Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro

The results reported here show some characteristics of adenylate cyclase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F⁻ under conditions where cyclic nucleotide degradative pathways are effectively inhibited.Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 × g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E₁ and E₂, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F_1α and F_2α. Stimulation by prostaglandin E₁ or E₂ is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E₁ is equal to that caused by optimal fluoride concentrations. Stimulation c caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.     

Beta-Galactosidase immunoassay for the measurement of cyclic AMP

The previously described method for phenotyping of alpha₁-antitrypsin (alpha₁-protease inhibitor, Pi) that utilizes separator isoelectric focusing on thin-layer agarose gel (A. R. Qureshi and H. H. Punnett, in Electrophoresis '81, 3rd International Conference on Electrophoresis, pp. 83–87 (1981)) has been improved to give a better resolution of Pi pattern. A shallow pH gradient in the region of the isoelectric point of Pi pattern was obtained by the use of N-(2-acetamido)-2 aminoethanesulfonic acid (1%) and serine (0.8%). The present technique can resolve the Pi alleles. The patterns of Pi phenotypes were found to be similar to those observed on acrylamide gels. The method is fast, reliable, and reproducible.     

Purification and properties of cyclic AMP dependent and independent protein kinases from rat pancreas.

Three protein kinases Ko, K1, and KII have been extracted from rat pancreas homogenate, Ko is not stimulated by cyclic AMP. K1 is poorly stimulated by cyclic AMP (1.3 times), Ku is highly stimulated (6 times). The specificity of KII with respect to various nucleotides and cyclic nucleotides has been determined. K1 and KII account for the total cyclic AMP dependent protein kinase activity in the homogenate.     

The permeability coefficient of albumin of the isolated rat mesentery Modification by some mediators of inflammation,cyclic AMP and calcium

In order to study the mechanisms whereby mediators of inflammation exert their exudative effects, we used isolated rat mesentery placed as a separation membrane between the two compartments of a diffusion cell. In this experimental arrangement, the permeability coefficient of albumin (P_A) can be easily computed from the equilibration rate of ¹²⁵I-labelled albumin added to one compartment. Histamine, bradykinin, serotonin and prostaglandins A₁, A₂, E₁, E₂, F_1α and F_2α all increased P_A to some extent, the maximal values being approx. +60%. Dibutyryl-cyclic AMP, theophylline and isoproterenol also increased P_A, thus suggesting involvement of cyclic AMP. Direct measurements of this nucleotide confirmed this hypothesis; furthermore, a linear relation between cyclic AMP levels and P_A could be demonstrated. In contrast, cyclic GMP is probably not involved in the control of P_A. Calcium-depleted tissues had a low P_A (approx. 40% below controls), and did not respond to exogenous prostaglandin E₁. These results suggest that transmesenteric passage of albumin is at least partly controlled by cyclic AMP and intracellular Ca²⁺ levels.     

Mammalian brain microtubules are sensitive to cyclic AMP in vitro

Microtubules assembled in vitro with ATP were depolymerized by the addition of cyclic AMP, which correlates with a stimulation of the endogeneous phosphorylation reaction. When assembled with GTP, however, microtubules were only sensitive to cyclic AMP when ATP was present. This nucleoside triphosphate induced the disassembly of microtubules in a concentration-dependent, cyclic nucleotide-stimulated manner. Since UTP, CTP and the nonhydrolyzable ATP analog adenosine-5'-(beta, gamma-methylene)triphosphate were without comparable effect, it was assumed that phosphorylation of the microtubule-associated proteins may represent a physiological mechanism by which microtubules in the living cell respond to external stimuli.     

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney

High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg²⁺, Mn²⁺, and Co²⁺ each supported activity, but highest activity was obtained with Mg². The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E_a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The K_m for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The K_i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K_m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K_i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.     

Influence of epidermal growth factor and cyclic AMP on growth and differentiation of palatal epithelial cells in culture

A serum-free, hormonally defined medium was developed which supports growth and differentiation in primary culture of epithelial cells from prefusion embryonic mouse palatal shelves. Using this culture system, medial epithelial programmed cell death was investigated. In the absence of EGF, medial epithelial cells undergo cell death and detach from the substratum by 24 hr of culture. The addition of EGF alone or in combination with various agents which increase intracellular cyclic AMP levels prevented medial epithelial cell death in both cell and organ culture. EGF appeared to exert its most dramatic effect in cell culture on growth and differentiation of the squamous oral epithelial cells. In addition, EGF and agents such as 8-bromo-cyclic AMP, dibutyryl cyclic AMP, or cholera toxin synergistically stimulated the appearance of a long-lived, rapidly proliferating cell type by Day 4 of culture. Our results suggest that both EGF and cyclic AMP together may be important in regulating proliferation of embryonic palatal epithelial cells.     

Quantitative analysis of cyclic AMP waves mediating aggregation in Dictyostelium discoideum

We have previously reported the detection of cAMP waves within monolayers of aggregating Dictyostelium discoideum cells (K. J. Tomchik and P.N. Devreotes, 1981, Science 212, 443-446). The computer-assisted analysis presented here of the fluorographic images of the cAMP waves reveals (1) all the waves have a consistent width and height; (2) cAMP concentrations within centers of concentric aggregation territories oscillate periodically while at spiral centers the concentration builds up to a plateau value within 2 mm; (3) cells within the region of intersection of two oppositely directed cAMP waves are stimulated to produce more cAMP than those responding to a single wave; (4) cells start to move when the cAMP level begins to increase and cease movement when the peak cAMP concentration reaches the cell.     

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.     

Dissociation of cyclic AMP accumulation from that of luteinizing hormone (LH) release in response to gonadotropin releasing hormone (GnRH) and cholera enterotoxin.

Anterior hemipituitaries from female rats were incubated $\text{in}\text{vitro}$ in Krebs Ringer bicarbonate buffer, pH 7.2 containing 2 mg/ml of glucose in the absence and in the presence of GnRH or cholera enterotoxin. Following this incubation, the pituitaries were separated from the medium and cAMP and LH were assayed in the tissue and the medium, respectively. Incubations with GnRH in the range of 25 ng/ml to 400 ng/ml resulted in increase in LH release into the medium. Cholera enterotoxin at a concentration of 1 μg/ml, by contrast, caused no release of LH into the medium, but caused a 5-fold increase in cAMP level and this effect was concentration dependent. Cholera enterotoxin did not interfere with the GnRH-mediated LH release. It is concluded from these experiments that the ability of GnRH to increase cAMP level may be independent of its ability to release LH.     

Correlation between changes in intracellular level of cyclic AMP, activation of cyclic AMP-dependent protein kinase, and the morphology of Chinese hamster ovary cells in culture.

The morphological conversion of Chinese hamster ovary cells induced by treatment with dibutyryl cyclic AMP is correlated with increases in the intracellular level of cyclic AMP and the activation of cyclic AMP-dependent protein kinase. When cholera toxin is used to induce the increase in intracellular cyclic AMP, a similar correlation is obtained. Treatment of cells with prostaglandin E₁, which causes a transient increase in intracellular cyclic AMP and a transient activation of protein kinase activity, does not result in the morphology change. From these studies we conclude that a stable activation of the cyclic AMP-dependent protein kinase, which results from an increase in intracellular cyclic AMP, induces the morphological conversion of Chinese hamster ovary cells through phosphorylation of one or more cellular components.     

Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.     

Regulation of abalone sperm cyclic AMP concentrations and the acrosome reaction by calcium and methylxanthines

When Ca2+ is added to abalone sperm (Haliotis rufescens) in Ca2+-free artificial seawater (CaFASW) to a final concentration of 9.6 mM a 4-fold elevation in sperm cAMP occurs within 15-30 sec. The methylxanthines, theophylline and 1-methyl-3-isobutylxanthine (MIX), also elevate sperm cAMP concentrations. In CaFASW, either compound causes up to a 3-fold increase in cAMP within 15-30 sec. MIX (150 microM), added to sperm in the presence of 9.6 mM Ca2+, elevates sperm cAMP 100-fold within 15-30 sec and also triggers the acrosome reaction (AR) in the same period. Under identical conditions theophylline (1.67 mM) is much less potent at elevating cAMP and inducing the AR. The effects of methylxanthines on cAMP of sperm incubated in the presence of Ca2+ appear to represent a potentiation by these compounds of the action of Ca2+. Neither compound induces the AR in the absence of Ca2+. All of the observed effects on sperm cAMP and the AR are dependent on Ca2+ and methylxanthine concentrations. Added cyclic nucleotides or their derivatives do not induce the AR in either the absence or presence of Ca2+. Experiments with isolated sperm heads and flagella indicate that the dramatic stimulatory response of sperm cAMP to Ca2+ plus MIX is present in the head region (acrosome, nucleus, midpiece) of the cell. The data suggest that the dramatic elevation of cAMP by MIX in the presence of Ca2+ may occur directly by an inhibition of phosphodiesterase activity and indirectly by an increase in cellular Ca2+. A strong temporal correlation between the cAMP elevation and the abalone AR exists, although cAMP elevation by itself does not act as the primary mediator of this exocytotic event.     

Regulation of ovarian steroidogenesis: The disparity between 125I-labelled choriogonadotropin binding,cyclic adenosine 3′,5′-monophosphate formation and progesterone synthesis in the rat ovary

The binding of ¹²⁵I-labeled human choriogonadotropin, formation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP), and synthesis of progesterone were examined in ovarian cells from immature rats. Collagenase dispersed ovarian cells were found to respond specifically to lutropin-like activity. The equilibrium dissociation constant (K_d) for the binding of ¹²⁵I-labelled choriogonadotropin was 1.7 · 10^1?10 M. Progesterone synthesis was increased at least 40-fold and cyclic AMP formation 10-fold in response to maximum hormonal stimulation. The concentration of choriogonadotropin which stimulated progesterone synthesis maximally in Eagle's minimum essential medium ?0.1% gelatin (2 ng/ml), resulted in minimal (less than 30% of maximum) increases in cyclic AMP accumulation and hormone bindind. Similarly, binding of choriogonadotropin was not saturated at a hormone concentration (50 ng/ml) that stimulated maximal cyclic AMP formation. These results are consistent with the existence of receptor reserve in the ovarian cell. A marked shift in the dose vs. response relationship for progesterone synthesis occurred when fetal calf serum was used to supplemen Eagle's minimum essential medium, however. Under these experimental conditions, progesterone synthesis reached a maximum at a hormone concentration of the same order of magnitude as did cyclic AMP formation. It is concluded that the degree of spare receptor effect observed may depend not only on an absolute amount of excess receptor, but also on the readiness of the system to respond in a given fashion.     

Effect of aging on neurotransmitter receptor binding in rat and human brain.

β-Adrenergic and GABA receptor binding were measured in brain areas of rats 3 to 24 months of age. While GABA receptor binding was not significantly different across age in any area, β-adrenergic receptor binding was significantly reduced in the cerebellum and brain stem, but not cerebral cortex, of 24-month-old animals. The loss in β-adrenergic receptor binding does not correlate in a temporal fashion with the reported decrease in norepinephrine-stimulated cyclic AMP accumulation in the cerebellum which occurs as early as 12 months of age. An age-related reduction in β-adrenergic binding was also noted in human cerebellar tissue obtained at autopsy, suggesting that the cerebellar dysfunction seen with aging may be related to a loss of cerebellar neurons which receive noradrenergic input.     

Effect of estradiol and progesterone on glucosamine 6-phosphate synthase in the uterus of rat

Injection of estradiol to ovariectomised rats caused significant increase in the activity of glucosamine 6-phosphate synthase of uterus. Progesterone did not cause any increase in the activity of the enzyme; however, it antagomised the effect of estradiol. It was observed that the enzyme is predominantly localised in the endometrium of uterus.     

Selective expression of type I and type II cyclic AMP-dependent protein kinases in subcellular fractions of concanavalin A-stimulated rat thymocytes

Total protein kinase activity and the expression of the type I and type II cyclic adenosine 3′:5′-monophosphate-dependent protein kinases were studied in subcellular fractions of rat thymocytes and the effect of concanavalin A treatment on protein kinase activity was assessed. At a concentration of 100 μ/ml of concanavalin A a marked decline of total nuclear protein kinase activity occurred which lasted approximately 20 to 90 min. Concomitantly, a twofold increase of total protein kinase activity in the 900g supernatant fraction was observed which lasted from 5 to 30 min. Studies using the heat-stable protein kinase inhibitor revealed that the concanavalin A-mediated activity changes were primarily due to changes of cAMP-dependent protein kinase activity, whereas cAMP-independent protein kinase activity remained unchanged. Analysis of the type I and type II cAMP-dependent protein kinase isozyme pattern before and after concanavalin A treatment revealed a selective change of the relative expression of isozyme activities. Whereas type I protein kinase was the major nuclear isozyme before concanavalin A treatment, nuclear type II cAMP-dependent protein kinase increased markedly with a concomitant loss of type I isozyme expression. In the 900g supernatant fraction, containing primarily the type II isozyme in unstimulated cells, concanavalin A treatment caused an increase of the expression of the type I isozyme. The concanavalin A-mediated relative changes of cAMP-dependent protein kinase isozyme expression were confirmed by photoaffinity labeling of the regulatory subunits R^I and R^II before and after concanavalin A stimulation. The intracellular concanavalin A-mediated isozyme changes were time dependent, exhibiting maximal effects about 20 min after concanavalin A addition. These results indicate that selective regulation of intracellular cAMP-dependent protein kinase isozyme expression may be a mechanism related to isozyme-specific phosphorylation of specific intracellular substrates in concanavalin A-activated thymocytes.