Development of hypothermia in rats at increase of [Ca2+] in the blood

In rats, data on influence of i. v. administration of calcium chloride on the level of [Ca2+] in the blood and on process of oppression ofthermoregulatory and respiratory functions in rats in hypothermia. 0.18 or 0.135 mmol Ca2+ on the 3rd minute from beginning of the administration increased [Ca2+] in the blood from 1.01 +/- 0.03 to 2.56 +/- 0.08 mM (or 2.27 +/- 0.06 mM). Then [Ca2+] was reduced gradually, in 20 minutes from administration, solution of CaCh [Ca2+] exceeded the initial level by 20-30 %. The increase of concentration of ionized calcium in the rat blood strengthened the cold oppression of breathing and cold shivering as compared with the control (administration of physiological solution). Arrest of breathing in rats after administration of CaCl2 solution occurred at higher rectal temperatures (21 +/- 0.03 degrees C) as compared with control experiments (18 +/- 0.4 degrees C), p < 0.05. It is suggested that increase of [Ca2+] in the blood strengthens effects of cold in the form of oppression of thermoregulatory and respiratory functions.     

Studies on the generation of Ca2+/calmodulin-independent activity of calmodulin-dependent protein kinase II by autophosphorylation. Autothiophosphorylation of the enzyme.

The mechanism for the generation of the Ca2+/calmodulin (CaM)-independent activity of calmodulin-dependent protein kinase II (CaM-kinase II) by autophosphorylation was studied by characterizing the autothiophosphorylated enzyme, which is resistant to hydrolysis. When CaM-kinase II was incubated with adenosine 5'-O-(thiotriphosphate) at 5 degrees C, the incorporation of thiophosphate into the enzyme occurred rapidly, reaching a maximum level within a few minutes, in parallel with increase in Ca2+/CaM-independent activity. The maximum level was 1 mol of thiophosphate per mol of subunit of the enzyme, and the thiophosphorylation occurred exclusively at Thr286 in the alpha subunit and Thr287 in the other subunits of the enzyme. These results, taken together, indicate that the autothiophosphorylation of Thr286/Thr287 of each subunit is involved in the generation of the Ca2+/CaM-independent activity. The activity of the autothiophosphorylated enzyme, when assayed in the presence of Ca2+/CaM, showed the same kinetic properties as did the Ca2+/CaM-dependent activity of the original non-phosphorylated enzyme, but when assayed in the absence of Ca2+/CaM, it showed the same Vmax as the Ca2+/CaM-dependent activity but higher Km values for protein substrates. Thus, the phosphorylation of Thr286/Thr287 of the subunit of the enzyme by autophosphorylation appears to not only enhance the affinity of its substrate-binding site for the protein substrate, although it is lower than that of the enzyme activated by the binding of CaM, but also convert the active site to the fully active state.     

Parallel changes in brain tissue blood flow and mitochondrial function during and after 30 minutes of bilateral forebrain ischemia in the gerbil

Forebrain ischemia was induced in Mongolian gerbils by bilateral occlusion of the common carotid arteries for 30 minutes. These animals do not have a complete circulus arteriosus Willisii. Mitochondria were prepared from the forebrain tissue at the end of the 30 minutes occlusion period as well as at different time points after the release of the occlusion. Tissue blood flow in the forebrain was also determined by measuring the brain tissue accumulation of 14C-iodoantipyrine. Tissue blood flow in the forebrain decreased from a control level of 1.43 +/- 0.03 ml/min/gr to 0.13 +/- 0.03 ml/min/gr by the 30th minute of ischemia, increased to 1.12 +/- 0.25 ml/min/gr after 5 minutes of reflow, but decreased again to 0.41 +/- 0.07 ml/min/gr after 1 1/2 hours of reflow. Oxygen consumption rate of mitochondria prepared from the forebrain (glutamate + malate as substrates in the presence of ADP) was 98 +/- 13 nmoles O2/min/mg protein in control animals, decreased to 61 +/- 9 nmoles O2/min/mg protein after 30 minutes of occlusion, recovered to 106 +/- 9 nmoles O2/min/mg protein during the first 30 minutes of reperfusion. During extended reperfusion, mitochondrial respiratory activity declined reaching 20 +/- 5 nmoles O2/min/mg protein after 5 1/2 hours of reperfusion. Respiratory control ratio of the mitochondria (relative increase of respiration upon addition of ADP) was 9.2 +/- 1.3 in control animals, 7.0 +/- 1.5 after 30 minutes of carotid occlusion, 9.0 +/- 1.2 after 30 minutes of reperfusion, and 5.8 +/- 0.8 after 5 1/2 hours of reperfusion. Superoxide dismutase activity of the forebrain mitochondria was 5.10 +/- 0.7 I.U./mg protein in control animals, decreased to 3.3 +/- 1.6 I.U./mg protein after 30 minutes of occlusion and remained at this level throughout the reperfusion period. These data confirm earlier reports that deterioration of mitochondrial function may contribute to the development of ischemic and post-ischemic brain tissue damage. It also appears possible that postischemic damage of mitochondrial function develops secondary to postischemic deterioration of tissue blood flow.     

Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the alpha subunit

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg much less than Lys much less than Gln much less than Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase alpha subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2(+)-independent kinase activity (5.46 +/- 0.88%) compared to the wild-type Arg283 (1.86 +/- 0.71%) and the Gln283 mutant (2.15 +/- 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2(+)-independent activity, the Arg283 kinase attained maximal Ca2(+)-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2(+)-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2(+)-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.     

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane Ca2(+)-ATPase

The role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane (SPM) Ca2(+)-ATPase was investigated by its reconstitution into different phospholipids. Retention of activity of the solubilized Ca2(+)-ATPase depended on addition of exogenous phospholipids. As the cholate concentration used for solubilization of native SPM increased, a larger excess of exogeneous phospholipids, relative to membrane protein, had to be added to maintain optimal activity. Highest ATP-dependent Ca2+ transport activity was obtained when reconstitution was carried out in calf brain phospholipids (BPLs) followed by soybean phospholipids (SPLs) and the lowest in egg PC; reconstitution at a 40:1 weight ratio of exogenous phospholipids to native SPM protein resulted in ATP-dependent Ca2+ transport of 40.0 +/- 4.16, 23.4 +/- 8.48, and 11.54 +/- 2.31 nmol of Ca2+ (mg of protein)-1 (5 min)-1, respectively. Partial substitution of egg PC with BPLs led to an increase in the activity of the reconstituted Ca2+ pump. The highest ATP-dependent Ca2+ uptake was obtained when ratios of 15:25 or 10:30 egg PC to BPLs were used. Testing the individual phospholipids participating in the BPL mixture showed that addition of PS to egg PC led to a consistent increase in Ca2+ pump activity. Substitution of 50% of the PC with PS resulted in a 3.8-fold higher ATP-dependent Ca2+ uptake than that obtained in egg PC alone. No other phospholipid tested--PE, SM, or PI--had a similar effect. Increasing the proportion of PS within the BPL mixture above its original content led to a gradual decrease in the reconstituted SPM Ca2+ pump activity. Enrichment of asolectin with PS led first to increased Ca2+ pump activity; then, as the proportion of PS increased, Ca2+ transport of the reconstituted pump decreased. An increased proportion of PE, SM, or PI within the BPLs or asolectin, above their original contents, resulted in decreased Ca2+ transport. These results indicate that optimal SPM Ca2+ pump activity requires the combined presence of a critical amount of PC and PS within the reconstituted membrane.     

Nerve growth factor treatment or cAMP elevation reduces Ca2+/calmodulin-dependent protein kinase III activity in PC12 cells

Ca2+/calmodulin-dependent protein kinase III (Ca2+/CaM kinase III) phosphorylates a protein of Mr = 100,000 (the 100-kDa protein), a major substrate for Ca2+/CaM-dependent protein phosphorylation found in many mammalian tissues and cell lines (Nairn, A.C., Baghat, B., and Palfrey, H.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7939-7943). Treatment of PC12 cells with nerve growth factor (NGF) or forskolin resulted in a decrease in the depolarization-dependent phosphorylation of the 100-kDa protein in intact cells and in a decrease in the Ca2+/CaM-dependent phosphorylation of the 100-kDa protein in cytosolic extracts. In experiments using cytosolic extracts, the initial effect of NGF on the phosphorylation of the 100-kDa protein was observed in less than 1 h, was maximal (70% decrease) after 12 h, and began to recover after 24 h. The effect of forskolin was more rapid and the maximal effect was greater (90-95% decrease). Decreased Ca2+/CaM kinase III activity was also found in PC12 cells treated with epidermal growth factor, 2-chloroadenosine plus isobutylmethylxanthine, or dibutyryl cAMP. The effect of forskolin did not reverse unless it was removed. Cycloheximide blocked the recovery of Ca2+/CaM kinase III activity observed following the removal of forskolin but did not affect the ability of forskolin to reduce kinase activity. Short-term treatment with phorbol ester had little effect on Ca2+/CaM kinase III activity; long-term treatment with phorbol ester, which results in the disappearance of enzymatically detectable protein kinase C, had no effect on the ability of NGF or 2-chloroadenosine to reduce Ca2+/CaM kinase III activity. The level of the 100-kDa protein as determined by immunological techniques was not changed by any treatment. These results suggested that the effect of treatment of PC12 cells with NGF or forskolin was to reduce the level of Ca2+/CaM kinase III per se.     

Vitamin D3 and 1,25-dihydroxyvitamin D3 stimulate the skeletal muscle-calcium mobilization in rachitic chicks

The Ca content in skeletal muscle relative to vitamin D3 intake was studied in chicks. It was found that the Ca content in rachitic chick muscle was significantly higher than normal and it decreased with vitamin D3 treatment. In 4-week-old chicks fed a vitamin D-deficient diet, the Ca content in leg muscle reached 9.86 +/- 1.07 mg/100 g wet wt, although in chicks receiving vitamin D3 in doses of 100 and 500 IU/kg diet, it was 7.80 +/- 0.72 and 6.08 +/- 0.61 mg/100 g wet wt, respectively. A single i.m. dose of 0.50 micrograms of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or vitamin D3 caused a dramatic decrease in the muscle Ca content by 3 to 6 h after the injection. A simultaneous rise in the Ca level in blood serum was observed. However, at this time the Ca binding protein content in duodenal mucosa and the stimulation of Ca absorption were negligible. These findings allow the conclusion that the vitamin D deficiency in chicks leads to a surplus Ca accumulation in skeletal muscle. The administration of vitamin D3 or its metabolites causes rapid Ca release during the first 6 h. This may be the source of the Ca level increase in blood serum. In this respect 1,25(OH)2D3 was much more effective than vitamin D3.     

Des-enkephalin-gamma-endorphin: bioavailability in rats following the subcutaneous and intramuscular route of administration

A pharmacokinetic study with [3H]des-enkephalin-gamma-endorphin (3H-DE gamma E) was performed in rats after the intravenous, subcutaneous and intramuscular route of administration. Disappearance of non-metabolized 3H-DE gamma E from blood upon intravenous dosing followed a biphasic decay with half-lives of 0.7 +/- 0.3 (+/- S.D.) min for the initial distribution phase and 6.3 +/- 2.7 min for the terminal elimination phase. The central and peripheral volumes of distribution were strikingly high (0.38 and 0.55 1 X kg-1, respectively). Extensive metabolism occurred already within the first minutes after injection. The blood clearance rate was found to be 0.29 +/- 0.12 1 X min-1 X kg-1, which value points to remarkable extrahepatic elimination of the neuropeptide. As compared to the intravenous route of administration, subcutaneous or intramuscular injection of 3H-DE gamma E resulted in low but longer-lasting peptide levels in blood. These levels reached already peak values at 2 min after both routes of administration and then declined to below the limit of detection at 2-3 h. The absolute bioavailability of DE gamma E after subcutaneous injection amounted to 30.9 +/- 16.3% (range 16.0-46.9%), whereas the bioavailability after intramuscular injection was observed to be 3.5 times lower (8.5 +/- 3.0%; range 4.6-12.0%). These data suggest that subcutaneous dosing of DE gamma E might be more effective in displaying CNS activity than the intramuscular route.     

Effect of acebutolol on plasma catecholamine concentrations in anesthetized dogs with ouabain-induced arrhythmias and its direct actions in vitro on the adrenal medulla

We have performed studies on blood hormone dynamics following intravenous administration of acebutolol, a newly synthesized beta-blocker, and its direct action on the adrenal medulla in vitro. Intravenous injection of acebutolol into anesthetized dogs almost doubled the plasma adrenaline and noradrenaline concentrations within 5 to 15 minutes, while renin activity was reduced to approximately two-thirds of the pre-administration level. When arrhythmia was induced in dogs with ouabain, the plasma adrenaline and noradrenaline levels increased to 220 +/- 109 and 392 +/- 84 pg/ml, respectively, from the basal levels of 44 +/- 24 and 140 +/- 43 pg/ml. The restoration of sinus rhythm following the administration of acebutolol was accompanied by a further increase in the plasma adrenaline and noradrenaline levels to 797 +/- 364 and 1226 +/- 263 pg/ml, respectively. A perifusion experiment indicated that acebutolol directly accelerated catecholamine release from the adrenal medulla in pigs.     

Inhibitory effects on phospholipase A2 and antivenin activity of melanin extracted from Thea sinensis Linn

Antivenin activity of melanin extracted from black tea (MEBT) was reported for the first time. The antagonistic effect of MEBT was evaluated for Agkistrodon contortrix laticinctus (broadbanded copperhead), Agkistrodon halys blomhoffii (Japanese mamushi), and Crotalus atrox (western diamondback rattlesnake) snake venoms administered i.p. to ICR mice. MEBT was injected i.p. immediately after the venom administration in dose of 3 mg per mouse in the same place of venom injection. MEBT demonstrated neutralization effect against all venoms tested. The greatest antivenin effect of MEBT was found against Japanese mamushi snake venom. In this case, half the mice died within 2.5 +/- 0.7 h after injection of 0.9 mg/kg of venom. An immediate injection of MEBT substantially reduced the toxic effect of venom and extended time at the 50% level of survival up to 52.3 +/- 2.3 h. The antivenin activity of MEBT is due to chelating of Ca++ and non-specific binding of phospholipase A2. The inhibitory effect of MEBT on phospholipase A2 assessed for different venoms was similar to that obtained with pure enzyme. Low toxicity of MEBT in combination with its antagonistic activity against different venoms may allow effective life-saving treatment against snakebites. Such application of MEBT is important when identification of the snake is impossible or if specific treatment is unavailable.     

Administration of parathyroid hormone, prostaglandin E2, or 1-alpha,25-dihydroxyvitamin D3 restores the bone inductive activity of rhBMP-2 in aged rats

Bone morphogenetic protein (BMP) induces bone formation in young rodents, but aging causes a reduction in the bone-forming ability of BMP. Most patients who require bone reconstruction are relatively old. Accordingly, we examined whether anabolic hormones could restore the bone inductive activity of rhBMP-2 in aged rats. rhBMP-2 in a carrier pellet was implanted subcutaneously in both 4- and 50-week-old female Wistar rats. PTH, PGE2, or 1,25(OH)2D3 was injected every day during the period of BMP implantation. The pellets were harvested, and were examined both histologically and biochemically 2 weeks after implantation. Bone-forming ability was measured by alkaline phosphatase (ALP) activity and calcium (Ca) content. Pellets in 50-week-old rats showed a significant reduction in bone formation compared to pellets in 4-week-old rats. However, daily injections of PTH into 50-week-old rats restored both ALP activity (103 +/- 4.6%) and Ca content (105 +/- 2.6%). 1,25(OH)2D3 and PGE2 also restored Ca content (103 +/- 4.5% and 98 +/- 3.8%, respectively) and stimulated ALP activity (142 +/- 2.3% and 133 +/- 3.6%). These results show that the administration of these hormones restores bone-forming ability in aged rats. A combination treatment of these hormones with rhBMP-2 might be applicable to the reconstruction of bone defects in elderly patients.     

Ischemia-Induced Loss of Brain Calcium/Calmodulin-Dependent Protein Kinase II

Forebrain ischemia in gerbils, produced by brief bilateral carotid occlusion, induced the dramatic loss of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) as determined by both kinase activity assays and western blot analysis. In cortex and hippocampus, cytosolic CaM-kinase II was completely lost within 2-5 min of ischemia. Particulate CaM-kinase II was more stable and decreased in level approximately 40% after 10 min of ischemia followed by 2 h of reperfusion. CaM-kinase II in cerebellum, which does not become ischemic, was not affected. The rapid loss of CaM-kinase II within 2-5 min was quite specific because cytosolic cyclic AMP kinase and protein kinase C in hippocampus were not affected. These data indicate that cytosolic CaM-kinase II is one of the most rapidly degraded proteins after brief ischemia. Because the multifunctional CaM-kinase II has been implicated in the regulation of numerous neuronal functions, its loss may destine the neuronal cell for death.     

Study on mechanisms of a haemostatic effect of 1 deamino-8-D-arginine vasopressin (desmopressin) in uraemic patients.

The effect of 1 deamino-8-D-arginine vasopressin (DDAVP) on blood platelet serotonin and some parameters of haemostasis was investigated. DDAVP was administered intravenously in a dose of 0.4 micrograms/kg BW to 16 uraemic patients maintained on chronic haemodialysis in a double-blind crossover study compared with placebo. The bleeding time was significantly shortened after DDAVP administration from 21.3 +/- 8 minutes to 11.5 +/- 6 minutes (p less than 0.001). VIII: Ag increased from 239.1 +/- 94% to 473 +/- 293% (p less than 0.01). Euglobulin lysis time was shortened from 238 +/- 101 to 148 +/- 84 minutes (p less than 0.005). The platelet serotonin level was significantly reduced from 532 +/- 141 to 366 +/- 88 ng/10(9) platelets (p less than 0.02). There were no changes in haematocrit, platelet count, VIII: C levels and blood serotonin concentrations after DDAVP administration. In placebo group there were no changes in all investigated parameters. Our data indicate that DDAVP shortens prolonged bleeding time in uraemic probably by means of the serotonergic mechanism. Further studies are needed to confirm this suggestion.     

Intranasal administration of ACTH(1-24) stimulates catecholamine secretion.

Previously, we reported that intranasal (IN) ACTH(1-24) administration stimulates adrenocortical steroid secretion in normal subjects. To determine the efficiency of transmucosal absorption of ACTH into the adrenal medulla, we measured serum cortisol, aldosterone, epinephrine, norepinephrine and dopamine levels after IN vs. intravenous (IV) administration of 250 microg ACTH(1-24) in 7 healthy adult men (mean age 21.7 +/- 1.2 yr; range, 21 - 24 yr). Blood was collected at 0, 30, 60 and 120 min after administration of ACTH(1-24), and the levels of adrenocortical steroids and catecholamines were measured by specific RIA and HPLC methods, respectively. There were no side effects associated with IN or IV ACTH administration. Consistent with the previous study, serum cortisol and aldosterone increased after IN administration of ACTH(1-24), peaking 30 min after administration. Sixty minutes after IN and IV administration of ACTH, epinephrine levels increased by 41.9 +/- 13.1 % and 63.3 +/- 11.8 %, respectively, and remained elevated throughout the sampling period. Thirty minutes after IN or IV administration of ACTH(1-24), plasma norepinephrine levels increased by 55.9 +/- 13.4 % and 73.7 +/- 15.0 %, respectively, peaking 30 min after ACTH(1-24) administration, and decreasing to basal levels within 60 min. Plasma dopamine levels did not change after IN administration of ACTH(1-24). Adrenocortical steroid and catecholamine levels did not increase after IN administration of saline. These results demonstrate that IN administration of ACTH(1-24) not only stimulates adrenocortical steroids, but also epinephrine and norepinephrine.     

Regulatory Properties of Calcium/Calmodulin-Dependent Protein Kinase II in Rat Brain Postsynaptic Densities

Calcium/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) contained within the postsynaptic density (PSD) was shown to become partially Ca2+-independent following initial activation by Ca2+/CaM. Generation of this Ca2+-independent species was dependent upon autophosphorylation of both subunits of the enzyme in the presence of Mg2+/ATP/Ca2+/CaM and attained a maximal value of 74 +/- 5% of the total activity within 1-2 min. Subsequent to the generation of this partially Ca2+-independent form of PSD CaM-kinase II, addition of EGTA to the autophosphorylation reaction resulted in further stimulation of 32PO4 incorporation into both kinase subunits and a loss of stimulation of the kinase by Ca2+/CaM. Examination of the sites of Ca2+-dependent autophosphorylation by phosphoamino acid analysis and peptide mapping of both kinase subunits suggested that phosphorylation of Thr286/287 of the alpha- and beta-subunits, respectively, may be responsible for the transition of PSD CaM-kinase II to the Ca2+-independent species. A synthetic peptide 281-309 corresponding to a portion of the regulatory domain (residues 281-314) of the soluble kinase inhibited syntide-2 phosphorylation by the Ca2+-independent form of PSD CaM-kinase II (IC50 = 3.6 +/- 0.8 microM). Binding of Ca2+/CaM to peptide 281-309 abolished its inhibitory property. Phosphorylation of Thr286 in peptide 281-309 also decreased its inhibitory potency. These data suggest that CaM-kinase II in the PSD possesses regulatory properties and mechanisms of activation similar to the cytosolic form of CaM-kinase II.     

Autoactivation of calmodulin-dependent protein kinase II by autophosphorylation

Autophosphorylation of calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) under limiting conditions (2 microM ATP) decreased progressively with increasing concentrations of a substrate, Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys (syntide-2), suggesting a competition between the substrate and the autophosphorylation site(s) of the enzyme. The rate and extent of the generation of Ca2+/CaM-independent activity of the enzyme by autophosphorylation were also decreased by the presence of syntide-2. The syntide-2 phosphorylation in the presence of Ca2+/CaM under the limiting conditions reached a steady state, after a lag, when the Ca2+/CaM-independent activity reached a plateau. A linear relationship was observed between the activities in the presence and absence of Ca2+/CaM of the enzyme which had undergone various degrees of autophosphorylation, and the extrapolation of activity in the absence of Ca2+/CaM to zero gave 15-20% of the maximum activity. The steady-state rate of syntide-2 phosphorylation in the presence of Ca2+/CaM by the enzyme that had not undergone prior autophosphorylation was decreased by high concentrations of syntide-2 which suppressed autophosphorylation as well as the generation of Ca2+/CaM-independent activity. These results suggest that although the nonautophosphorylated enzyme possesses a basal low level of Ca2+/CaM-dependent activity, autophosphorylation is required for full activation.     

Concentration of components of the adenylate system in heavy and light fractions of the sarcoplasmic reticulum of skeletal muscles and sensitivity of these fractions to the effects of imidazole-containing compounds and caffeine]

Fragments of sarcoplasmic reticulum from rabbit sceletal muscles sedimented within the range from 2000 g to 8000 g (heavy fraction) and 8000 g to 40000 g (light fraction) and washed with 0.6 M KCl, were practically free of adenylatecyclase activity. Phosphodiesterase cAMP was not found in the light fraction, while its activity in the heavy fraction was 500 pmol of cAMP/min per mg of protein. Both fractions contain bound cAMP (1-2 pmol/mg of protein) and specific sites of cAMP binding, the binding constant being approximately 10(6)M-1. The number of binding sites is 60 pmol/mg of protein for the heavy and 30 pmol/mg of protein for the light fractions. The level of phosphodiesterase activity in the heavy fraction correlates with its sensitivity to imidazole, anserine and caffeine. Imidazole and anserine increase in 1.5-1.8 times the value of Ca2+/ATP in the heavy fraction and produce no effect on Ca2+ transport by the light fraction. Caffeine decreases almost twice the Ca2+/ATP value in the heavy fraction and has practically no effect on Ca2+ absorption by enzymes of the light reticulum fraction. Imidazole and anserine activate membrane-bound phosphodiesterase, while caffeine inhibits it. It is suggested that structural rearrangements of membrane-bound phosphodiesterase under the effect of caffeine, imidazole and anserine are responsible for changes in the efficiency of Ca2+ transport by fragments of the heavy reticulum fractions.     

Calmodulin-dependent regulation of Ca,Mg-ATPase activity in plasma membranes of the swine myometrium

Highly purified plasma membrane (PM) preparations of pig myometrium were found to contain 0.91 +/- 0.22 microgram calmodulin per mg of PM protein. Treatment of membranes with 1 mM EGTA in the presence of 0.2 M NaCl causes the diminution of the calmodulin content down to 3% of the original level. The activity of Ca, Mg-ATPase is thereby decreased by 40%. Exogenous calmodulin restores the enzyme activity up to 1.94 +/- +/- 0.30 mumol Pi/mg protein/hour. The maximal activation of Ca, Mg-ATPase is observed with 10(-7) M calmodulin. Calmodulin increases the total ATPase activity of myometrium PM without affecting the Mg-ATPase activity. Trifluoroperazine (20 microM) diminishes the activating effect of exogenous calmodulin on Ca, Mg-ATPase. Calmodulin stimulates Ca, Mg-ATPase at low concentrations of Ca2+(10(-8)-10(-6) M) by decreasing Km for Ca2+ from 0.4.10(-6) M to 2.10(-8) M as well as by increasing Vmax--from 0,8 to 1.42 mumol Pl/mg protein/hour. It is supposed that the activating effect of calmodulin on Ca, Mg-ATPase is based on electrostatic interactions of Ca2+-free calmodulin with the enzyme.     

Proteolytic activity in intact sheets of polarized epithelial cells as determined by a cell-permeable fluorogenic substrate

The purpose of the present investigation was to develop a system for continuous evaluation of extralysosomal proteolytic activity and its regulation in polarized epithelial cells. Filter inserts containing a tight monolayer of primary cultured pig thyrocytes were placed in a thermostated aluminium block. The cell-permeable, fluorogenic calpain and proteasome substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was added to the apical buffer and fluorescence changes were continuously measured via the fibre optics of a luminometer held at a fixed distance from the cell layer. Basal proteolytic activity was reduced by 60-70% by the proteasome inhibitor lactacystin. Proteolysis was increased within a few minutes after application of Ca(2+)-mobilizing agents (ionomycin, 4-bromo-A23187, thapsigargin and maitotoxin). Forskolin and staurosporine also enhanced the proteolytic activity. We conclude that Ca(2+)mobilization, and possibly also changes of protein kinase activity, rapidly increase non-lysosomal proteolysis in the intact thyroid epithelium.