Prostaglandin E2 binding and cyclic AMP production in isolated bone cells

The binding of prostaglandin E2 (PGE2) to bone cells was studied to provide direct evidence for the existence of specific receptors in bone. Bone cells were isolated by collagenase digestion of fetal and newborn rat calvaria. Isolated cells were incubated with 3H-PGE2 and collected on Millipore filters. Specific binding was determined by subtracting the binding that occurred with 10(-6) M non-radioactive PGE2 and 3H-PGE2 from that with 3H-PGE2 alone. With heterogeneous cell preparations and at PGE2 concentrations from 10(-9) - 1.7 X 10(-8) M at 37 degrees C, specific binding reached steady state within 10 min. Bound 3H-PGE2 was displaced by the addition of increasing amounts of unlabeled PGE2. Inhibition of PGE2 binding was observed with PGE1 and the endoperoxide analog, U44069, but not with PGF2 alpha, a lipopolysaccharide, or 13,14-dihydro 15-keto PGE2. Studies with bone cell populations, obtained by sequential digestions, indicated that an osteoclastic population binds 30-fold more PGE2 than osteoblastic cells. Scatchard analyses revealed that the osteoclastic cells have an affinity constant for PGE2 binding similar to that obtained with heterogeneous populations. However, the PGE2 binding capacity in this osteoclastic population was fivefold greater than in the heterogeneous population. The osteoclastic population responded with an increase in cyclic AMP to lower concentrations of PGE2 than the osteoblastic populations. These studies suggest that differences in the binding capacity of PGE2 receptors exist among bone cell-types and that these differences are reflected in the cellular cyclic AMP response.     

Prostaglandin E2 binding and cyclic AMP production in isolated bone cells

Summary The binding of prostaglandin E₂ (PGE₂) to bone cells was studied to provide direct evidence for the existence of specific receptors in bone. Bone cells were isolated by collagenase digestion of fetal and newborn rat calvaria. Isolated cells were incubated with³H-PGE₂ and collected on Millipore filters. Specific binding was determined by subtracting the binding that occurred with 10⁻⁶ M non-radioactive PGE₂ and³H-PGE₂ from that with³H-PGE₂ alone. With heterogeneous cell preparations and at PGE₂ concentrations from 10⁻⁹ − 1.7 × 10⁻⁸ M at 37°C, specific binding reached steady state within 10 min. Bound³H-PGE₂ was displaced by the addition of increasing amounts of unlabeled PGE₂. Inhibition of PGE₂ binding was observed with PGE₁ and the endoperoxide analog, U44069, but not with PGE_2α, a lipopolysaccharide, or 13,14-dihydro 15-keto PGE₂. Studies with bone cell populations, obtained by sequential digestions, indicated that an osteoclastic population binds 30-fold more PGE₂ than osteoblastic cells. Scatchard analyses revealed that the osteoclastic cells have an affinity constant for PGE₂ binding similar to that obtained with heterogeneous populations. However, the PGE₂ binding capacity in this osteoclastic population was fivefold greater than in the heterogeneous population. The osteoclastic population responded with an increase in cyclic AMP to lower concentrations of PGE₂ than the osteoblastic populations. These studies suggest that differences in the binding capacity of PGE₂ receptors exist among bone celltypes and that these differences are reflected in the cellular cyclic AMP response.     

Hypocalcemia and nephrogenous cyclic AMP production in critically ill or injured patients.

Critically ill or injured patients often have impaired cardiovascular function. Since low ionized calcium levels can cause such changes, serum calcium and urine calcium were measured in a prospective study involving 28 criticially ill or injured patients and 16 normal controls. Serum protein levels were also measured to calculate "corrected" total calcium levels. Ionized calcium levels are difficult to measure. Since ionic hypocalcemia is thought to increase the "nephrogenous production" of cyclic AMP, cyclic AMP levels were measured in the blood and urine of these patients and the "nephrogenous" cyclic AMP calculated from the creatinine clearance. The mean total serum calcium in these patients was 7.7 +/- 0.8 mg/dl (S.D.). This was significantly lower (p less than 0.001) than our controls (9.6 +/- 0.6). When corrected for hypoproteinemia, the mean serum calcium (8.7 +/- 0.8) was still significantly lower (p less than 0.005) than control (9.4 +/- 0.5). The mean urine calcium excretion in the patients (56 +/- 66 mg/100 ml G.F.R.) was lower, but not significantly so, than in the controls (84 +/- 44 mg/100 ml G.F.R.). The "apparent nephrogenous" cyclic AMP in the study group was 2,731 +/1 1,451 pm/ml/100 ml G.F.R. The nephrogenous cyclic AMP had a negative correlation (r =-0.45) with "corrected" total calcium levels. Thus "total," "corrected" total, and "ionized" calcium levels appear to be reduced in the majority of critically ill or injured patients studied. The clinical implications of these findings and the potential value of serial cyclic AMP determinations in blood and urine will be discussed.     

Effects of cyclic AMP and dibutyryl cyclic AMP on cerebral hemodynamics and metabolism in the baboon.

Adenosine 3',5'-cycle monophosphate (cyclic AMP) (0.5 mg/kg) was infused into the carotid artery of baboons anesthesized with sodium pentobarbital, causing a biphasic increase in cerebral blood flow (CBF) and reduction in cerebrovascular resistance (CVR) associated in each phase with stimulation of cerebral metabolism evidenced by increased cerebral oxygen consumption (COMRO2) and cerebral glucose consumption (CMRG1). Intracarotid dibutyryl cyclic AMP (0.5 mg/kg) caused a monophasic increase in CBF and reduction of CVR but failed to alter cerebral metabolism. This may be due to its rapid removal from the circulation with ineffective passage across the blood-brain barrier since intracisternal infusion of dibutyryl cyclic AMP caused sustained increase in CBF, CMRO2 and CMRG1 and reduction in CVR. Intracarotid AMP (0.4 mg/kg) and adenosine (0.3 mg/kg) caused an immediate and more marked increase in CBF and decrease in CVR unassociated with cerebral metabolic change making it unlikely that the observed effects of cyclic AMP can be attributed to its breakdown products. Cyclic AMP or its dibutyryl derivative may alter cerebral metabolism secondary to neuronal activation but increase in glucose/oxygen utilization ratio after intracarotid cyclic AMP and intracisternal dibutytyl cyclic AMP also suggests an influence on enzymatic regulation of glucose metabolism.     

Effect of pertussis toxin on parathyroid hormone-stimulated cyclic AMP production in cultured kidney cells

The effect of pertussis toxin, which inactivates the guanine nucleotide binding regulatory proteins Gi and Go on cAMP production in response to parathyroid hormone PGE2 or forskolin, was examined in confluent opossum kidney (OK) cells. This effect was compared with that caused by dexamethasone. The response to PTH was increased in cells preincubated with either agent. The effect of pertussis toxin was selective for PTH, since cAMP production in response to neither PGE2 nor forskolin was increased. In contrast, the response to forskolin was enhanced in dexamethasone-treated cells. These results indicate that both stimulatory and inhibitory guanine nucleotides binding regulatory proteins modulate PTH-induced cAMP production in OK cells. Moreover, pertussis toxin and dexamethasone appear to affect different levels of the PTH-receptor-adenylate cyclase complex.     

The cyclic AMP response to glucagon. Comparison of tissue and plasma cyclic AMP levels in the rabbit.

The effects of glucagon on tissue and plasma cyclic AMP levels have been investigated in rabbits anesthetized with urethane. Glucagon (2 nmole/kg.) caused at least a twofold increase in hepatic cyclic AMP, which reached a peak within two minutes and declined to basal values after 40 minutes. Plasma cyclic AMP also increased at least twofold, reaching a peak at 10 minutes and declining to basal values after 60 minutes. Glucagon (20 nmole/kg.) stimulated hepatic and plasma cyclic AMP in a manner indistinguishable from that observed at the lower dose. Hepatectomy abolished the plasma cyclic AMP responses to glucagon, and no significant stimulation of cyclic AMP concentration was noted in the heart, adipose tissue, small bowel, or kidney. Cyclic AMP hydrolysis was estimated in blood taken before and after administration of glucagon. Glucagon (2 nmole/kg.) increased cyclic AMP hydrolysis slightly, but this was explained by the raised cyclic AMP levels. By contrast, cyclic AMP hydrolysis increased two-to-threefold in blood taken 20 and 40 minutes after glucagon (20 nmole/kg.). The higher dose of glucagon also stimulated cyclic AMP hydrolysis in crude liver homogenate, which could not be explained by increases in cyclic AMP concentration. The increase in cyclic AMP hydrolysis observed in blood and liver may partly explain the failure to show additional stimulation of hepatic and plasma cyclic AMP levels with the higher dose of glucagon. Despite the changes in cyclic AMP hydrolysis, a highly significant correlation was observed in individual rabbits between the hepatic and plasma cyclic AMP responses to glucagon (2 and 20 nmole/kg.), when these were calculated as incremental areas above mean basal levels. It is suggested that measurement of plasma cyclic AMP levels after stimulation by glucagon may be an accurate index of the hepatic cyclic AMP response to glucagon in vivo.     

Distribution of cyclic AMP in human seminal plasma and its relation to sperm progressive motility

Organelles and amorphous substance (pellet II) isolated from human seminal plasma contained 3'5' AMP (cyclic AMP, cAMP) in manifold smaller amounts than did the particle-free seminal plasma. The amount of cAMP associated with pellet II did not differ significantly between normospermic and oligozoospermic or teratozoospermic ejaculates. In analyses of split ejaculate fractions, the distribution of cAMP coincided with that of fructose and protein (but not with the Mg2+- and Ca2+-dependent ATPase activity or with zinc), indicating secretion of cAMP by the seminal vesicles. The distribution profiles of cAMP in the various ejaculate fractions were similar for particle-free seminal plasma and for pellet II material. The cAMP contents of the fractions were compared with sperm motility in the same fractions. An inverse relationship was found, with the first three fractions displaying higher sperm motility and the last three fractions higher cAMP content.     

Urinary excretion of cyclic AMP in hyperparathyroidism.

The urinary excretion of adenosine 3', 5'-monophosphate (cAMP) was investigated in 15 subjects with primary hyperparathyroidism prior to parathyroidectomy and in 13 of them also after the operation. In comparison with healthy control subjects the cAMP excretion was raised in 8 of the patients pre-operatively and after operation all values had become normal. The discriminatory value of the cAMP analyses seemed to be increased by relating the cAMP values to the urinary excretion of calcium. With the applied methods determination of urinary cAMP was superior to a radioimmunoassay of parathyroid hormone in recognizing patients with primary hyperparathyroidism. In normal subjects it appeared that the cAMP excretion expressed per gram creatinine was somewhat higher in women than in men.     

Levels of plasma cyclic AMP in cardiac surgery

The actions of hormones such as catecholamines, vasopressin and growth hormones are mediated by a common intracellular second messenger, cyclic AMP (adenosine 3',5'-monophosphate). The effects of cardiac surgery on plasma cyclic AMP were studied in 32 adults patients with aorta-coronary bypass or with valvular disease. Blood specimens were obtained before operation, at the beginning and at the end of the cardiopulmonary bypass, 1, 3, 6, 9, 12, 24, 48, 72, 168 hours after surgery. The plasma cyclic AMP level during cardiac operation was elevated above the preoperative level. High levels of plasma cyclic AMP were found both in the aorta-coronary bypass and in the valvular disease immediately after the end of cardiopulmonary bypass. The plasma cyclic AMP level in patients undergoing aorta-coronary bypass with aortic clamping time more than 60 minutes was 38.6 +/- 11.7 pmol/ml, compared to 25.6 +/- 6.6 pmol/ml with aortic clamping time less than 60 minutes immediately after the end of cardiopulmonary bypass. In patients undergoing valve replacement and/or commissurotomy with aortic clamping time more than 60 minutes, the plasma cyclic AMP level immediately after the end of cardiopulmonary bypass was 113 +/- 63.3 pmol/ml, compared to 45.4 +/- 10.3 pmol/ml with aortic clamping time less than 60 minutes (p less than 0.01, Student's t test). During 24 hours after cardiac surgery, the plasma cyclic AMP concentration returned to normal range. It is considered that the plasma cyclic AMP level reflects the risk of cardiac surgery in response to homeostatic derangement.     

The role of renal prostaglandin E as a possible modulator of cyclic AMP production in nephrotic syndrome

Urinary prostaglandin E(PGE) and cyclic AMP (cAMP) excretions were studied by radioimmunoassay in children with nephrotic syndrome and in a control population. In cases with nephrotic syndrome there was a significant elevation in urinary PGE excretion and cAMP excretion was urinary osmolality (U_osm) and the ratio urine to plasma osmolality (U_osm/P_osm); and a negative correlation between urinary cAMP excretion and urine volume. A negative correlation was observed between the values of PGE excretion and urinary cAMP. These data confirmed the role of PGE as a modulator of cAMP production, which was inhibited in the nephrotic syndrome.     

Ethanol increases and vitamin D metabolites decrease the production of cyclic AMP by canine renal cortical membranes

Summary Ethanol 0.16% increased cyclic AMP production by canine renal cortical membranes in the basal state and when challenged with different parathyroid hormone or fluoride concentrations. 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) 40 pM completely inhibited this effect of ethanol and reversed cyclic AMP production to the level observed in buffer alone. The same inhibitory effect was observed with 25OHD₃ and with 24,25-dihydroxycholecalciferol (24,25(OH)₂D₃). The inhibitory effect was related to the vitamin D metabolites' concentration and was maximal for 160 pM; it was independent of their biological activity. This suggests that the effect is mediated through an interaction with the membrane lipids. The effect of vitamin D metabolites on cyclic AMP production was also observed in the presence of serum proteins and should be taken into account if unextracted plasma is assayed in the renal cortical membrane system for PTH bioactivity.     

Hypercalcemia and urinary excretion of cyclic AMP in bronchogenic carcinoma

Plasma calcium and phosphate levels and the cyclic AMP/creatinine ratio in the urine were studied in 23 patients with bronchogenic carcinoma. Hypercalcemia was found in 4 patients (17.4%), confirming the frequency of this abnormality observed by others. However, only one patient had both hypercalcemia and hypophosphatemia. In 4 patients with hypercalcemia and no bone metastases, surgical removal of the tumor was followed by return of the plasma calcium level to normal, suggesting that the hypercalcemia was caused by ectopic secretion of a parathormone-like substance by the neoplasm. Contrary to what might be expected, the urinary cAMP/Cr ratio was abnormally low in most patients, including 3 of the 4 patients with hypercalcemia. Moreover, the cAMP/Cr ratio in the urine did not decrease after removal of the tumor. This finding suggests another difference between PTH-like substances of neoplastic origin and natural PTH if, indeed, a PTH-like substance was responsible for the hypercalcemia.

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Résumé Chez 23 malades atteints de cancer bronchique, nous avons étudié la calcémie, la phosphorémie et le rapport d'excrétion urinaire AMP cyclique/ créatinine. Comme d'autres auteurs, nous avons constaté la fréquence de l'hypercalcémie: 4 cas, soit 17.4%. Mais un seul malade avait à la fois une hypercalcémie et une hypophosphorémie. Chez 4 patients hypercalcémiques sans métastase osseuse, l'exérèse de la tumeur a été suivie d'un retour à la normale du calcium plasmatique. Ceci suggère que l'hypercalcémie résulte d'une sécrétion ectopique, par la tumeur, d'une pseudo-parathormone. Pourtant, dans la plupart des cas et chez 3 des 4 patients hypercalcémiques, le rapport urinaire AMP cyclique/ créatinine est anormalement bas. De plus, ce rapport ne diminue pas après ablation de la tumeur. Ces observations suggèrent, une fois de plus, que la parathormone naturelle et les pseudo-parathormones d'origine néoplasique sont des substances différentes, si l'on admet que l'hypercalcémie est bien due à cette sécrétion hormonale.

     

Oral calcium tolerance and urinary cyclic AMP in urolithiasis.

Oral calcium tolerance and urinary cyclic AMP testing was used in the evaluation of 61 unselected patients with stones. The oral calcium tolerance test was easy to perform and was useful in defining several distinct metabolic abnormalities contributing to calculous formation. Oral calcium tolerance testing is more precise than twenty-four-hour urinary calcium determination and should provide a means of determining proper medical treatment of urolithiasis. Urinary cyclic AMP was disappointing as a measure of parathormone activity.     

Parathormone and chronic kidney disease

The serum parathyroid hormone (PTH) rises in chronic kidney disease (CKD) and induces renal bone disease as well as other organ damage. The bone disease guidelines were released by the K-DOQI in 2003 in order to help physicians to improve bone management at all different CKD stages. However, many different PTH commercial assays are available today and some questions are raised concerning the interpretation, the validity and the practical choice of these different measurements. After reviewing PTH biosynthesis and metabolism, we will describe the regulation of different PTH fragments (particularly 1-84 and 7-84) and the various types of PTH assays. In compromised clinical situations, bone biopsy still remains the golden standard assessment of bone disease, and it will be helpful to clarify the interest of new 3rd generation PTH measurements. At present, we do not dispose of valid therapeutic recommendations using 3rd generation tests, as well as the relevance of the ratio PTH 1-84/7-84.     

Benzo(B)thiophene-2-carboxylic acid: calcium uptake and cyclic AMP production in isolated bone cells

The purpose of the present study was to investigate the mechanism of action on bone of Benzo(B)Thiophene-2-Carboxylic Acid (BL-5583). BL-5583, at a dose range of 0.01-100 micrograms/ml, inhibited spontaneous as well as A23187 and PTH-induced bone resorption in tissue culture. This compound also decreased calcium uptake in both osteoclastic and osteoblastic enriched bone cell populations obtained by sequential collagenase digestion of 1-2 day newborn rat calvariae. The decrease occurred after a 5 min. incubation with 45Ca and BL-5583. The effective dose range was 0.01-100 micrograms/ml. No effect on leucine incorporation or lactic acid production by bone cells was observed. BL-5583 also induced a transient decrease in calcium uptake in skin cells isolated from fetal rats by collagenase digestion, suggesting a lack of tissue specificity for this compound. No effect on cyclic AMP in isolated bone cells was observed with the same dose range that produced a calcium effect.     

Benzo(B)Thiophene-2-Carboxylic acid: Calcium uptake and cyclic AMP production in isolated bone cells

Summary The purpose of the present study was to investigate the mechanism of action on bone of Benzo(B)Thiophene-2-Carboxylic Acid (BL-5583). BL-5583, at a dose range of 0.01–100 μg/ml, inhibited spontaneous as well as A23187 and PTH-induced bone resorption in tissue culture. This compound also decreased calcium uptake in both osteoclastic and osteoblastic enriched bone cell populations obtained by sequential collagenase digestion of 1–2 day newborn rat calvariae. The decrease occurred after a 5 min. incubation with⁴⁵Ca and BL-5583. The effective dose range was 0.01–100 μg/ml. No effect on leucine incorporation or lactic acid production by bone cells was observed. BL-5583 also induced a transient decrease in calcium uptake in skin cells isolated from fetal rats by collagenase digestion, suggesting a lack of tissue specificity for this compound. No effect on cyclic AMP in isolated bone cells was observed with the same dose range that produced a calcium effect.     

Bronchodilator drug efficacy via cyclic AMP.

Cyclic adenosine 3', 5'-monophosphosphate (cyclic AMP) as measured by radioimmunoassay is found in diced rat lung in an amount approximating one picomole per milligram of wet weight lung tissue. Incubation of rat lung with adrenaline, a beta adrenergic agent, produced a rapid increase in cyclic AMP, 100% increase at 15 seconds and 340% at 2 minutes. Isoprenaline was more stimulatory than adrenaline; noradrenaline was less stimulatory, and ephedrine produced a negligible effect. The methylxanthines, caffeine and theophylline, produced an increase in cyclic AMP concentration. Of these, caffeine was more potent, and synergism with adrenaline was demonstrated. The beta adrenergic blocking agent, propranolol, completely inhibited the expected rise in cyclic AMP secondary to adrenaline stimulation. In contrast, the alpha blocker, phentolamine, produced no effect. This animal model offers evidence that adrenergic agents and methylxanthines act to increase cyclic AMP in lung tissue. It is likely that many of the beneficial effects of these drugs in pulmonary patients occur through similar changes and modulation of the cyclic AMP system.