Stimulation of cyclic AMP production by vasoactive agents in cultured bovine aortic and pulmonary artery endothelial cells

Summary The ability of selected vasoactive agents to influence cyclic AMP levels of confluent, early-passaged bovine calf aortic and pulmonary artery endothelial cells was investigated. Among the agents tested, only the catecholamines (isoproterenol, epinephrine, nonrepinephrine) and prostaglandins (PGE₁, PGE₂, PGF_2a) resulted consistently in increased cyclic AMP production in both cell populations. The degree of cyclic AMP stimulation obtained with other vasoactive compounds (angiotensins I and II, bradykinin, and serotonin) tended to be either very small or difficult to reproduce. Isoproterenol stimulation was blocked completely by propanolol, a β-blocking agent, but not by phentolamine, an α-blocking agent. These results reveal that bovine calf aortic and pulmonary artery endothelial cells are responsive to catecholamines and prostaglandins, and therefore presumably possess both sensitive adenylate cyclases and plasma membrane receptors for these compounds. This work was supported by a Young Investigator Grant HL-21189 from the National Institutes of Health, United States Public Health Service.     

Stimulation of cyclic AMP production by vasoactive agents in cultured bovine aortic and pulmonary artery endothelial cells

The ability of selected vasoactive agents to influence cyclic AMP levels of confluent, early-passaged bovine calf aortic and pulmonary artery endothelial cells was investigated. Among the agents tested, only the catecholamines (isoproterenol, epinephrine, norepinephrine) and prostaglandins (PGE1, PGE2, PGF2 alpha) resulted consistently in increased cyclic AMP production in both cell populations. The degree of cyclic AMP stimulation obtained with other vasoactive compounds (angiotensins I and II, bradykinin, and serotonin) tended to be either very small or difficult to reproduce. Isoproterenol stimulation was blocked completely by propanolol, a beta-blocking agent, but not by phentolamine, and alpha-blocking agent. These results reveal that bovine calf aortic and pulmonary artery endothelial cells are responsive to catecholamines and prostaglandins, and therefore presumably possess both sensitive adenylate cyclases and plasma membrane receptors for these compounds.     

Role of phosphodiesterase in cyclic AMP signaling in cultured rat granulosa cells

Inactivation of the cyclic nucleotide signal in granulosa cells depends on a complex array of cyclic nucleotide phosphodiesterases (PDE). In order to examine the role of PDE in cyclic AMP (cAMP) signaling in granulosa cells, the present study examined the expression of PDE4D proteins and regulation of cAMP-PDE activities in cultured rat granulosa cells. The results of immunoblot analyses showed that two predominant PDE4D subtypes of approximately 80 and 70 kDa appeared when immature rat granulosa cells were treated with FSH. However, these two new subtypes presumed to be PDE4D proteins were not influenced by treatments of DETA/NO, cGMP and PKB inhibitor, LY294002. Immature rat granulosa cells treated with medium alone displayed low cAMP-PDE activity throughout 48 h of culture while those treated with FSH (2 ng.mL-1) showed a marked increase in cAMP-PDE activity between 6 and 12 h of culture, followed by a decline. The findings from the present study indicate that the increased cAMP-PDE activity by FSH is mainly related to the changes of PDE4D protein levels. However, the inhibitory effects of NO on cAMP accumulation in rat granulosa cells are not via the increased cAMP-PDE activity.     

Ethanolamine metabolism in cultured bovine aortic endothelial cells

The role of extracellular ethanolamine in phospholipid synthesis was examined in cultured bovine aortic endothelial cells. Serine and ethanolamine were both readily accumulated by these cells and incorporated into phospholipid. Exposing cells to extracellular ethanolamine for 4-6 weeks had no effect on cell growth, yet increased the phosphatidylethanolamine content of these cells by 31% as compared to control cells. The intracellular content of ethanolamine was measured by high performance liquid chromatography, and results showed that the ethanolamine-treated cells contained a significantly greater amount of free ethanolamine compared to control cells (0.62 +/- 0.07 nmol/mg of protein versus 0.27 +/- 0.05 nmol/mg of protein, respectively). Ethanolamine-treated cells also had decreased accumulation and incorporation into lipid of [3H]ethanolamine throughout a 48-h incubation and increased K'm and V'max parameters of ethanolamine transport as compared to control cells. Studies were also done to examine the effect of ethanolamine on the generation of free ethanolamine from phosphatidylserine. In pulse-chase experiments with [3H]serine, a physiological concentration of ethanolamine (25 microM) decreased the amount of 3H-labeled phosphatidylethanolamine produced from 3H-labeled phosphatidylserine by 12 h as compared to the amount of 3H-labeled phosphatidyl-ethanolamine produced in the absence of ethanolamine in the chase incubation. Furthermore, ethanolamine-treated cells accumulated 20% less labeled ethanolamine in the aqueous pool from [3H]serine after 24 h of incubation than did control cells. These results can be explained by isotope dilution with the ethanolamine pool that accumulates in these cells with time when exposed to media supplemented with a physiological concentration of ethanolamine and by an effect of ethanolamine on ethanolamine generation from phosphatidylserine. The results show that an extracellular source of ethanolamine significantly influences the phospholipid metabolism of cultured bovine aortic endothelial cells.     

Plasminogen activator activity by cultured bovine aortic endothelial cells

This communication reports the observations that bovine aortic endothelial cells (EC) in culture during their log phase of growth secrete plasminogen activator. Hydrocortisone, dibutyryl cAMP, theophylline, colchicine and cycloheximide, dependent upon concentration, inhibit plasminogen activator activity. Several substances associated with inflammation and thrombosis, such as thrombin, serotonin,catecholomines, histamine, vasopressin, endotoxin, and indomethacin, at the concentrations tested, did not significantly alter plasminogen activator activity when compared with controls.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

Ethanolamine and choline transport in cultured bovine aortic endothelial cells

The transport of the polar head groups, ethanolamine and choline, was examined in cultured bovine aortic endothelial cells. Both ethanolamine and choline are taken up by high- and low-affinity systems. The K'm and V'max for the Na+-dependent, high-affinity ethanolamine and choline transport system are 3.0 and 3.0 microM and 5.4 and 7.3 pmol/mg protein/min, respectively. Ethanolamine and choline competitively influence one another's transport as the presence of 50 microM ethanolamine increases the K'm but not the V'max of choline uptake. Likewise, 50 microM choline increases the K'm but not the V'max of ethanolamine transport. The concentration of ethanolamine that inhibits maximal velocity of 5 microM choline by 50% is 9.7 microM, while 12 microM choline inhibits 5 microM ethanolamine maximal velocity by 50%. Uptake of both head groups is only partially Na+-dependent and is inhibited similarly by 2-methylethanolamine and 2,2-dimethylethanolamine at all concentrations examined. Hemicholinium-3, a classic inhibitor of high-affinity, Na+-dependent choline transport, reduces both ethanolamine and choline accumulation in a concentration-dependent fashion, but has a greater effect on choline transport at higher concentrations. The major portion of these data is consistent with our hypothesis that the uptake of physiological concentrations of ethanolamine and choline may occur through the same transport system. However, the results of the effect of hemicholinium-3 and the extent of Na+-dependency of choline and ethanolamine uptake could be interpreted as meaning that separate transport systems for choline and ethanolamine exist which cross react or that a single transport system exists which has separate active sites for the two compounds.     

Protein activator of cyclic AMP phosphodiesterase and cyclic nucleotide phosphodiesterase in bovine retina and bovine lens. Activity, subcellular distribution and kinetic parameters.

We have examined the activity of cyclic AMP phosphodiesterase, cyclic GMP phosphodiesterase and the protein activator of cyclic AMP phosphodiesterase in various anatomic and subcellular fractions of the bovine eye. Cyclic GMP hydrolysis was 1.6--12 times faster than hydrolysis of cyclic AMP in the subcellular fractions of the retina and in the precipitate of the rod outer segment. An opposite pattern was seen in the bovine lens, where the hyrolysis of cyclic AMP occurred 17 and 169 times faster than that of cyclic GMP in the supernatant and precipitate of lens, respectively. The activity of cyclic AMP phosphodiesterase was not affected by ethylene-glycol bis(beta-aminoethylether)-N,N'-tetraacetic acid in any fractions except in the retinal supernatant, suggesting that the phosphodiesterase exists primarily as a Ca2+-independent, activator-independent form. However, the protein activator of cyclic AMP phosphodiesterase existed in all fractions examine. A complex kinetic patternwas observed for both cyclic AMP and cyllic GMP hydrolysis by the 105000 times g lens supernatant. The Michaelis constants for both cyclic AMP (1.3-10(-6) and 9.I-10(-6) M) and cyclic GMP (1.04-10(6) AND 1.22 10(-5) M) appeared to be similar.     

Dibutyrylic cyclic AMP and theophylline inhibit proliferation of accessory cells in primary cultures of adrenomedullary cells

Summary Studies on isolated adrenal chromaffin cells in primary cultures may be seriously hampered by the presence of non-chromaffin, mainly fibroblast-like cells, which always occur in dissociates of adrenal medullary tissue and often outnumber the chromaffin cells by the end of the first week of culture, when no measures are taken to control their proliferation. The present study offers a new means to inhibit effectively the proliferation of these accessory cells by treating the cultures with dibutyrylic cyclic AMP (dbcAMP, 0.1 or 0.01 mM) and equimolar amounts of the phosphodiesterase inhibitor theophylline. With this treatment cultures of young rat adrenal chromaffin cells remain virtually free of accessory cells for two weeks of culture. Cultures of bovine adrenomedullary cells retain their initial amounts of non-chromaffin cells, which largely depends upon whether the primary cell suspensions have undergone differential plating prior to seeding. Suppression of accessory cell proliferation with dbcAMP and theophylline is partly due to maintaining differentiation of cortical cells, which otherwise dedifferentiate into rapidly dividing fibroblast-like elements. However, a more direct action of dbcAMP on accessory cells in terms of growth control is also conceivable. DbcAMP and theophylline in the doses applied do not impair the viability, ultrastructure and catecholamine-storing capacity of cultured chromaffin cells.     

Effects of phosphodiesterase inhibitors, imidazole and phosphate on cyclic CMP phosphodiesterase are different from those on cyclic AMP and cyclic GMP phosphodiesterases.

The effects of various agents on the newly identified cyclic CMP phosphodiesterase (C-PDE) in crude extracts of a number of rat tissues and on the enzyme partially purified from the rat liver were examined. Papaverine and 1-methyl-3-isobutylxanthine were without effects on C-PDE at concentrations that inhibited up to 90% of cyclic AMP phosphodiesterase (A-PDE) and cyclic GMP phosphodiesterase (G-PDE) activities. When assayed using 1 micron substrates, theophylline inhibited C-PDE to a lesser extent than A-PDE and G-PDE. 2'-Deoxy cyclic AMP (specific A-PDE inhibitor) and 2'-deoxy cyclic GMP (specific G-PDE inhibitor) were relatively poor and non-specific inhibitors for C-PDE. Imidazole, while augmenting the high Km A-PDE and G-PDE from the liver but not from the heart, was without effect on the liver C-PDE but stimulated the heart C-PDE. Potassium phosphate was more specific in inhibiting C-PDE than A-PDE and G-PDE. The present findings suggest that C-PDE represents a potential site of specific pharmacological regulations, and that C-PDE may be a separate enzyme distinguishable from the purine cyclic nucleotide class of phosphodiesterases.     

Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium

Bovine aortic endothelial cells were cultured in medium containing [3H]glucosamine and concentrations of [35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of [3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower [35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal [35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in [3H]chondroitin [35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in [3H] heparan [35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation.     

Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine-evoked release of adenosine from spinal cord synaptosomes.

The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1-100 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta-methylene ADP and GMP, which inhibits ecto-5'-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of alpha,beta-methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

Activation of cultured bovine aortic endothelial cells by extracellular pyrimidine triphosphate.

1. The addition of ATP to cultured bovine aortic endothelial cells induced the increase in intracellular free calcium concentration ([Ca2+]i) and thereby activated the sodium/proton exchanger and the prostacyclin production in a similar dose-dependent manner, as observed by the addition of ATP. 2. Other nucleoside triphosphates also activated the cells and the potency orders of the nucleotides were ATP greater than UTP greater than ITP greater than CTP greater than GTP for all the responses. 3. Pretreatment of the cells with UTP desensitized the response to ATP and the pretreatment of ATP desensitized the response to UTP. 4. The responses to ATP and UTP were inhibited by neither pertussis nor cholera toxin. 5. The receptor for UTP, however, may be a distinct pyrimidinoceptor different from the purinoceptor of the cells for ATP, because the 50% effective concentration of UDP was much larger than that of UTP, while ATP and ADP were essentially equipotent ligands to the endothelial cells.     

Insulin control of cyclic AMP phosphodiesterase

Cyclic AMP phosphodiesterase (PDE) is an enzyme involved in cellular homeostasis of cyclic AMP. It exists as multiple isozymes in cells, but only the high affinity, membrane-bound isozyme is sensitive to hormonal modulation. Several isozymes or isoforms of the low Km PDE have been detected. Data suggest that several mechanisms exist for hormonal modulation of PDE. Activity of the low Km PDE species may be modulated by phosphorylation/dephosphorylation, phospholipid substrate concentration, insulin second messenger, cyclic GMP, guanine nucleotide binding proteins, calmodulin, or aggregation/disaggregation of monomeric forms. Modulation of PDE isoforms by different hormones may be through different regulatory components or mechanisms.     

The subcellular localization of calmodulin, cyclic AMP phosphodiesterase, and adenylate cyclase in bovine adrenal medulla

The subcellular localization of calmodulin, cyclic nucleotide phosphodiesterase, and adenylate cyclase was studied in bovine adrenal medulla. Approximately 70% of the calmodulin and 90% of the cAMP phosphodiesterase activities were found colocalized in the cytoplasm. The subcellular distribution of adenylate cyclase closely paralleled the distribution of acetylcholinesterase, a marker for plasma membranes. The fraction of calmodulin which is particulate in nature has a distribution profile very similar to that of adenylate cyclase. The chromaffin granule fraction contained only 0.86% of the total cAMP phosphodiesterase, 0.41% of the total adenylate cyclase, and 1.4% of the total calmodulin.