Urinary excretion of calcium, hydroxyproline and 3',5'-cyclic adenosine monophosphate in primary hyperparathyroidism.

Urinary excretion of calcium (Ca), hydroxyproline (Hyp) and 3',5'-cyclic adenosine monophosphate (cAMP) was measured during fasting, and in the afternoon, over a 3 day period. Twelve hyperparathyroid patients, of whom 6 were re-studied after successful parathyroid surgery, and 10 control subjects participated, and were maintained on a collagen free diet for the duration of the study. Expressed as creatinine ratio values, Hyp was significantly higher in the morning than during the afternoon, whereas the Ca excretion pattern showed low morning and high afternoon values for all groups. cAMP excretion did not change during the two sampling periods. Large day to day variations for each parameter were observed in the individual patient. The value of cAMP measurements in the diagnosis of primary hyperparathyroidism was confirmed. The results may imply that a diurnal variation in Hyp excretion exists in primary hyperparathyroidism and that food intake produces a suppression of Hyp excretion, possibly secondary to suppression of parathyroid function or, in our view, to increased calcitonin excretion.     

Calcium-membrane interactions in the myocardium: effects of ouabain, epinephrine and 3',5'-cyclic adenosine monophosphate

Cardiac microsomes, which represent an enriched but not pure preparation of the heart's sarcoplasmic reticulum, can remove calcium from solution by 2 kinetically dissimilar mechanisms. In the presence of adenosine triphosphate (ATP), Ca⁺⁺ is taken up by cardiac microsomes by a process designated Ca-binding, which exhibits saturation kinetics. The rate and extent of Ca-binding, and the high affinity of the Ca-binding sites could allow this process to cause the intact cell to relax. When anions that permit Ca⁺⁺ to be precipitated within the mlcrosomal vesicles are included along with ATP, much larger amounts of Ca⁺⁺ are taken up by cardiac microsomes. This second process, designated Ca-uptake, does not follow saturation kinetics. Instead, the rate of Ca-uptake increases linearly with increasing Ca⁺⁺ concentration until Ca-uptake becomes inhibited at higher Ca⁺⁺ concentrations.The finding of 2 kinetically distinct Ca⁺⁺ transport processes in cardiac microsomes, both of which are highly active in the micromolar range of Ca⁺⁺ concentration, suggests that Ca⁺⁺ movements in the intact myocardial cell may be controlled by 2 mechanisms. It is suggested that one of these, possibly manifest in vitro as Ca-binding, represents an intracellular release site that initiates systole by delivering Ca⁺⁺ to the contractile proteins. The second process, possibly manifest in vitro as Ca-uptake, is suggested to represent the uptake of Ca⁺⁺ into an intracellular storage site whose Ca⁺⁺ content indirectly determines the amount of Ca⁺⁺ that is delivered to the contractile proteins. These 2 intracellular Ca⁺⁺ pools can be tentatively related to Ca⁺⁺ movements into and out of the myocardial cell, permitting the formulation of a model by which a number of inotropic interventions might modulate myocardial contractility.Cardiac glycosides had no detectible effect on either cardiac microsomal Ca-binding or Ca-uptake. Cyclic adenosine monophosphate (cAMP), which by itself was without effects on cardiac microsomes, more than doubled the rate of Ca-uptake in the presence of a cyclic AMP-dependent protein kinase. The resulting increase in rate of Ca-uptake could explain the actions of epinephrine to enhance contractility at the same time that systole is abbreviated.     

Mitogen-activated protein kinase-dependent stimulation of proliferation of rat lactotrophs in culture by 3',5'-cyclic adenosine monophosphate.

Intracellular cAMP regulates cell proliferation as a second messenger of extracellular signals in a number of cell types. We investigated, by pharmacological means, whether an increase in intracellular cAMP levels changes proliferation rates of lactotrophs in primary culture, whether there are interactions between signal transduction pathways of cAMP and the growth factor insulin, and where the dopamine receptor agonist bromocriptine acts in the cAMP pathway to inhibit lactotroph proliferation. Rat anterior pituitary cells, cultured in serum-free medium, were treated with cAMP-increasing agents, followed by 5-bromo-2'-deoxyuridine (BrdU) to label proliferating pituitary cells. BrdU-labeling indices indicative of the proliferation rate of lactotrophs were determined by double immunofluorescence staining for PRL and BrdU. Treatment with forskolin (an adenylate cyclase activator) or (Bu)2cAMP (a membrane-permeable cAMP analog) increased BrdU-labeling indices of lactotrophs in a dose- and incubation time-dependent manner. The cAMP-increasing agents were also effective in increasing BrdU-labeling indices in populations enriched for lactotrophs by differential sedimentation. The stimulatory action of forskolin was observed, regardless of concentrations of insulin that were added in combination with forskolin. Inhibition of the action of endogenous cAMP by H89 or KT5720, a protein kinase A inhibitor, attenuated an increase in BrdU-labeling indices by insulin treatment. On the other hand, the specific mitogen-activated protein kinase inhibitor PD98059, which was effective in blocking the mitogenic action of insulin, markedly suppressed the forskolin-induced increase in BrdU-labeling indices. (Bu)2cAMP antagonized not only inhibition of BrdU labeling indices but also changes in cell shape induced by bromocriptine treatment, although forskolin did not have such an antagonizing effect. These results suggest that: 1) intracellular cAMP plays a stimulatory role in the regulation of lactotroph proliferation; 2) cAMP and insulin/mitogen-activated protein kinase signalings require each other for their mitogenic actions; and 3) the antimitogenic action of bromocriptine is, at least in part, caused by inhibition of cAMP production.     

Effects of antibody to renal plasma membrane on urinary excretion of adenosine 3',5'-monophosphate and phosphate induced by parathyroid hormone infusion in rats

Studies were made on the effects of pretreatment of rats with antibody to renal cortical plasma membrane (anti-RPM) on the urinary excretion of cAMP and phosphate induced by infusion of parathyroid hormone (PTH). With PTH infusion, the increase in the urinary excretion of cAMP and phosphate were not parallel. Infusion of 1-40 mg anti-RPM inhibited PTH-induced increase in urinary phosphate excretion but not the increase in urinary cAMP. Infusion of 100 mg of anti-RPM inhibited PTH-induced increases in the excretion of both substances. Infusion of nonimmunized rabbit immunoglobulin (IgG; normal IgG) did not affect PTH-induced increases in the excretion of either cAMP or phosphate. In vitro in isolated renal tubules, with 10 mg/ml anti-RPM, the increases in cAMP content induced by PTH were significantly less than those seen with lower concentrations of this antibody or with normal IgG.     

Progesterone increases basal 3',5'-cyclic adenosine monophosphate formation and down-regulates the agonist-induced inositol phosphates generation in human term placenta.

Whether the placenta is a target tissue for estrogens and progesterone, and their putative mechanism of action, is still a controversial question in the literature. The effect of progesterone and estradiol on 3',5'-cyclic adenosine monophosphate (cAMP) and inositol phosphates generation in human term placenta was investigated. Placental explants were incubated in vitro for up to 48 h in the absence and in the presence of estradiol, progesterone or both steroids (0.1 mumol/l final concentration in all cases), and were stimulated with terbutaline, a beta-adrenergic agonist, (0.1 mmol/l) or angiotensin II (1 mumol/l). The cAMP content was measured by a competitive protein binding assay, and the generation of labelled inositol phosphates formation in explants prelabelled with 3H-myo-inositol was measured by anion exchange chromatography. Progesterone increased significantly basal cAMP concentrations in comparison with control or estradiol-treated tissues (169 +/- 13, 72 +/- 8, and 69 +/- 2 pmol/g wet wt tissue, mean +/- SEM, respectively). However, following terbutaline stimulation cAMP levels (mean +/- SEM) increased to similar values under all conditions (182 +/- 33, 197 +/- 36, and 237 +/- 17 pmol/g wet wt tissue for control, estradiol-, and progesterone-treated tissues, respectively). Angiotensin II stimulated inositol phosphates generation in placental explants by an average of fivefold, but this increase was significantly reduced in the presence of progesterone (5.2 +/- 0.7, 3.7 +/- 0.4, and 2.2 +/- 0.3 fold increase vs non-angiotensin-stimulated tissues, for control, estradiol-, and progesterone-treated placenta, mean- +/- SEM, respectively). These data suggest that progesterone modulates the formation of second messengers in human placenta at term.     

Enhanced effectiveness of pulsatile 3',5'-cyclic adenosine monophosphate in stimulating prolactin and alpha-subunit gene expression.

cAMP is involved in the regulation of secretory activity in lactotrope, thyrotrope, and gonadotrope cells. The present study examined whether pulsatile or intermittent changes in cAMP are more effective than a continuous stimulation in increasing pituitary hormone gene expression. Pituitaries from adult female rats were dissociated, plated for 48 h (7-8 x 10(6) cells per well) to allow attachment to Matrigel-coated plastic coverslips, then inserted into perifusion chambers (five to eight chambers per group). After 24 h of treatment, the cells were recovered, RNA extracted, and messenger RNAs (mRNAs) determined by dot blot hybridization. Perfused cells were exposed to either hourly pulses of monobutyryl cAMP (Bt cAMP, 0.01, 0.1, or 1 mM; 1 mM butyrate pulses to controls), or continuously to forskolin (10 microM). Bt cAMP pulses increased both PRL and alpha-subunit mRNAs, maximal after the 0.1 mM dose for PRL (51% increase vs. butyrate controls) and after the 1 mM dose for alpha (60% increase). However, forskolin was ineffective in increasing PRL or alpha mRNA concentrations. TSH, LH, and FSH beta-subunit mRNAs were not altered by Bt cAMP pulses or forskolin. To confirm the different effects of pulsatile vs. continuous cAMP on PRL and alpha-subunit mRNAs, the response to pulsatile 8-bromo cAMP (1 mM) or Bt cAMP (0.5 mM) was compared to continuous Bt cAMP (0.5 mM). PRL and alpha-subunit mRNAs were increased by both cAMP analogs given in a pulsatile manner but not by continuous Bt cAMP. PRL and LH secretory responses (determined in perifusate samples after 2 h and 22 h of treatment) revealed that both PRL and LH release was increased by cAMP stimulation, given either in a pulsatile or continuous manner. These results show that PRL and alpha-subunit gene expression were sensitive to changes in cAMP stimulation, whereas that of TSH, LH, and FSH beta were unaltered. Only intermittent cAMP stimuli were effective in increasing PRL and alpha mRNAs. These data suggest that pulsatile fluctuations in intracellular cAMP may be essential for maximal expression of the PRL and alpha genes. Thus, intermittent changes in intracellular second messengers may be a necessary part of the pathway involved in the transduction of signals from the plasma membrane to the nucleus.     

Adenosine 3'5'-cyclic monophosphate dependent protein kinase in human adrenocortical tumors.

Protein kinase activity has been studied in four human adrenocortical tumors and compared to the one of the normal human adrenal. In two cases where the lack of action of ACTH was related to an anomaly of ACTH receptor, the protein kinase activity was normal. In the other two cases the ACTH receptor was normal, but the protein kinase activity was different from that of the normal adrenal. In one of these cases where the steroidogenesis response of isolated tumor cells to ACTH and DcAMP was higher than in normal adrenal, basal and cAMP stimulated protein kinase activities were significantly higher than those of the normal adrenal, but the activation constants of both nucleotides were similar to those of the normal gland. In the other case, the basal and the cAMP stimulated protein kinase activities were significantly lower, as well as the activation constant of cAMP. However, the binding affinity of 3H-cAMP was normal. Normal adrenal cytosol contains three protein kinases, as resolved by DEAE-cellulose, two of which designated I and II, are cAMP-dependent. The DEAE-cellulose chromatography of the last tumor showed a loss of isoenzyme II. In addition, the protein kinase eluted at the same molarity as that of isoenzyme I of the normal adrenal was not activated by cAMP. Therefore, the lack of response to ACTH of some adrenocortical human tumors may be attributed either to an anomaly of the ACTH receptor or to some defect of the cAMP-dependent protein kinase.     

Divalent cations modulate PTH-dependent 3',5'-cyclic adenosine monophosphate production in renal proximal tubular cells.

The kidney and parathyroid gland play key roles in calcium (Ca++) homeostasis. Recent data suggest that the kidney, in addition to being a primary target for PTH, also recognizes changes in the concentration of extracellular Ca++, thereby modulating hormone-dependent cAMP production, 1,25-dihydroxyvitamin D synthesis, and renin secretion. In this study, we examined: 1) the effects of varying concentration of divalent cations on PTH-dependent cAMP production in renal proximal tubular cells; and 2) the mechanisms by which extracellular Ca++ exerts its inhibitory effects on cAMP production. Single cell suspensions composed of 80-90% proximal tubular cells were prepared from cortical homogenates by collagenase digestion and sieving. In the presence of 1 mM isobutylmethylxanthine, cAMP content was measured by RIA in 5-15 min incubations and showed a 5- to 6-fold increase in response to PTH (10(-11) -10(-6) M). Increasing extracellular Ca++ and magnesium (Mg++) from 0 and 0.5 mM, respectively, to 5.0 mM inhibited PTH-dependent (3 x 10(-9) M) cAMP production by 54 +/- 4% and 47 +/- 6%, respectively. The half maximal inhibitory concentration for both Ca++ and Mg++ was 0.9 mM. In addition, increasing extracellular barium (Ba++) or strontium (Sr++) from 0-10 mM inhibited PTH-dependent (3 x 10(-9) M) production by 54 +/- 7% and 62 +/- 6% with half of the maximal observed inhibition at 2.2 and 2.7 mM, respectively. The inhibition of PTH-dependent cAMP production by 2.5 mM Ca++ was not reversed by the calcium channel blockers diltiazem or verapamil (10(-4) M). However, changes in intracellular calcium may play some role in the inhibitory effects of Ca++ on cAMP production, since ionomycin (10(-6)-10(-5) M) lowered PTH-dependent cAMP production by 25-36%. Our data suggest that the proximal tubular cell can sense physiologically relevant changes in Ca++, providing a potential mechanism for the modulation of 1,25-dihydroxyvitamin D production or other tubular functions relevant to fluid and mineral homeostasis.     

Stimulatory effect of vanadate on 3',5'-cyclic guanosine monophosphate-inhibited low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads.

When isolated rat fat pads were incubated with vanadate, the low Michaelis-Menten constant (Km) cAMP phosphodiesterase (PDE) activity in the microsomal fraction was increased in a time- and dose-dependent manner with vanadate. 3',5'-Cyclic GMP inhibited the vanadate-stimulated PDE activity with similar profile to the insulin-stimulated one. The stimulatory effect of vanadate was inhibited by inhibitors of tyrosine kinases such as amiloride, biochanin A, and genistein to various extents. Vanadate and insulin both showed the full effect in the absence of either K+, N+, or Ca2+ in the medium, while preincubation of the fat pads with a chelator of intracellular Ca2+ inhibited the vanadate action in a dose-dependent manner. The insulin action was not inhibited by it at tested concentrations. These results suggest that the vanadate action, in contrast to the insulin one, is dependent on the intracellular level of Ca2+. Preincubation of the fat pads with inhibitors of protein kinase C such as 1-(5-isoquinoline sulfonyl)-2-methylpiperazine (H-7) and staurosporine inhibited, in part, the vanadate action but did not inhibit the insulin one. Furthermore, vanadate increased the protein kinase C activity in fat pads but insulin did not increase. H-7 and amiloride showed a significant inhibition of stimulation of protein kinase C activity by vanadate. These results suggest that vanadate stimulates, in part, the 3',5'-cyclic GMP-inhibited low Km cAMP PDE activity in the microsomal fraction of fat pads through the activation of tyrosine kinase and protein kinase C-mediated processes.     

Dog thyroid cells in monolayer tissue culture: adenosine 3', 5'-cyclic monophosphate response to thyrotropic hormone.

A simple, rapid, and efficient method is described for establishing dog thyroid cells in tissue culture. Thyroid cell yield from a small amount of tissue (1 g) is high and viability is excellent. The adenosine 3',5-cyclic monophosphate (cAMP) response to thyrotropin (TSH) was investigated in these thyroid cells. Peak cAMP values were achieved after 10-15 min of TSH stimulation (in the presence of 0.5 mM 3-isobutyl-1-methyl-xanthine, MIX), with a subsequent decline to about half the maximal value after approximately 4 hours. This decline in cAMP concentration was associated with the development of refractoriness to TSH stimulation. Half-maximal stimulation of thyroid cell cAMP content was observed at a TSH concentration of between 1 and 2 mU/ml. Maximal cAMP values achieved were approximately 30-fold greater than basal values in the presence of MIX. The threshold of sensitivity of the cAMP response to TSH was very low, with significant stimulation being observed at a TSH concentration of 5-10 muU/ml. As determined by double reciprocal plots, the net cAMP response to TSH appeared to represent a single function over the entire TSH concentration range tested.     

Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin.

Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.     

3',5'-Cyclic adenosine monophosphate activation in osteoblastic cells: effects on parathyroid hormone-1 receptors and osteoblastic differentiation in vitro.

PTH has anabolic and catabolic effects in bone through activation of the PTH-1 (PTH/PTHrP) receptor and the cAMP/protein kinase A pathway. The effects of agents that regulate cAMP in nontransformed osteoblasts in relation to cell differentiation have not been described. The purpose of this study was to determine the effects of PTH fragments with differing cAMP-stimulating activity, and nonPTH cAMP regulators on PTH-1 receptor expression and activity, and osteoblast differentiation in vitro using MC3T3-E1 and primary rat calvarial cells. PTH (1-34), but not PTH (53-84), (7-34), or PTHrP (107-139) treatment (24 h) resulted in down-regulation of steady-state messenger RNA for the PTH-1 receptor. Forskolin (a stimulator of cAMP accumulation) also down regulated the PTH-1 receptor, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (an inhibitor of adenylyl cyclase) had no effect. Similarly, PTH (1-34) treatment for 48 h abolished PTHrP binding to cell surface receptors; however, neither the PTH analogs nor the cAMP regulating agents altered PTH binding or numbers of binding sites on osteoblastic cells. Basal levels of cAMP were reduced in cultured cells treated for 6 days with PTH (7-34) or THFA compared with controls. In contrast, PTH-stimulated cAMP levels were significantly increased in cultures treated with PTH (7-34) and THFA for 6 days during osteoblast differentiation and were decreased in cultures treated with PTH (1-34) and forskolin compared with controls. To evaluate effects of the cAMP pathway on osteoblast differentiation, cultures were treated continuously with PTH analogs and cAMP regulators during an 18-day differentiation regime, total RNA was isolated at multiple time points, and Northern blot analysis for osteocalcin (OCN) was performed. THFA and PTH (7-34)-treated cultures had increased OCN expression; whereas, PTH (1-34) and forskolin reduced OCN expression. Interestingly, PTH (7-34) and THFA-treated cultures had increased mineralized nodule formation, in contrast to PTH (1-34) and forskolin treatment, which reduced nodule formation. Similarly, calcium accumulation in cultures was significantly increased in the PTH (7-34) and THFA-treated cultures and reduced in the PTH (1-34) and forskolin-treated cultures. These data demonstrate that agents that increase cAMP down regulate PTH-1 receptor messenger RNA and inhibit osteoblast differentiation in vitro. Agents that reduce or block adenylyl cyclase or cAMP activity do not alter PTH-1 receptor expression or binding, but have striking effects on promoting osteoblast differentiation. We conclude that many effects of PTH on osteoblasts may be mimicked or antagonized by agents that alter cAMP activity and bypass the PTH-1 receptor.     

Time-dependent stimulation of insulin exocytosis by 3',5'-cyclic adenosine monophosphate in the rat islet beta-cell

Isolated rat islets were exposed to cAMP-elevating agents and/or nutrients. Insulin exocytosis subsequently triggered by a depolarizing concentration of K(+) or a stimulatory concentration of glucose was employed as an index of time-dependent potentiation (TDP). Stimulatory concentrations (>or=5.5 mM) of glucose caused TDP, and 6 micro M forskolin (an activator of adenylyl cyclase) significantly enhanced it (3.1-fold at most). Forskolin produced an 8.0-fold increase in islet cell cAMP; however, it returned to the baseline after washout by the time of stimulation of exocytosis. Two millimoles of dibutyryl cAMP (a cAMP analog), 0.1 mM isobutylmethylxanthine (a phosphodiesterase inhibitor), and 100 nM glucagon-like peptide-1 (an incretin hormone) also enhanced glucoseinduced TDP. The time-dependent effect of cAMP was not attenuated by protein kinase A inhibitors (200 micro M adenosine 3',5'-cyclic monophosphothioate, Rp isomer, and 10 micro M H89). Although glucose-induced TDP was attenuated by NaN(3) (a mitochondrial poison) and cerulenin (an inhibitor of protein acylation), cAMP enhancement of it was unaffected by these agents. In conclusion, cAMP time-dependently stimulates insulin exocytosis, provided the extracellular glucose concentration is equivalent to or higher than ambient plasma levels. Protein kinase A, mitochondrial metabolism, and protein acylation are not involved in this cAMP action. Incretin stimulation of insulin exocytosis may occur in part via this mechanism.