ca2+ synergistically increase glycogen phosphorylase activity in rat hepatocytes.     

Atrial natriuretic peptide secretion during development of the rat supraoptic nucleus

Since a relationship between atrial natriuretic peptide and oxytocin was recently demonstrated in the heart (Gutkowska et al., 1997), the aim of this study was to determine whether a relationship between the two peptides is present also in the rat hypothalamus. For this purpose, we measured ANP-ontogeny in the rat hypothalamus immunohistochemically and compared it with oxytocin-ontogeny which we previously studied. The results showed that the ANP-peptide and mRNA-ANP start at the 18th day of the fetal life. Our earlier data for oxytocin in the rat hypothalamus showed that only mRNA-oxytocin appeared the 18th day of foetal life (Farina Lipari et al., 2001); thus, at the 18th day of foetal life, mRNA-ANP, ANP-peptide and mRNA-oxytocin are present. We conclude that in the hypothalamus, differently from that in the heart, ANP might play a role on the synthesis of the oxytocin since ANP and its mRNA appear earlier than oxytocin.     

The phorbol ester induced atrial natriuretic peptide secretion is stimulated by forskolin and Bay K8644 and inhibited by 8-bromo-cyclic GMP

The role of intracellular signals in the regulation of atrial natriuretic peptide (ANP) release was studied using the isolated perfused rat heart. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to activate the protein kinase C pathway, produced a dose-dependent increase in perfusate ANP immunoreactivity. Bay k8644, a putative calcium channel activator, and forskolin, which stimulates adenylate cyclase, induced a sustained increase in ANP secretory rate. TPA in combination with either Bay k8644 or forskolin induced higher ANP secretion than the calculated additive value for each agent. 8-bromo-cyclic GMP and sodium nitroprusside, when given alone, had no effect on ANP secretion, but delayed the TPA-stimulated increase in perfusate ANP. ANP secretion appears therefore to be mediated both by the phosphoinositide and the cAMP system, whereas the cGMP pathway may be inhibitory.     

Atrial natriuretic peptide induces natriuretic peptide receptor-cGMP-dependent protein kinase interaction

Circulating natriuretic peptides such as atrial natriuretic peptide (ANP) counterbalance the effects of hypertension and inhibit cardiac hypertrophy by activating cGMP-dependent protein kinase (PKG). Natriuretic peptide binding to type I receptors (NPRA and NPRB) activates their intrinsic guanylyl cyclase activity, resulting in a rapid increase in cytosolic cGMP that subsequently activates PKG. Phosphorylation of the receptor by an unknown serine/threonine kinase is required before ligand binding can activate the cyclase. While searching for downstream PKG partners using a yeast two-hybrid screen of a human heart cDNA library, we unexpectedly found an upstream association with NPRA. PKG is a serine/threonine kinase capable of phosphorylating NPRA in vitro; however, regulation of NPRA by PKG has not been previously reported. Here we show that PKG is recruited to the plasma membrane following ANP treatment, an effect that can be blocked by pharmacological inhibition of PKG activation. Furthermore, PKG participates in a ligand-dependent gain-of-function loop that significantly increases the intrinsic cyclase activity of the receptor. PKG translocation is ANP-dependent but not nitric oxide-dependent. Our results suggest that anchoring of PKG to NPRA is a key event after ligand binding that determines distal effects. As such, the NPRA-PKG association may represent a novel mechanism for compartmentation of cGMP-mediated signaling and regulation of receptor sensitivity.     

Atrial natriuretic peptide in hypoxia

A growing number of mammalian genes whose expression is inducible by hypoxia have been identified. Among them, atrial natriuretic peptide (ANP) synthesis and secretion is increased during hypoxic exposure and plays an important role in the normal adaptation to hypoxia and in the pathogenesis of cardiopulmonary diseases, including chronic hypoxia-induced pulmonary hypertension and vascular remodeling, and right ventricular hypertrophy and right heart failure. This review discusses the roles of ANP and its receptors in hypoxia-induced pulmonary hypertension. We and other investigators have demonstrated that ANP gene expression is enhanced by exposure to hypoxia and that the ANP so generated protects against the development of hypoxic pulmonary hypertension. Results also show that hypoxia directly stimulates ANP gene expression and ANP release in cardiac myocytes in vitro. Several cis-responsive elements of the ANP promoter are involved in the response to changes in oxygen tension. Further, the ANP clearance receptor NPR-C, but not the biological active NPR-A and NPR-B receptors, is downregulated in hypoxia adapted lung. Hypoxia-sensitive tyrosine kinase receptor-associated growth factors, including fibroblast growth factor (FGF) and platelet derived growth factor (PDGF)-BB, but not hypoxia per se, inhibit NPR-C gene expression in pulmonary arterial smooth muscle cells in vitro. The reductions in NPR-C in the hypoxic lung retard the clearance of ANP and allow more ANP to bind to biological active NPR-A and NPR-B in the pulmonary circulation, relaxing preconstricted pulmonary vessels, reducing pulmonary arterial pressure, and attenuating the development of hypoxia-induced pulmonary hypertension and vascular remodeling.     

Atrial natriuretic peptide in infants and children

Atrial natriuretic peptide (ANP) was measured in the plasma of 192 normal infants and children aged 1 day to 18 years. Plasma ANP was high during postnatal adaptation, particularly in premature infants. In 96 infants and children aged 4 months to 18 years, plasma ANP was similar to values obtained in 7 healthy adult volunteers (23.9 +/- 11.9 vs. 25.7 +/- 4.6 fmol/ml). There was no significant relationship between ANP and age. ANP is elevated about twofold in full-term neonates being 3-4 days of age, and returned to normal thereafter. It is concluded that ANP is raised during the postnatal adaptation. This hormone is possibly involved in the postnatal volume contraction and may antagonize vasoconstrictor hormones that are elevated during the postnatal period.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.     

Calcium-dependent activation of lymphocytes by ionophore, A23187, and a phorbol ester tumor promoter

The phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore, A23187, have similar effects on many different cells. For example, both show mitogenic and comitogenic activities for lymphocytes. It had been suggested that some of TPA's effects are due to its ability to act as a calcium ionophore. In order to test this idea, we compared the ability of TPA and ionophore to synergize with concanavalin A (Con A) in a two-phase system of lymphocyte mitogenesis. We found that ionophore was most comitogenic with Con A when present in the early phase of stimulation. TPA was only comitogenic when present in the late phase. Ionophore and TPA could not replace one another in the system. However, both ionophore and TPA together could replace Con A and stimulate DNA synthesis when they were presented to the cells in the sequential order of ionophore followed by TPA. Both compounds required the presence of external calcium to be effective.     

The phorbol ester TPA enhances A23187--but not carbachol- and high K+-induced catecholamine secretion from cultured bovine adrenal chromaffin cells

The effect of the phorbol ester TPA on catecholamine secretion was studied in cultured bovine adrenal chromaffin cells. The pretreatment of chromaffin cells with TPA caused the enhancement of catecholamine secretion induced by the calcium ionophore, A23187. By contrast, neither carbachol- nor high K+-induced secretion was changed by TPA pretreatment. These results support the concept that protein kinase C plays an important role as a factor transducing the Ca2+ signal to the exocytotic process of catecholamine secretion in bovine adrenal chromaffin cells.     

Atrial natriuretic peptide effect on NADPH-diaphorase in rat intestinal tract.

Histochemical reaction of NADPH-diaphorase (NOS-NADPH-d) was used to identify NO synthesis. A 30-min 0.1 microg microg/kg/min ANP infusion led to about a 10% and 35% increase in small and large intestine enterocytes stain respectively. This increase was abolished by a bolus of 1 mg/kg L-NAME before ANP infusion in small intestine, and partially abolished it in colon. Incubation of small and large intestine with 0.5 microM ANP increased stain at about 20%. In both tissues the preincubation with 0.1 mM L-NAME abolished the ANP effect. Incubation with 0.1 mM 8-Br-cGMP enhanced staining about 70% and 30% in small and large intestine respectively. Our results show that ANP enhances NOS-NADPH-d activity, suggesting that ANP stimulates NO synthase in enterocytes by L-arginine-NO pathway. 8-Br-cGMP mimicked the effect of ANP described above. Therefore, the guanylate cyclase-coupled natriuretic receptors, NPR-A and NPR-B, probably mediate this ANP effect.     

Interleukin 2 mRNA induction in human lymphocytes: analysis of the synergistic effect of a phorbol ester and phytohemagglutinin

Induction of interleukin 2 (IL2) mRNA synthesis in human tonsillar lymphocytes was studied by quantifying the relative levels of IL2 mRNA in the lymphocytes stimulated under various conditions by the dot hybridization method. A remarkable increase of IL2 mRNA was induced by stimulation with phytohemagglutinin (PHA) in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The kinetic study revealed that the IL2 mRNA level of the lymphocytes increased from 2 h of culture, reached a maximal level at 12 h, maintained a relatively high level until 48 h and then sharply decreased by 72 h after the stimulation. Inhibition experiments with actinomycin D showed that the increase was due to a transient synthesis of the mRNA after the stimulation, which almost stopped by 12-16 h. DNA synthesis and cell division were not necessary for the induction of IL2 mRNA production but the induction was inhibited by dexamethasone, showing that the production was mainly associated with the G1 phase of the cell cycle. Two-step culture experiments showed that prior exposure of the lymphocytes to TPA for 1 h at 37 degrees C resulted in a remarkable increase of IL2 mRNA on subsequent stimulation with PHA. This suggests that TPA induces certain changes in the biochemical pathway of signal transduction so that the cells can be triggered to express IL2 gene by subsequent stimulation with mitogen.