Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg²⁺ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.     

The involvement of cyclic AMP-dependent protein kinase(s) in the induction of ornithine decarboxylase in the regenerating rat liver and in the adrenal gland after unilateral adrenalectomy

We investigated the temporal relationship between the level of cyclic AMP, the activation of cyclic AMP-dependent protein kinase(s), and the induction of ornithine decarboxylase in two important rapid growth systems: the regenerating rat liver and the remaining adrenal gland following unilateral adrenalectomy. There was a biphasic increase in the aactivity of ornithine decarboxylase at 4 and 14 h following partial hepatectomy. The concentration of cyclic AMP increased 2-fold compared to sham-operated animals within 2–3 h, returned to baseline by 8 h, and was elevated again 3-fold by 12 hours. The activation of cyclic AMP-dependent protein kinase(s) occured in a similar biphasic manner. From a control activity ratio (?cAMP/+cAMP) of 0.4, values for total soluble kinase activation reached 0.75 at both 2 and 14 h. After a delay of 2 h following unilateral adrenalectomy, ornithine decarboxylase activity in the remaining adrenal gland increased 15–20-fold of control level by 8 h. Cyclic AMP concentrations in the adrenal were elevated 3.5-fold within 30 min. The protein kinase activation increased from 0.25 to nearly a totally activated state of 1.0 within 1 h, decline to 0.4 by 2 h, and returned to the level of the sham-operated controls at 8 h. In both the rat liver in response to partial hepatectomy and the adrenal gland undergoing hypertrophy, cyclic AMP-dependent protein kinase(s) was markedly activated prior to increases in ornithine decarboxylase activity.     

Modified cyclic nucleotide systems in Morris hepatoma 3924A favoring expression of cyclic GMP effect

Modifications in the cyclic nucleotide systems favoring the expression of cyclic GMP effects were found to occur in the transplanted fast-growing Morris hepatoma 3924A. These included: (a) a decreased level of cyclic GMP phosphodiesterase and an increased level of cyclic AMP phosphodiesterase; (b) a disproportionately increased level of cyclic GMP-dependent protein kinase relative to that of cyclic AMP-dependent protein kinase; (c) a disproportionately increased level of stimulatory modulator of cyclic GMP-dependent protein kinase relative to that of inhibitory modulator of cyclic AMP-dependent protein kinase; and (d) an increased level of phosphoprotein phosphatase.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.     

Effect of triamcinolone treatment, starvation and diabetes on rat liver protein kinase activity

Triamcinolone administration for 3 days reduced protein kinase activity in normal and adrenalectomized rats by about one third. This was accompanied by a similar decrease in cyclic[³H] AMP-binding capacity. Alloxan diabetes reduced protein kinase activity as well as cyclic[³H] AMP-binding capacity by about 40%. Administration of insulin to diabetic rats produced an insignificant increase in protein kinase activity and did not improve cyclic[³H] AMP-binding capacity. In fasted rats, there was a gradual decrease in protein kinase activity to about one half of initial activity on the fourth day, whereas the decrease in cyclic[³H] AMP-binding capacity was less marked. The effect of starvation was observed in normal as well as adrenalectomized rats, indicating that the decrease due to fasting is not mediated by glucocorticoids. The findings suggest that the maintenance of liver protein kinase activity is under hormonal and nutritional control. This represents a long-term regulatory step at the stage of protein phosphorylation in addition to the rapid effect of hormones on cyclic AMP levels.     

The effect of turpentine-induced inflammation on rat liver glycosyltransferases and Golgi complex ultrastructure

The effect of vasopressin on the toad urinary bladder has been shown to be mediated by cyclic AMP. It has been assumed that, as demonstrated for other systems, this involves activation of cyclic AMP-dependent protein kinase. In order to test this hypothesis we investigated the effect of vasopressin on cyclic AMP-dependent protein kinases in epithelial cells of toad bladders. About 80% of protein kinase activity and cyclic AMP-binding capacity was found to be in the cytosol. DEAE-cellulose chromatography showed a pattern of 15--20% type I and 80--85% type II cyclic AMP-dependent protein kinase. Cytosolic kinase was activated 3--4-fold by cyclic AMP with half-maximal activation at 5 . 10(-8) M. Similarly, half-maximal binding of cyclic AMP occurred at 7 . 10(-8) M. Incubation of toad bladders in Ringer's solution containing 0.1 mM 3-isobutyl-1-methylxanthine, prior to homogenization and assay, showed stable cyclic AMP-binding capacity and protein kinase ratio --cyclic AMP/+cyclic AMP. Exposure of bladders to 10 mU/ml of vasopressin for 10 min caused intracellular activation of protein kinase and decrease in cyclic AMP-binding capacity that were maintained for at least 30 min. Incubation of bladders with increasing concentrations of vasopressin (0.5--100 mU/ml) resulted in a discrepancy between a progressive increase in cyclic AMP levels and a levelling off at 10 mU/ml of vasopressin for the changes in protein kinase ratio and cyclic AMP-binding capacity. The increase in kinase ratio was due to higher activity in the absence of exogenous cyclic AMP and was fully inhibitable by a specific protein kinase inhibitor. Using Sephadex G-25-CM50 column chromatography for separation of holoenzyme and free catalytic subunit we demonstrated that the activation of protein kinase in the vasopressin-treated bladders is due to intracellular dissociation of the kinase. These results show that the effect of vasopressin on the toad bladder involves activation of a cytosolic cyclic AMP-dependent protein kinase. The time course and the dose-response curve of the kinase activation closely parallel vasopressin's effect on osmotic water flow.     

Modified cyclic nucleotide systems in Morris hepatoma 3924A favoring expression of cyclic GMP effect.

Modifications in the cyclic nucleotide systems favoring the expression of cyclic GMP effects were found to occur in the transplanted fast-growing Morris hepatoma 3924A. These included: (a) a decreased level of cyclic GMP phosphodiesterase and an increased level of cyclic AMP phosphodiesterase; (b) a disproportionately increased level of cylic GMP-dependent protein kinase relative to that of cyclic AMP-dependent protein kinase; (c) a disproportionately increased level of stimulatory modulator of cyclic AMP-dependent protein kinase relative to that of inhibitory modulator of cyclic AMP-dependent protein kinase; and (d) an increased level of phosphoprotein phosphatase.     

Effect of aflatoxin B1 on phosphoinositide signal transduction pathway during regeneration of liver cells following partial hepatectomy.

Aflatoxin B1 (AFB1) when administered to partially hepatectomised rats 4 hr prior to sacrifice, activated the signalling pathway in regenerating rat liver. The activity of phosphatidylinositol (PI) kinase was found decreased at 30 min but increased at 24 hr and returned to normal at 48 hr. At 30 min, inositol-1,4,5-triphosphate (IP3) level increased significantly whereas diacylglycerol (DAG) level dropped. However, at 24 hr and 48 hr, DAG and IP3 showed the same trend i.e. an increase in their levels. Phosphatidylinositol-4-phosphate levels were found to increase at 24 hr. Protein kinase C (PKC), activity from the particulate fraction was significantly inhibited at 30 min, followed by increase in activity at 24 hr and return to normal at 48 hr. Cytosolic PKC showed a decrease at 24 hr and a significant increase at 48 hr. At the peak of DNA synthesis (24 hr) following partial hepatectomy, all these signalling steps had earlier been found to be inhibited, but the present study shows that aflatoxin B1 administration 4 hr prior to sacrifice reverses the action. Activation of PKC by aflatoxin B1, during regeneration of liver cells when PKC in normally inhibited, may possibly create conditions conducive to carcinogenesis.     

Protein Kinase Activities in Neurospora crassa

Several protein kinase activities have been found in 105,000g supernatant of Neurospora crassa mycelia grown up to the logarithmic phase. By chromatography on DEAE-cellulose the following enzyme activities have been resolved: (i) a cyclic AMP-dependent protein kinase (peak I kinase) eluting at 0.20 m NaCl, more active with histone than with phosvitin (it was inhibited by both a thermolabile fraction having cyclic AMP-binding activity and a thermostable inhibitor isolated from 105,0005g mycelial supernates), (ii) a cyclic nucleotide-independent protein kinase (peak II kinase) eluting at 0.35 m NaCl, also more active with histone than with phosvitin (this kinase was not inhibited by the fraction having cyclic AMP-binding activity but it was sensitive to the thermostable inhibitor); and finally, (iii) a protein kinase eluting at 0.43 m NaCl (peak II kinase), with similar activity toward histone and phosvitin, insensitive to cyclic nucleotides and to fractions carrying cyclic AMP-binding capacity (this kinase activity also resulted insensitive to the thermostable inhibiting factor).     

Subcellular alterations in cyclic AMP-binding and protein kinase activities during ontogeny in the rat cerebellum

Ontogenic relationships between levels of cyclic AMP-binding activity and protein kinase activity were examined in subcellular fractions of the cerebellum during the first 3 weeks of neonatal life. A progressive increase in cyclic AMP levels was paralleled by an increase in cyclic AMP bindign by the nuclear and cytosol fractions, but not by the mitochondrial or microsomal fractions. Utilization of heat-stable protein kinase inhibitor permtited distinction of the cyclic AMP-dependent from the cyclic AMP-independent form of the protein kinase population. Cyclic AMP-dependent protein kinase increased between days 4 and 20 to represent a progressively greater proportion of the protein kinase population. In all subcellular fractions alterations of cyclic AMP-dependent protein kinase during neonatal development paralleled changes in binding of cyclic AMP to protein in these fractions. In both the nuclear and cytosol fractions cyclic AMP-dependent protein kinase activity increased progressively between days 4 and 20, i.e. 64 ± 6 to 176 ± 16 and 79 ± 12 to 340 ± 12 pmol/min per mg protein, respectively. Cyclic AMP-dependent protein kinase activity in the mitochondrial fraction declined during the postnatal period studied, and in the microsomal fraction it rose to a non-sustained peak at 14 days and fell thereafter. Unlike the cyclic AMP-dependent form, cyclic AMP-independent protein kinase activity did not follow the ontogenetic pattern of cyclic AMP-binding activity. The specific activity of nuclear cyclic AMP-independent protein kinase did not change during days 4–20, and a non-sustained rise of cyclic AMP-independent protein kinase activity in both cytosol and microsomal fractions during the 7th–12th day tended to parallel more closely known patterns of postnatal proliferative growth. The findings reported herein indicate that the ontogenic pattern of cyclic AMP-dependent protein kinase varies between different subcellular fractions of the neonatal cerebellum, that these patterns parallel the changes in cyclic AMP-bidign activity, and suggest that the component parts of the cyclic AMP system may develop as a functional unit.     

Cyclic AMP-binding and cyclic AMP-dependent protein kinase activities in the cytosol of differentiating bone marrow erythroblasts.

Cytosolic cyclic AMP-binding capacity and cyclic AMP-dependent protein kinase activity have been studied in relation to differentiation and maturation of rabbit bone marrow erythroblasts. Using cells fractionated by velocity sedimentation at unit gravity, it was found that both activities decreased in dividing cells when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative, but only quantitative, changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent KD for the interaction of binding proteins with cyclic AMP was 4 X 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and in their affinity for cyclic AMP. Plasma membranes of erythroblasts were also able to bind cyclic AMP but only in dividing cells.     

Altered interaction between binding and catalytic subunits of a cyclic AMP-stimulated protein kinase from hepatoma cells.

Binding activity obtained from an established line of hepatoma tissue culture (HTC) cells has a lower apparent affinity for cyclic AMP at physiological pH than has the analogous binding activity from rat liver. However, the apparent binding affinity of HTC preparations can be reversibly increased by adding NaCl or guanidine · HCl. In the presence of such activating substances, a macromolecular inhibitory activity has been chromatographically separated from the cyclic AMP-binding activity. Removal of this inhibitory component causes the apparent affinity of the cyclic AMP-binding activity from HTC cells to increase and resemble that observed with liver preparations. Before treatment with salt, the inhibitory activity seems to be physically associated with the binding activity. Adding the isolated inhibitory component back to a suitably activated binding preparation from HTC cells results in a decrease in the apparent affinity for cyclic AMP. The isolated inhibitory component is devoid of cyclic AMP-binding and cyclic AMP phosphodiesterase activities and has an apparent minimal molecular weight of about 30,000 by gel filtration. It possesses protein kinase activity and seems to be identical to the catalytic subunit of a cyclic AMP-stimulated protein kinase on the basis of chromatographic properties and sensitivities to heat and low pH. This catalytic subunit represents only a minor portion of total cellular protein kinase activity and is also present in liver extracts. However, the binding activity from liver is not inhibited significantly under conditions where the binding from HTC cells is affected by the catalytic subunit. The difference in this inhibitory response between liver and HTC preparations appears to reflect differences in the cyclic AMP-binding proteins themselves.     

Cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and phosphodiesterases in arteries and veins of dogs. Distribution and effects of arteriovenous fistula and arterial occlusion.

Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.     

Cyclic GMP-dependent and cyclic AMP-dependent protein kinases,protein kinase modulators and phosphodieterases in arteries and veins of dogs Distribution and effects of arteriovenous fistula and arterial occlusion

Possible involvement of cyclic GMP-dependent and cyclic AMP-dependent protein kinases, protein kinase modulators and cyclic nucleotide phosphodiesterases in functions of vascular tissues were investigated in the dog. All of the above activities, localized in the smooth muscle-rich inner layer of the blood vessels, were found to be higher in the arteries than in the veins. The peripheral arteries were disproportionately richer in cyclic GMP-dependent protein kinase (as indicated by high ratios of cyclic GMP-dependent to cyclic AMP-dependent protein kinase) than were the veins, with the exception of the pulmonary artery, an atypical arterial tissue exposed to low blood pressure. Interestingly, the protein kinase ratio for the aorta, an artery with no significant role in blood pressure regulation, was not higher than that for the vena cava. Creation of femoral arteriovenous fistulae in the dogs led to preferential reductions in the cyclic GMP-dependent enzyme activity both in the proximal and distal arteries, whereas it was elevated in the stressed vein distal to the anastomotic site. The cyclic GMP-dependent enzyme was preferentially reduced in the saphenous artery distal to occlusion. Changes in the cyclic GMP-dependent enzyme activity appeared to precede gross atrophy or hypertrophy of the vessels. It is suggested that the vascular cyclic GMP-dependent protein kinase may be closely related to peripheral resistance and its regulation.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.