Cyclic AMP stimulated protein kinase activity within the secretory vesicle fraction of rat islets.

The isolated rat islet secretory vesicle fraction (24,000xg) was incubated with gamma [³²P ATP] and the TCA-insoluble material was pelleted. The pellet was acid hydrolyzed, lyophilized, redissolved, then subjected to two-dimensional chromatographic separation. Two labeled compounds were identified, i.e., phosphoserine and inorganic phosphorus. With the addition of cyclic AMP (3.5 × 10^?6M), there was a 235% increase in phosphoserine radioactivity (P<.01) within endogenous protein subunits. When histones were added to the incubation media, the addition of cyclic AMP resulted in a threefold increase in phosphoserine radioactivity in the TCA-insoluble material (P<.01). A comparison was made of cyclic AMP-stimulated protein kinase activity in the homogenate and various islet subcellular fractions. Cyclic AMP-stimulated protein kinase activity is associated with the 24,000xg (secretory vesicle) fraction.     

Cyclic AMP in plasmacytoma cells

Summary In Balb/c plasmacytoma cells (MOPC 315) the synthesis and secretion of IgA in vitro was suppressed by prior incubation of the tumor cells with anti-Balb/c alloantibody. In tumor cells so treated, the levels of cyclic AMP were found to be increased. The alloantibody, either as alloantiserum or ascitic fluid globulins from allogeneic strains of mice immunized with spleen cells, had been differentially absorbed with Balb/c spleen cells. Such absorption reduces the cytotoxic antibody effect to below detectable levels, as measured by Trypan Blue exclusion, but permits retention of substantial levels of the suppressive antibody effect. The range of dilutions of alloantibody causing the increase in cAMP corresponded with those that caused suppression of IgA secretion. When tumor cells were exposed to agents that increased the level of cAMP either endogenously or exogenously, suppression of secretion of IgA was found, similar to that caused by the alloantibody. When submaximal concentrations of suppressive antibody were combined with submaximal levels of the cAMP-increasing agents greater degrees of suppression were found, suggesting an additive effect. No cytotoxic effect on these tumor cells, as indicated by Trypan Blue exclusion, was caused by these cAMP-increasing agents at the concentrations shown, either alone or in combination with the suppressive antibody.     

Characterization of the cytoplasmic androgen receptor of rat seminal vesicle

Postmitochondrial supernatant (PMS) (1) has been prepared from the homogenate of rat seminal vesicles and the characteristics of the binding reaction of 5-dihydrotestosterone (DHT) to the cytoplasmic androgen receptor have been studied using a charcoal adsorption procedure.

At 0°C apparent equilibrium of binding is reached between 60 and 90 min of incubation but no exchange of bound (³H)DHT can be observed in the presence of a 100-fold excess of unlabelled DHT.

Saturation analysis shows a single class of independent binding sites for DHT with an apparent dissociation constant of 1 nM at 0°C and 2 nM at 25°C. Concentration of binding sites is in the range of 25–80 fmoles/mg protein.

When not occupied by DHT the receptor molecules are inactivated spontaneously following first order reaction kinetics. A rate constant of 0.27 hours⁻¹ at 0°C was determined for the inactivation reaction.

In the (³H)DHT-binding reaction testosterone and 19-nortestosterone are even more efficient competitors than unlabelled DHT, while hydrocortisone does not compete at all. On the other hand significant binding of (³H) testosterone could not be demonstrated.

The (³H)DHT-receptor complex is precipitated from the cytosol by 0 to 33% saturation of ammonium sulphate and sediments as a single, 3.1 S peak in sucrose gradients prepared in 0.4 M NaCl.     

Cyclic AMP in the cell cycle of Physarum polycephalum

Cyclic AMP, [³H]thymidine incorporation, and DNA content were measured in the cell cycle of Physarum polycephalum. A sensitive radioimmunoassay was employed to assay cyclic AMP so that plasmodia could be assayed individually. In contrast to previously published results (Lovely, J.R. and Threlfall, R.J. (1976) Biochem. Biophys. Res. Commun. 71, 789–795), no pre-mitotic peak of cyclic AMP was detected. In seven experiments levels of cyclic AMP showed only small changes in individual experiments and ranged from 1–6 pmol/mg protein in different experiments. When plasmodia in the immediate premitotic period were collected on the basis of nuclear mitotic morphology, no evidence of a peak of cyclic AMP was found. Light was found to increase plasmodial cyclic AMP in a rapid, transient fashion. However, the brief exposure of cell cycle samples to light during collection did not induce any apparent cell cycle specific peaks of cyclic AMP. Although the occurrence of extremely rapid transient peaks of cyclic AMP in the cell cycle cannot be ruled out, it appears likely that the P. polycephalum cell cycle can proceed normally without major changes in cyclic AMP.     

Internal surface and fine structure of the rat seminal vesicle.

The seminal vesicle of the rat was studied with scanning and transmission electron microscopy. The internal surface of the organ is partitioned into small areas by a system of elevated ridges of connective tissue and covered by a columnar epithelium. This consists of small basal cells, presumably reserve elements, and larger ones containing the typical cell organelles involved in protein synthesis. The surface of the cell is covered with slender microvilli, varying in height and number from region to region. It is suggested that they are involved in the maintenance of hydration and/or regulation of low molecular weight substances in the secretion product. The latter develops from small granules which pass the apical cell border and then fuse together as larger drops.     

Autophagy in the epithelial cells of murine seminal vesicle in vitro

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pretreatment in a medium containing 0.03% Triton X-100. In addition to the significant increase in the cytoplasmic volume fraction and the mean size of autophagic vacuoles, the appearance of shorter or longer smooth membrane pairs located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological features observed after application of various fixation methods, freeze-substitution and freeze-fracture techniques show that they are unclosed nascent isolation membranes, representing a unique class of intracellular membranes. They may grow around the nucleus, leading to its complete autophagic sequestration and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes, leading to their regression with a halving time of 7 min. In contrast, these inhibitors cause extremely fast shrinking of nascent isolating membranes, leading to their complete disappearance within 7 min. We propose that the early events of autophagy involve three main steps: initiation, growth and closure, and suggest that the growth of nascent isolation membranes is reversible i.e. the membranes may be subject to disassembly before their closure is completed. Bending and closure of the isolation membrane and the stability of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by phosphoinositide kinases as indicated by wortmannin sensitivity.     

Cyclic AMP levels during the maturation of Xenopus oocytes

While several studies have suggested that the induction of oocyte maturation results from a transient decrease in cAMP levels, attempts to demonstrate such a change have led to inconsistent results with respect to whether or not a decrease occurs as well as timing of the decrease. In this report the results of experiments designed to demonstrate small changes in cAMP content in Xenopus laevis oocytes are presented and a statistically significant 20% decrease in cAMP content from between 2 and 50 min postprogesterone addition is found. The cAMP content subsequently rises to 12% higher than control levels and then becomes indistinguishable from control values for the remainder of the maturation period.     

Cyclic changes in levels of cyclic AMP and cyclic GMP in frog myocardium during the cardiac cycle

The phase diagrams of binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine, and cholesterol and dimyristoyl phosphatidylcholine in the presence of excess water have been investigated using spin labels.     

Cellular autophagocytosis in mouse seminal vesicle cells in vitro

Cellular autophagocytosis was observed in mouse seminal vesicle cells incubated in vitro up to 8 h in medium 199 or Krebs-Ringer bicarbonate buffer. During the first 2 h of incubation, early forms of autophagic vacuoles were seen in the cells, advanced forms containing degraded material began to cumulate later. After 6--8 h, early vacuoles occurred sparsely, while advanced forms were detected in a great number. During the first 2 h of incubation, we often observed smooth surfaced membrane pairs between the cisternae of rough surfaced endoplasmic reticulum resembling isolating membranes of autophagic vacuoles. They varied in size and shape from short, straight cisternae to long, curved ones, almost completely encircling areas of the cytoplasm. Based on these observations, we propose a tentative scheme of the formation of autophagic vacuoles, viz., the short, straight cisternae would represent the first stage in the development of an autophagic vacuole, while the curved sack-like forms are interpreted as successive steps leading to the complete sequestration of an area of the cytoplasm.     

Cyclic AMP calcium and the growth of mastocytoma cells

Arresting P815 mastocytoma cell growth with N6, O2'-dibutyryladenosine 3':5' cyclic monophosphate (db cAMP) and theophylline increased 45Ca2+ uptake and efflux by the cells (i.e, Ca2+ cycling) without altering cytoplasmic free Ca2+ concentrations or the amount or distribution of protein kinase C in the cells. Attempts to identify the Ca2+ channels involved using a wide variety of drugs were unsuccessful. However, the inhibitory effect of db cAMP on growth was greatly increase in medium containing low Ca2+ concentrations, confirming that interactions between Ca2+ and cyclic AMP can affect mastocytoma cell growth.     

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.     

Cyclic AMP, cyclic GMP and glucose utilization by diabetic rat fat cells

Fat cells from epididymal adipose tissue from normal and streptozotocin-diabetic rats were studied to determine glucose utilization and cyclic nucleotide levels. Diabetic rat fat cells present a higher cAMP content (P less than 0.05) compared with controls. Addition of insulin decreases within 10-min incubation the cAMP content in both normal and diabetic cells (P less than 0.05). However, the value obtained in the latter remains by 25% higher than that of normal cells not exposed to insulin. No changes in cGMP were detected. Pretreatment of the diabetic animals during two days with propranolol (1 mg kg body wt-1 day-1) induces the decrease to normal levels of the fat cell cAMP content. However, it persists the impairment on glucose utilization observed in fat cells from diabetic animals. It seems that the increase in the intracellular amount of cAMP found in fat cells from diabetic rats is not involved, at least directly, to the impaired glucose utilization found in the diabetic state. Furthermore, through an unknown mechanism, pretreatment with propranolol can induce a drop in fat tissue cAMP toward normal values without normalizing glucose utilization.