Uptake and catabolism of N6, 2'-O-dibutyryl cyclic AMP by the perfused heart

Dibutyryl cyclic AMP when administered to perfused rat hearts produced increased contractile tension and increased intracellular levels of cyclic AMP. When [³H]dibutyryl cyclic AMP was administered, it was taken up by the heart in extremely small amounts. The material was present in the tissue throughout the duration of the inotropic response. The compound was slowly degraded to monobutyryl cyclic AMP and traces of cyclic AMP. It is suggested that N⁶-monobutyryl-cyclic AMP, or possibly cyclic AMP, may mediate the effects of the dibutyryl analog.     

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 microM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol.min(-1).mg protein(-1) in normoxic hearts and from 5 to 55 pmol.min(-1).mg protein(-1) in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity (A0.5) was 3 +/- 1 microM for hypoxic hearts and 28 +/- 13 microM for normoxic hearts. The A0.5 for alpha2-isoform AMPK activity was 2 +/- 1 microM for hypoxic hearts and 13 +/- 8 microM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK alpha-subunit. In potassium-arrested hearts perfused with variable O2 content, alpha-subunit Thr172 phosphorylation increased at O2 < or = 21% even though [AMP] was <0.3 microM. Thus hypoxia or O2 < or = 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].     

Histamine receptors and cyclic AMP

The identification and characterization of histamine receptors in the organ systems of various species has been made possible in recent years by the introduction of relatively selective agonists and antagonists of H1 and H2 receptors. H2 receptors have now been clearly demonstrated in gastric mucosa, heart, rat uterus, brain, and adipose tissue. Less well-defined H2 receptor systems have also been described in the vasculature, bronchioles, and other smooth muscles as well as in the thyroid gland and lymphocytes. In tissues where it has been examined a close correlation between H2 receptors and the adenylate cyclase--cyclic AMP system has been found. With the exception of the central nervous system stimulation of H1 receptors does not seem to be involved with cyclic AMP. In the case of the brain the H1 receptor stimulation of adenylate cyclase can be differentiated from H2 receptor stimulation of the enzyme by the use of blocking agents and by the fact that the H1 receptor response is enhanced in the presence of adenosine. Studies of the involvement of histamine with the adenylate cyclase--cyclic AMP system have been concentrated on such tissues as gastric mucosa, heart, rat uterus, brain, and adipose tissue. The present review will concentrate on the literature concerning those tissues.     

Changes in AMP deaminase activities in the hearts of diabetic rats

AMP deaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantitated by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile change to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP Km = 1.78, stimulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria AMP Km = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP Km = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP Km = 0.97, inhibited by ATP, NAD(P).     

Glycogen synthase kinases. Distribution in mammalian tissues of forms that are independent of cyclic AMP.

Extracts of rat tissues contain kinases which catalyze the conversion of glycogen synthease from the glucose 6-phosphate-independent (I) form to the glucose 6-phosphatate-dependent (D) form. These kinases were stimulated by adenosine 3':5' monophosphate (cyclic AMP). The glycogen synthase kinase activity ratio (activity in the absence of cyclic AMP divided by activity in the presence of cyclic AMP) varied from 0.28 to 0.97. The activity ratio for histone kinase in the same extracts ranged from 0.11 to 0.29. The levels of glycogen synthase kinase varied by a factor of 80 in the following rat tissues (given in order of decreasing enzyme activity): kidney, liver, stomach mucosa, lung, brain, heart, skeletal muscle, and adipose tissue. In the same tissues the levels of histone kinase varied by only a factor of 6 and did not correlate with the levels of glycogen synthase kinase. A modification of the method of Walsh et al. ((1971) J. Biol. Chem. 246, 1977-1985) was developed for purification of the heat-stable inhibitor of cyclic AMP-dependent protein kinases (inhibitor). The modified procedure resulted in good yields of highly purified inhibitor and was much simpler than the previously described procedure. This inhibitor completely inhibited cyclic AMP-dependent histone kinase activity of the extracts but much of the glycogen synthase kinase activity was not inhibited. The portion of glycogen synthase kinase that was insensitive to the inhibitor was: stomach mucosa, 95%; brain, 90%; liver, 82%; kidney, 81%; lung, 68%; adipose tissue, 65%; skeletal muscle, 63%; and heart, 54%. This histone kinase activity in the extracts and hte ratio of glycogen synthase kinase to histone kinase activity of purified catalytic subunit of the cyclic AMP-dependent protein kinase was used to calculate for each extract the glycogen synthase kinase activity contributed by the cyclic AMP-dependent protein kinase. Based on these calculations, the portion of the glycogen synthase kinase which was due to kinases independent of cyclic AMP was: kidney, 97%; liver, 91%; lung, 89%; brain, 87%, heart, 85%; stomach mucosa, 84%; adipose tissue, 38%; and skeletal muscle, 33%. A significant portion of the glycogen synthase kinase activity, but virtually none of the cyclic AMP-dependent histone kinase activity, of these extracts could be adsorbed to phosphocellulose columns. Liver extracts contained, in addition, a form of glycogen synthase kinase which was not adsorbed to phosphocellulose and which could be separated from the cyclic AMP-dependent protein kinase by additional chromatography. These studies demonstrate that kinases independent of cyclic AMP account for most of the glycogen synthase kinase activity of many tissues. The widespread distribution and high concentrations of these enzymes suggest that they are of physiological importance.     

Depolarizing agent-induced cyclic AMP accumulation in isolated rat spinal cord

The stimulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation by the depolarizing agents K+, ouabain and veratridine, was studied in rat and guinea pig spinal cord tissue slices. Significantly increased accumulation of cyclic AMP was produced by each of the agents in a concentration-dependent manner. Veratridine and ouabain were equipotent (EC50 = 5 x 10(-5)M) and approximately 500 fold more potent than K+ (EC50 = 10(-2)M). Depolarizing agent-induced cyclic AMP accumulation in slices from guinea pig spinal cord was approximately double the response in rat spinal cord. Maximum stimulation occurred within 2.5 min of incubation with these agents and lasted for at least 30 min. Regional studies demonstrated that the maximal accumulation of cyclic AMP occurred to a greater degree in tissue slices from the dorsal section of spinal cord from both rat and guinea pig. Whereas the ouabain and veratridine stimulatory responses are completely dependent on extracellular Ca++, the K+ response is only partially dependent. Stimulation due to ouabain and veratridine is dependent, and K+ is independent, of release of neurohumoral substances such as norepinephrine or adenosine from spinal neurons. These experiments indicate the possible modulatory role of depolarization-linked events in regulating the spinal cord cyclic AMP system.     

AMP deaminases of rat small intestine

Phosphocellulose column chromatography revealed the existence of two forms of AMP deaminase both in whole tissue and in the intestinal epithelium. AMP deaminase I, which eluted from the column as a first activity peak, exhibited hyperbolic, nonregulatory kinetics. The substrate half-saturation constants were determined to be 0.3 and 0.7 mM at pH 6.5 and 7.2, respectively, and did not change in the presence of ATP, GTP and Pi. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and Pi. The S0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S0.5 decreased to 2.5 mM and caused the sigmoidicity to shift to hyperbolic. The ATP half-activation constant was increased 9-fold in the presence of GTP and was not affected by Pi. Mg2+ significantly altered the effects exerted by nucleotides. The S0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and Pi. When MgATP was present, AMP deaminase II from rat small intestine was less susceptible to inhibition by GTP and Pi. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in Vmax observed in the presence of MgCl2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. GuoPP[NH]P at low concentrations, in contrast to GTP, did not affect the enzyme and even activated it at concentrations above 0.2 mM. We postulate that AMP deaminase II may have a function similar to that of the rat liver enzyme. The significance of the existence of an additional, non-regulatory form of AMP deaminase in rat small intestine is discussed.     

MICROASSAY OF ADENOSINE-3′,5′-MONOPHOSPHATE (CYCLIC AMP) IN BRAIN AND OTHER TISSUES BY THE LUCIFERIN-LUCIFERASE SYSTEM1

Abstract— A simple, sensitive and specific method for assaying cyclic AMP in various tissues is reported. Cyclic AMP was isolated from contaminating nucleotides and was converted to ATP with a phosphodiesterase-myokinase-pyruvate kinase system. The ATP was determined enzymically in a liquid scintillation counter by the firefly luciferin-luciferase technique. This procedure was capable of detecting as little as 5 × 10^?14 mol of cyclic AMP and could therefore be used for analyses on less than 1 mg of brain. The assay was reproducible and linear over a wide range of tissue concentrations. In the rat, the highest levels of cyclic AMP (2.7–4.2 pmol/mg wet wt. of tissue) were present in the pineal, heart, pituitary, thyroid, cerebellar cortex, kidney, adrenal, liver and pyloric region of the stomach; intermediate levels (1.5–2.7 pmol/mg wet wt. of tissue) were found in testis, skin, aorta, intestine, submaxillary gland, spleen, muscle and cerebral cortex, moderately low levels (1.0–1.5 pmol/mg wet wt. of tissue) were found in lung, trachea and greater curvature of the stomach; whereas low levels (0.15–0.60 pmol/mg wet wt. of tissue) were found in adipose tissue.     

Release of AMP and adenosine from rat heart mitochondria

Release of AMP and adenosine from rat heart mitochondria was studied. The rate of appearance of extramitochondrial adenosine was independent of the extramitochondrial phosphate concentration between 5 and 20 mM. In the absence of exogenous, respiratory substrates or in the presence of glutamate/malate plus rotenone, the rate of appearance of adenosine was relatively low when phosphate was not added. The appearance of extramitochondrial AMP + adenosine was found to be directly proportional to the extra-mitochondrial phosphate concentration. Zn2+ (10 mM) decreased the rate of adenosine appearance by 90% and increased the rate of AMP appearance 6-fold. The mitochondrial preparations dephosphorylated exogenous AMP; this activity was inhibited by 10 mM Zn2+. We conclude that the adenosine appearing in the extramitochondrial space was not due to a direct release from the matrix, but instead was due to adenine nucleotide release with subsequent conversion to adenosine in the extramitochondrial space.     

Specific antibodies and the selective inhibitor ICI 118233 demonstrate that the hormonally stimulated ''dense-vesicle'' and peripheral-plasma-membrane cyclic AMP phosphodiesterases display distinct tissue distributions in the rat.

Polyclonal-antibody preparations DV1 and PM1, raised against purified preparations of rat liver insulin-stimulated 'dense-vesicle' and peripheral-plasma-membrane cyclic AMP phosphodiesterases, were used to analyse rat liver homogenates by Western-blotting techniques. The antibody DV1 identified only the 63 kDa native subunit of the 'dense-vesicle' enzyme, and the antibody PM1 only the 52 kDa subunit of the plasma-membrane enzyme. These antibodies also detected the subunits of these two enzymes in homogenates of kidney, heart and white adipose tissue from rat. Quantitative immunoblotting demonstrated that the amount of these enzymes (by wt.) varied in these different tissues, as did the expression of these two enzymes, relative to each other, by a factor of as much as 7-fold. The ratio of the dense-vesicle enzyme to the peripheral-plasma-membrane enzyme was lowest in liver and kidney and highest in heart and white adipose tissue. ICI 118233 was shown to inhibit selectively the 'dense-vesicle' cyclic AMP phosphodiesterase in liver. It did this in a competitive fashion, with a Ki value of 3.5 microM. Inhibition of tissue-homogenate cyclic AMP phosphodiesterase activity by ICI 118233 was used as an index of the contribution to activity by the 'dense-vesicle' enzyme. By this method, a tissue distribution of the 'dense-vesicle' enzyme was obtained which was similar to that found by using the immunoblotting technique. The differential expression of isoenzymes of cyclic AMP phosphodiesterase activity in various tissues might reflect a functional adaptation, and may provide the basis for the different physiological actions of compounds which act as selective inhibitors.     

Rat osteogenic sarcoma cells:effects of some prostaglandins, their metabolites and analogues on cyclic AMP production.

Cyclic AMP production by freshly isolated cells, from a 32P-induced transplantable rat osteogenic sarcoma, was stimulated by PGE1, PGE2 and to a less extent by PGF2alpha and PGA2. In the case of PGE2, the cyclic AMP content of cells was maximal within 5 min. The 13,14-dihydroderivatives of PGE1, PGE2 and PGF2alpha had approximately 40% of the activity of the parent prostaglandin whilst, in every case, the metabolites (15-keto and 13,14-dihydro-15-keto) had very little activity. Two prostaglandin endoperoxide analogues (U44069 and U46619) had only 10% of the activity of an equimolar dose of PGE2. The data presented in this paper demonstrates similarities between the responses of these cells and cells derived from bony tissue in terms of the ability of prostaglandins to stimulate bone resorption in tissue culture.     

Cyclic AMP metabolism by swine adipocyte microsomal and plasma membranes

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5'-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 microM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 microM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5'-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.     

Isolation and regulation of piglet cardiac AMP deaminase

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a K_m of 2 mM for 5-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the K_m of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD⁺, whereas, the K_m was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in V_max with no change in K_m. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.     

Dibutyryl cyclic AMP increases phosphodiesterase activity in the rat heart

The influence of increasing the in vivo concentration of cyclic AMP on the activity of cyclic nucleotide phosphodiesterase (PDE) in rat heart was investigated. One, three, and five hourly injections of 5.0 mg dibutyryl (Bt2) cyclic AMP significantly increased the activity of PDE in the supernatant fraction of rat heart using 1.0 microM cyclic AMP as the assay substrate concentration. When 100 microM cyclic AMP was used in the assay reaction, increases in enzymes activity were seen following five and eight nucleotide injections. The nucleotide-induced increase in PDE activity was dose dependent. When the five-injection protocol was used, PDE activity remained elevated for at least 4 h, while activity had returned to control levels within this time when two hourly injections were used. The nucleotide stimulation of PDE activity was blocked by cycloheximide. Five hourly infections of Bt2 cyclic AMP increased PDE activity in the liver and fast-twitch red muscle. A reduction in PDE activity in fast-twitch white muscle was seen following nucleotide injections. These findings are consistent with the hypothesis that prolonged elevations in the intracellular concentration of cyclic AMP cause an elevation in myocardial PDE activity. The increased activity seems to be the result of protein synthesis. These data suggest that cyclic AMP contributes significantly in regulating its own metabolism in the rat heart.     

Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration

AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5-7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and (31)P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr(172) on AMPK-alpha, and phosphorylation of Ser(79) on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.     

Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.     

Effects of prostaglandins on rat renal adenylate cyclase-cyclic AMP systems.

The effects of prostaglandin (PG) E1, E2, A1, F1alpha, F2alpha or D2 on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AMP systems were examined. While high concentrations (8X10-4M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE1 was the only prostaglandin to stimulate at 10-7M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10-4M. This effect of PGA's was limited to the outer medulla. PGD2 was the least stimulatory. Observations with renal slices yielded qualitatively similar results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O2 deprivation (5% O2) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O2 compared to 95% O2. However, in the inner medulla PGE stimulation was observed only at 5% O2 and not 95% O2. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O2. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.     

Evidence that endogenous cyclic AMP does not modulate serum sulfation factor action on embryonic chicken cartilage

The role of cartilage cyclic AMP as a mediator or modulator of serum sulfation factor (SSF) action on embryonic chicken cartilage was assessed. Media with concentrations of rat serum (7.5%) sufficient to maximally stimulate chondromucoprotein synthesis as measured by ³⁵SO₄ incorporation did not change cartilage cyclic AMP levels. Theophylline (2.5mM) doubled cyclic AMP in cartilage incubated in media but had no effect on ³⁵SO₄ incorporation. In media containing 5% rat serum, theophylline at 0.5, 1.5 and 2.5mM caused a similar and significant rise in tissue cyclic AMP but only 2.5mM inhibited SSF stimulated ³⁵SO₄ incorporation. The data indicate that cartilage cyclic AMP neither mediates nor modulates SSF action on cartilage chondromucoprotein synthesis.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Cyclic AMP formation and morphology of myocardial cells isolated from adult heart: effect of Ca2+ and Mg2+.

Adult rat heart cells were isolated by perfusion with a calcium-free phosphate buffer containing collagenase. Optimal conditions gave a high proportion of elongated cells. Isoprenaline increased cydic AMP content linearly, with ED50 (dose effective in 50% of the population) about 10(-7) M. Ca2+ made the cells spherical, and it nearly abolished cyclic AMP response as did lack of Mg2+.