Post-radiation disorders in the cyclic AMP system of mouse spleen and thymus lymphoid cells]

Early alterations in the enzyme activities controlling cyclic adenosine-3',5'-monophosphate (cAMP) metabolism after the irradiation (800 rad) of mice were found in the lymphoid cells of the spleen and thymus. Postradiation disturbances in these activities' ration induced alterations in cAMP steady-state concentrations in the cell. It was also demonstrated that irradiation reduced lymphoid cell ability to accumulate cAMP in response to isoproterenol administration.     

Effect of thymalin on the function of the blood kallikrein-kinin system in thymectomized rats

The effects of thymus ablation and injection of thymalin on the blood plasma kallikrein-kinin system were studied. Thymus ablation was followed by activation of kinin formation, evidenced by an elevation of the total kallikrein activity and drop of the kininogen level. Injection of thymalin into thymectomized animals makes the characteristics under study return to normal.     

Multiple forms of cyclic nucleotide phosphotransferases in mouse thymocytes

The presence of cAMP- and cGMP- specific phosphodiesterases in mouse thymocytes has been demonstrated. Sucrose density gradient centrifugation revealed two peaks corresponding to cAMP-phosphodiesterase (3.6S and 6.0S) and one peak corresponding to cGMP-phosphodiesterase (6.6S). Gel filtration demonstrated high molecular forms of the enzymes; according to the rechromatography data, cAMP and cGMP phosphodiesterases are represented by oligomers containing subunits with Ms 45 000 and 160 000 respectively. Incubation of thymocyte lysates for 24-48 hours led to a decrease of the number of high molecular weight forms and an increase in the number of low molecular weight forms paralleled with a rise in V and Km values.     

Effect of adenosine on cyclic nucleotide levels in brain structures

Adenosine influence on cAMP and cGMP levels in cortex, hypothalamus, hippocampus and cerebellum was studied. It was established that adenosine and inhibitor of its reuptake--dipyridamole change cyclic nucleotide levels in some structures of brain (intraperitoneal injection). It was shown that cAMP and cGMP were in reciprocal relations in cortex, but not in hypothalamus, hippocampus and cerebellum.     

Effect of calcium on cyclic nucleotide phosphodiesterase in parathyroid tissue

The hydrolysis of cyclic AMP and cyclic GMP by homogenates of normal bovine parathyroid gland and human parathyroid adenomas was decreased by EGTA. When supernatants were chromatographed on DEAE-cellulose it was found that sheep brain calmodulin in the presence of calcium stimulated cyclic AMP and cyclic GMP phosphodiesterase activity. The response to calmodulin in two human parathyroid adenomas was less than that in normal bovine parathyroid. Calmodulin was detected in heat-treated supernatants of 11 parathyroid adenomas by its ability to activate calmodulin-free sheep brain phosphodiesterase. The results suggest a role for calcium in the hydrolysis of cyclic nucleotides in parathyroid tissue.     

Calcium and cyclic nucleotide regulation in incubated mouse retinas.

When retinas from dark-adapted C57BL/6 mice were incubated in the dark for 5 min at 37 degrees C in Earle's medium, they contained 80-120 pmol/mg protein of cGMP and about 13 pmol/mg protein of cAMP. When the incubation in darkness was in calcium-deficient Earle's medium with 3 mM EGTA, a 10-20 fold increase occurred in the cGMP level, peaking at 2-3 min, but no change occurred in cAMP. This elevated level fell in 3 min to normal dark levels on return to normal Earle's medium, but was still about three times that of control levels after 15 min in EGTA-containing solution. Bright light after 2 min of dark incubation of dark-adapted retinas resulted in a 40-50% fall in cGMP, and bright light sharply reduced the elevated dark cGMP level of retinas in calcium-deficient media with 3 mM EDTA. However, no depression of normal dark levels of cGMP has thus far been obtained by increasing external calcium levels, even in the presence of the ionophore A23187. All the above phenomena involving dark cGMP levels and calcium are similar in Earle's medium with 100 mM of K+ substituted for Na+. Congenic rodless (rd/rd) mouse retinas have less than 5% of control cGMP and show only traces of calcium sensitivity. Thus, the above phenomena in controls are likely to be largely occurring in rods. The data suggest a dependency of the dark cGMP level on the calcium level, but that the light-induced fall in cGMP may largely be calcium insensitive.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes

Guinea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E₁, D₁, F_1α, E₂, D₂, or F_2α. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment.Maximum stimulation of cAMP levels was observed with PGD₂, smaller increases with PGE₂ and relatively transient rises with PGF_2α which were of low significance, but confirm earlier data. Similar results were observed with PGD₁, PGE₁, and PGF_1α with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD₂ and PGE₂. With PGF_2α, maximum cGMP levels were noted after some delay.All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD₂ treatment.     

Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes.

Ginea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase of growth with prostaglandins E1, D1, F1 alpha, E2, D2, or F2 alpha. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment. Maximum stimulation of cAMP levels was observed with PGD2, smaller increases with PGE2 and relatively transient rises with PGF2 alpha which were of low significance, but confirm earlier data. Similar results were observed with PGD1, PGE1, and PGF1 alpha with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD2 and PGE2. With PGF2 alpha, maximum cGMP levels were noted after some delay. All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGD2 treatment.     

Effect of lipids on the binding of calmodulin with cyclic nucleotide phosphodiesterase

The effects of various lipids on calmodulin interaction with Ca-dependent phosphodiesterase were investigated. Palmitic, myristic and stearic acids increased the enzyme activity; the degree of the enzyme activation by calmodulin was decreased thereby. Oleic acid produced a weak activating effect on phosphodiesterase but completely blocked calmodulin action. The effects of the fatty acids under study were reversible, the activation constant was equal to 10(-4)-5 X 10(-4) M. In the presence of Ca2+ phosphoinositides and fatty acids changed the fluorescence intensity of dansyl-labelled calmodulin; in the absence of Ca2+ the lipids did not affect protein fluorescence. The lipids had no influence on the protein affinity for Ca2+. During chromatography of phosphodiesterase on calmodulin-Sepharose the enzyme was eluted from the column both in the presence of EGTA and palmitic acid. It was concluded that fatty acids prevent the formation of the calmodulin - phosphodiesterase complex. This effects may both be due to the lipid binding to the enzyme and to calmodulin.     

Age-related alterations in cyclic nucleotide phosphodiesterase activity in dystrophic mouse leg muscle

Previous reports have described both increased and decreased cyclic nucleotide phosphodiesterase (PDE) activity in dystrophic muscle. Total PDE activity was measured in hind leg muscle from a mouse model of Duchenne muscular dystrophy (mdx) and a genetic control strain at 5, 8, 10, and 15 weeks of age. Total PDE activity declined in fractions isolated from mdx muscle over this time period, but was stable in fractions from control mice. Compared with age-matched controls, younger mdx muscle had higher cAMP and cGMP PDE activity. However, at 15 weeks, fractions from both strains had similar cGMP PDE activity and mdx fractions had lower cAMP PDE activity than controls. Particulate fractions from mdx muscle showed an age-related decline in sensitivity to the PDE4 inhibitor RO 20-1724. A similar loss of sensitivity to the PDE2 inhibitor erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA) was seen in a particulate fraction from mdx muscle and to a lesser degree in control muscle. These results suggest that the earlier disagreement regarding altered cyclic nucleotide metabolism in dystrophic muscle may be due to changes with age in PDE activity of dystrophic tissue. The age-related decline in particulate PDE activity seen in dystrophic muscle appears to be isozyme-specific and not due to a generalized decrease in total PDE activity.     

Effect of somatostatin on the proliferation of mouse spleen lymphocytes in vitro

The effect of somatostatin on the spontaneous proliferation of mouse spleen lymphocytes was investigated in vitro. The rate of 3H-thymidine incorporation was used as an index of lymphocyte proliferation. Somatostatin in a concentration of 10(-7) M enhanced the lymphocyte proliferation and abolished the antiproliferative effect of rat hypothalamic extract. Lower concentrations of somatostatin slightly decreased the lymphocyte DNA synthesis.     

Effect of spleen and bone marrow regeneration on the number of hematopoietic colonies in mouse spleen]

The spleen (2/3) was removed in CBA male mice (the 1st group); in the 2nd group the bone marrow from the right posterior shin was removed. The hemopoietic splenic colonies were counted on the 8th day after the lethal irradiation and injection of 1 X 10(-6) nucleated cells of the intact spleen. A significant increase of the number of colonies in comparison with their number in control intact mice was observed. The authors suppose that this increase could also be caused by the local influence of the regenerating stroma of the spleen and by some stimulating factor discharge by the regenerating hemopoietic tissue.     

Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system

Concentrations of cAMP (cyclic adenosine 3′,5′-monophosphate) and cGMP (cyclic guanosine 3′,5′-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4–6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or Na Fstimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.     

Calcium-dependent protein modulator of cyclic nucleotide phosphodiesterases from mouse epidermis.

1. A heat-stable modulator protein was partially purified from mouse epidermis. The protein stimulated modulator-depleted cyclic AMP phosphodiesterase from bovine brain in the presence of Ca2+. 2. DEAE-cellulose chromatography of epidermal extracts demonstrated the presence of two main phosphodiesterase activities that hydrolysed both cyclic AMP and cyclic GMP. A minor peak was eluted between 0.1 and 0.3 M-sodium acetate and a major peak was eluted between 0.3 and 0.45 M-sodium acetate. 3. Cyclic AMP phosphodiesterase activity eluted at low salt concentrations was markedly activated by the epidermal modulator protein in the presence of Ca2+. Storage of the enzyme led to a decrease in its sensitivity to the protein modulator. 4. Treatment of mouse skin with the tumour promoter 12-O-tetradecanoylphorbol 13-acetate, which leads to an increase in epidermal cyclic nucleotide phosphodiesterase activity, did not alter the amount of modulator present in soluble epidermal extracts. The tumour promoter decreased the amount of modulator extractable from particulate epidermal preparations with Triton X-100.