Presence Of Theophylline Research Articles

Fluorimetric-flow injection determination of theophylline based on its inhibitory effect on immobilized alkaline phosphatase

A fluorimetric method for the determination of low levels of theophylline based on the inhibition of the enzyme activity of alkaline phosphatase is proposed. The approach used is a flow-injection configuration where bovine alkaline phosphatase is immobilized on controlled pore glass. The enzymatic reaction is based on the hydrolysis of 4-methylumbelliferone phosphate with fluorimetric monitoring of the 4-methylumbelliferone formed at λ ex = 365 nm and λ em = 445 nm. The presence of theophylline in the samples inhibits the catalytic effect of alkaline phosphatase. The linear determination range was found to be between 0.1 and 200 μmol l −1, with a relative standard deviation < 3.5%. The main common interferent species in serum were tested and they did not produce disturbance at their normal concentration in serum. The applicability of the method was checked by adding the analyte to human serum. The recoveries found were between 93 and 105%.

Theophylline and hypoxic ventilatory response in the rat isolated brainstem-spinal cord

We have used the isolated brainstem-spinal cord preparation of the neonatal rat to study the effects of theophylline on the ventilatory response to hypoxia. The brainstem-spinal cord was isolated from neonatal rats (0–4 days) and superfused with mock cerebrospinal fluid (CSF), equilibrated with a gas mixture (F O 2 , 0.90; F CO 2 , 0.02; F N 2 , 0.08; control CSF) at 27°C. We recorded phrenic nerve discharge from C4 roots, using suction electrodes, and measured respiratory frequency ( fR) and the amplitude of the integrated phrenic neurogram (∫ phr). We examined how theophylline and the specific adenosine antagonist, 8-p-sulfophenyltheophylline (SPT), modify the ventilatory response to hypoxia. The response during superfusion with hypoxic CSF (F O 2 , 0.06) consisted of a marked decrease in fR (to 60% of control) and a slight decline in ∫ phr (to 85% of control). By contrast, in the presence of theophylline (30 mg/L = 165 μM) and SPT (5 mg/L = 15 μM) in the superfusate hypoxia reduced fR only moderately (to 87% of control) and exerted virtually no effect on ∫ phr (105% of control). Theophylline and SPT attenuated the rate of decrease in fR and completely blocked the decrease in ∫ phr. There was no difference between the effects of theophylline and those of SPT. The results suggest that theophylline attenuates hypoxic respiratory depression, and that this effect is mediated by the blockade of adenosine.

Gonadotropin Releasing Hormone Stimulation of Cyclic 3′,5′-AMP in the Pituitary Cell of a Teleost (Channa punctatus, Bloch) Requires Extracellular Calcium: Its Relationship to Gonadotropin Release

Pituitary cells were prepared by enzymatic dispersion and incubated in vitro . To observe the effect of gonadotropin releasing hormone (GnRH) and Ca2+ on the murrel pituitary cyclic 3′,5′-AMP (cAMP), cells were dispersed by 0.3% collagenase plus 0.05% trypsin in Earle's minimum essential medium without Ca2+ and a considerably high yield of viable cells were obtained. Addition of a murrel, Channa punctatus, GnRH (cGnRH, 10 μg/incubation) to pituitary cell incubation (6 × 104 cells/well) containing 4 mM theophylline, a phosphodiesterase (PDE) inhibitor, stimulated cAMP accumulation in the pituitary cell 2.4-fold and its release into the medium about 2-fold as compared to control. The extent of stimulation was greatly increased on addition of Ca2+ (2 mM/incubation) with cGnRH: accumulation 5.8-fold and release 3.7-fold, respectively, in comparison to control. A time-course study with cGnRH (20 μg/incubation) plus Ca2+ (2 mM/incubation) on pituitary cell cAMP accumulation showed that the peak of cAMP level was reached at 15 min and remained at the same level until 60 min in the presence of theophylline; this peak was drastically reduced (5-fold) at 30 min in the absence of theophylline, indicating rapid hydrolysis of cAMP by PDE. Ca2+ -augmented cGnRH stimulatory effect on cAMP accumulation and release could be significantly (P < 0.01) inhibited by verapamil (3 μ M/incubation), a specific calcium channel blocker, suggesting requirement of extracellular Ca2+ influx in this process. Calmodulin (CaM), a Ca2+ carrier protein, addition to cGnRH and Ca2+ incubation further augmented the increase of cellular accumulation of cAMP and its release by 39.5 and 45%, respectively, in comparison to cGnRH and Ca2+ (both were statistically significant, P < 0.01). CaM effect could be blocked by calmidazolium (1 μ M/incubation), a specific inhibitor of CaM, indicating specificity of the stimulatory action of CaM. Addition of radioiodinated 125I-CaM, in the presence of Ca2+ or cGnRH plus Ca2+ resulted in the binding of 125 I-CaM to pituitary cells and to the pellet of the lysed cells. 125 I-CaM specifically binds to pituitary cell plasma membrane preparation and saturation of 125I-CaM binding occurred at 9 ng of 125 I-CaM. To investigate whether cGnRH plus Ca2+ -stimulation of pituitary cells cAMP is linked to gonadotropin (GtH) release, similar protocols were followed. It was found that GtH release was augmented to 7-fold by cGnRH plus Ca2+, which was inhibited by verapamil and stimulated by CaM in a similar manner as observed in the case of cAMP. A time-course study with forskolin (40 μ M/incubation) or dibutyryl cAMP (dbcAMP, 0.5 mM/incubation) in the presence of theophylline (4 mM/incubation) showed that GtH release at 5 and 15 min remained unaltered, while at 30 and 60 min a highly significant increase occurred in comparison to control (P < 0.01). A time kinetics study with cGnRH plus Ca2+ demonstrated a sharp increase in GtH release at 15 min, reaching a peak at 60 min. Results therefore indicate that augmentation of GtH release by cGnRH and Ca2+ at 15 min may not be related to cAMP while later stimulation of GtH release is dependent on cAMP.

Inhibitory effect of zinc-chelating dipeptide on parathyroid hormone-stimulated osteoclast-like cell formation in mouse marrow cultures: involvement of calcium signaling.

A possible mechanism of zinc action inhibiting the PTH-induced osteoclast-like cell formation in mouse marrow culture system in vitro was investigated. Bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone-resorbing agent parathyroid hormone (1-34) (PTH). Osteoclast-like cell formation was estimated with staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The effect of zinc sulfate (10(-6) M) or beta-alanyl-L-histidinato zinc (AHZ; 10(-6) M) inhibiting the PTH (10(-8) M)-induced osteoclast-like cell formation was clearly seen in the absence or presence of theophylline (10(-4) M). However, zinc compounds did not inhibit the stimulatory effect of dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP; 10(-4) M) on osteoclast-like cell formation. The stimulating effect of PTH (10(-8) M) on osteoclast-like cell formation was clearly weakened (about 50%) in the presence of EGTA (1.0 mM) or dibucaine (10(-5) M). Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator (10(-8) to 10(-6) M), clearly stimulated osteoclast-like cell formation. PMA effect was inhibited by the presence of AHZ (10(-6) M) or zinc sulfate (10(-8) M). However, the inhibitory effect of zinc compounds was not seen in the presence of both PTH (10(-8) M) and PMA (10(-6) M). The present findings suggest that zinc compounds inhibit PTH-stimulated osteoclast-like cell formation mediated through the Ca(2+)-dependent activation of protein kinase C.

Inhibitory effect of sodium nitroprusside on the relaxing action of isoproterenol in isolated rat urinary bladder

1. Isoproterenol caused relaxation of rat detrusor muscles contracted by electrical stimulations. Sodium nitroprusside (SNP) and methylene blue inhibited the relaxation induced by isoproterenol without affecting forskolin-induced relaxation. 2. In the presence of theophylline, zaprinast still potentiated isoproterenol-relaxation. In the presence of butoxamine, a selective β 2-adrenoceptor inhibitor, but not metoprolol, a selective β 1-adrenoceptor inhibitor, SNP still inhibited isoproterenol-relaxation. 3. SNP inhibited the relaxation to dobutamine, a β 1-adrenoceptor agonist, but not that to terbutaline, a β 2-adrenoceptor agonist. The relaxation to dobutamine was also inhibited by methylene blue. Further, in the presence of theophylline, zaprinast still potentiated dobutamine-relaxation. 4. Isoproterenol increased tissue level of cGMP, which was inhibited by propranolol. 5. These results suggest a possibility that, in rat detrusor muscles, isoproterenol causes relaxation due to increases in the level of both cAMP and cGMP.

Theophylline suppresses the release of tumour necrosis factor-alpha by blood monocytes and alveolar macrophages.

The purpose of this study was to evaluate the effect of theophylline on tumour necrosis factor-alpha (TNF-alpha) release by human blood monocytes (BMo), and rat BMo and alveolar macrophages (AM). BMo and AM were incubated in the absence or presence of theophylline, and the cell-free supernatants were harvested and tested for TNF-alpha activity by bioassay. Theophylline dose-dependently reduced TNF-alpha release by human BMo: significant inhibition was observed at 100 microns (41 +/- 5.9% of controls) and at 50 microns (59 +/- 4.8% of controls), while the inhibitory activity of theophylline at 10 microns (71 +/- 8.9% of controls) was not statistically significant. This activity was maximal at 2 h and declined at 4 h (59 +/- 5.2% of controls) and 24 h (89 +/- 3.1% of controls). Northern analysis performed on ribonucleic acid (RNA) extracted from human BMo demonstrated that theophylline was able to reduce TNF-alpha gene expression. Comparable levels of inhibition of TNF-alpha release were observed on rat BMo and AM (rat BMo 42 +/- 4.9% of controls; rat AM 38 +/- 1.7% of controls), suggesting that BMo and AM are equally susceptible to suppression of TNF-alpha release induced by theophylline. These results indicate that theophylline suppresses TNF-alpha release by mononuclear phagocytes. Since TNF-alpha is involved in the pathogenesis of bronchial hyperresponsiveness and asthma, our results suggest that the therapeutic activity of theophylline might be partly related to its effect on TNF-alpha release.

In vitro fertilization of bovine oocytes in the presence of theophylline

To determine the effect of theophylline on in vitro fertilization, in vitro matured bovine oocytes were co-incubated with sperm from four different bulls. Dose response was varied among individual bulls, but theophylline at 2.5–5 mM enhanced sperm fertilization of cumulus-intact oocytes by accelerating the time of fertilization in vitro. For cumulus-free oocytes, no beneficial effect of theophylline on sperm fertilizing ability was observed even when sperm were pre-incubated with theophylline for 4–5 h before insemination. It is suggested that sperm might be capacitated by cumulus cells surrounding the oocyte, and that theophylline could increase the probability of sperm contact with the cumulus-oocyte complexes and might facilitate cumulus-induced capacitation and/or zona-induced acrosome reaction. In the presence of theophylline, sperm penetration into cumulus-intact oocytes required 4 h. The rate of fertilization varied among individual bulls, and reached a maximum level at 12–20 h after insemination. Subsequent developmental capacity of fertilized oocytes was also determined after fertilization by co-culture with bovine oviduct epithelial cells. Seven days after in vitro fertilization 18.1–22.7% of the inseminated oocytes had developed into blastocysts (150–160 cells).

The negative inotropic effects of amiodarone on isolated guinea pig heart: A possible role of Na +-Ca 2+ Exchange

1. The negative inotropic effects of amiodarone (AM) were studied in isolated, isometrically contracting ventricular papillary muscles from guinea pigs. 2. AM, 4.4 × 10 −5 M, significantly decreased ouabain 10 −6 M)-induced increase in the developed tension. 3. Manganese (10 −2 M), a partial blocker of Na +-Ca 2+ exchange, attenuated the AM's negative inotropy. 4. Theophylline (1.5 × 10 −2 M), an inhibitor of phosphodiesterase, produced a marked increase in the tension (about twice compared to the ouabain effect). 5. However, the magnitude of decrease by AM in the tension in the presence of theophylline was similar to that in the case of ouabain. 6. Tetrodotoxin (TTX) decreased the contraction by about a half, and then subsequent addition of AM in the presence of TTX led to a further decrease in the tension. 7. Eventually co-existence of TTX and AM led to a decrease in tension of same degree, compared to the decrease in tension by AM alone. 8. The results suggest that a large portion of negative inotropic action of AM may, at least, reflect interference with the Na +-Ca 2+ exchange mechanism. 9. This interference with the Na +-Ca 2+ exchange mechanism may exert a strong negative inotropic effect of the drug, in combination with a decrease in Ca 2+ influx via Ca 2+ channels and/or an impairment of Ca 2+-sequestration.

Epinephrine induces beta-adrenergic desensitization and differentiation of HL-60 cells.

The HL-60 cell line was cultured in a serum-free medium and exposed to various concentrations of EPI. The effects on cell growth, differentiation and beta-adrenergic response were followed during the culture period of 72 h. Short-term exposure (3 min) to EPI (1 nM-1 mM) in the presence of theophylline (4 mM) caused a dose-dependent increase of cAMP levels with a maximum of 1500% above basal levels. When the cells were exposed to EPI (1 nM-10 microM) for 72 h, a dose-dependent increase of cAMP levels with a maximum of 60% above basal levels. Sustained exposure to EPI generated a dose-dependent desensitization of the beta-adrenergic signal system. After EPI treatment for 72 h, IPR (10 microM for 3 min) in the presence of theophylline (4 mM) increased cAMP-levels by only 80% above baseline level (cAMP levels after maintained exposure to EPI), compared to 1080% above unstimulated level in control cells. The alpha-adrenergic receptor blocker PHENT (10 microM) did not affect baseline cAMP level or IPR-dependent cAMP response, but a mixture of EPI and PHENT increased the response to IPR. The HL-60 cell growth was not influenced by EPI. However, after repeated exposure to EPI for 72 h a concentration-dependent increase of HL-60 differentiation was demonstrated. Differentiation was not influenced by PHENT. These results suggest a differentiation induction due to a beta-adrenergic-induced cAMP elevation.

Epithelial ion transport in the ileal J-pouch after proctocolectomy in the rat.

In ulcerative colitis and polyposis coli, creation of an ileal pouch with ileoanal anastomosis after proctocolectomy has become a common surgical method. The aim of our study was to characterize the adaptation of the epithelial ion transport function in the pouch by using electrophysiologic techniques. Proctocolectomy and ileoanal anastomosis was performed in rats either with (pouch) or without (control) creation of an ileal J-pouch. To characterize the epithelial barrier function, impedance analysis was performed 6 months after surgery. Epithelial resistance was 29 +/- 2 omega.cm2 in controls and was unchanged in the pouch (28 +/- 4 omega.cm2; NS). In contrast, subepithelial resistance increased from 33 +/- 3 omega.cm2 to 54 +/- 5 omega.cm2 (P < 0.01) owing to work hypertrophy of the muscle layers in the pouch. To detect changes in net ion transport, ion tracer fluxes were measured in the Ussing chamber. In control ileum, absorptive net Na and Cl fluxes of similar magnitude were present, indicating that electroneutral NaCl absorption was the predominant ion transport system. Neither Isc nor net Na and Cl fluxes were significantly altered in the pouch. Glucose-coupled Na absorption was measured as the 3-o-methyl-glucose-induced increase in Isc. Km remained unaltered, while Vmax decreased from 7.5 +/- 2.1 mu eq.h-1 cm-2 in controls to 1.7 +/- 0.8 mu eq.h-1 cm-2 (P < 0.05) in the pouch. Then, maximal transport capacity for electrogenic Cl secretion was measured as the Cl-induced increase in Isc blockable by serosal bumetanide (in the presence of theophylline and prostaglandin E1).(ABSTRACT TRUNCATED AT 250 WORDS)

Possible transsynaptic cholinergic neuromodulation by ATP released from ileal longitudinal muscles of guinea pigs

The effects of α, β-methylene ATP (α, β-mATP) and β, γ-methylene ATP (β, γ-mATP) on endogenous acetylcholine (ACh) release evoked by electrical nerve stimulation were evaluated in guinea-pig ileal longitudinal muscles. Release of ACh was measured with an HPLC-electrochemical detector system and release of ATP by luciferinluciferase essay. Electrically evoked endogenous ACh release was reduced by both α, β-mATP and β, γ-mATP at concentrations of 3 and 30 μM. The inhibitory effect of α, β-mATP (30 μM) on ACh release was not detectable in the presence of theophylline (100 μM), a P 1-purinoceptor antagonist, that itself enhanced ATP release. When exogenuos ATP (0.1 μM) was added to the bath in which the ileal segment was suspended, it was rapidly metabolized, presumably by ecto-ATPase, and disappeared from the medium within 15 min. At 30 μM, α, β-mATP induced ATP release in a suramin-sensitive but Ca 2+-and atrophine-insensitive manner, suggesting P 2-receptor-mediated release of ATP from the smooth muscle. We conclude from these findings that α, β-mATP and, probably, also β, γ-mATP, do not reduce ACh release by direct stimulation of presynaptic P 1-purinoceptors, and that endogenous ATP released postjunctionally by these ATP analogs is decomposed metabolically to adenosine in the synapse and this adenosine triggers P 1-purinoceptor sensitive neuromodulation of cholinergic transmission.

Cyclic adenosine monophosphate accumulation and β-adrenergic binding in unweighted and denervated rat soleus muscle

Unweighting, but not denervation, of muscle reportedly “spares” insulin receptors, increasing insulin sensitivity. Unweighting also increases β-adrenergic responses of carbohydrate metabolism. These differential characteristics were studied further by comparing cyclic adenosine monophosphate (cAMP) accumulation and β-adrenergic binding in normal and 3-day unweighted or denervated soleus muscle. Submaximal amounts of isoproterenol, a β-agonist, increased cAMP accumulation in vitro and in vivo (by intramuscular [IM] injection) to a greater degree ( P < .05) in unweighted muscles. Forskolin or maximal isoproterenol had similar in vitro effects in all muscles, suggesting increased β-adrenergic sensitivity following unweighting. Increased sensitivity was confirmed by a greater receptor density (B max) for [ 125I]iodo-(−)-pindolol in particulate preparations of unweighted (420 · 10 −18 mol/mg muscle) than of control or denervated muscles (285 · 10 −18 mol/mg muscle). The three dissociation constant ( K d ) values were similar (20.3 to 25.8 pmol/L). Total binding capacity (11.4 fmol/muscle) did not change during 3 days of unweighting, but diminished by 30% with denervation. This result illustrates the “sparing” and loss of receptors, respectively, in these two atrophy models. In diabetic animals, IM injection of insulin diminished cAMP accumulation in the presence of theophylline in unweighted muscle (−66% ± 2%) more than in controls (−42% ± 6%, P < .001). These results show that insulin affects cAMP formation in muscle, and support a greater in vivo insulin response following unweighting atrophy. These various data support a role for lysosomal proteolysis in denervation, but not in unweighting, atrophy.

Determination of Theobromine, Theophylline, and Caffeine in Various Food Products Using Derivative UV-Spectrophotometric Techniques and High-Performance Liquid Chromatography

Abstract Accurate, sensitive and precise derivative ultraviolet Spectrophotometric and high-performance liquid chromatographic (HPLC) methods are described for the simultaneous determination of three natural methyl xanthines (theobromine, theophylline and caffeine) in various food products. The derivative UV-spectro-photometry, with zero crrosing technique of measurement was selected for determination of theobromine and theophylline. First-derivative procedure (D1) was adopted for theobromine determination in the presence of theophylline and caffeine; and second-derivative procedure (D2) was adopted for theophylline determination in the presence of theobromine and caffeine. Caffeine was determined by compensation method using the zero order spectrophotometry. The second method was based on HPLC on a reversed phase column, using a mobile phase of 0.01 M sodium acetate solution: acetonitrile (85:15%) at PH 4.0 with detection at 272 nm. Both methods show good linearity, precision and reproducibility. The developed methods proved to be applicable for the determination of methyl xanthines in various food products. The validity of the proposed methods were tested by analyzing laboratory-prepared mixtures containing the three components in various proportions and by analyzing several food products spiked with a known concentration of the three components.

Depression of miniature endplate potential frequency by acetylcholine and its analogues in frog.

1. Acetylcholine (ACh), 7.5 x 10(-5) M, and carbachol, 5 x 10(-6) M (CCh) depressed the frequency of miniature endplate potentials (m.e.p.ps) in the frog (Rana temporaria) sartorius neuromuscular junction with active acetylcholinesterase to about 50-55% of the controls. 2. A similar depression was produced by the nicotinic agonists, nicotine, suberyldicholine and tetramethylammonium. 3. The muscarinic agonists, oxotremorine, methylfurmethide and methacholine were without effect on m.e.p.p. frequency. The muscarinic antagonist, atropine and the nicotinic antagonist, (+)-tubocurarine, had no effect on the depression of m.e.p.p. frequency evoked by CCh. 4. The ganglionic blockers, benzhexonium and IEM-1119, were also without effect on the CCh-evoked depression of m.e.p.p. frequency. 5. Pretreatment of muscles with anticholinesterases did not prevent the CCh-induced drop in m.e.p.p. frequency. 6. The effect of CCh was proportionally the same as in the controls in preparations where the m.e.p.p. frequency was changed by elevation of K+ and in the presence of theophylline, noradrenaline, dibutyryl adenosine 3':5'-cyclic monophosphate (db cyclic AMP) and db cyclic GMP. 7. An inhibitor of Na+,K(+)-ATPase, ouabain, 5 x 10(-5) mol l-1, prevented or reversed the depression of m.e.p.p. frequency by CCh. However, the depression was present in a nominally K(+)-free medium. Insulin and adrenaline, which are considered to be Na+,K(+)-ATPase activators, were without effect on depression of m.e.p.p. frequency. 8. The depression of m.e.p.p. frequency by 5 x 10(-6) M CCh was the same at temperatures between 5 and 30 degrees C with a Q10 near to 1.0. When threshold amounts of CCh were used (6 x 10-7 and 3 x 10-7 M), the depression was less at higher temperatures.9. The receptive structures responsible for the CCh (or ACh)-evoked depression of m.e.p.p. frequency differ pharmacologically from muscarinic, nicotinic ganglionic and neuromuscular junction ACh-receptors as well as from the synaptic cholinesterase, in contrast to previous reports (Duncan & Publicover, 1979).The low temperature-dependence points to the possibility that physical rather than biochemical processes are limiting in this presynaptic effect of cholinomimetics.

Theophylline, fatigue, and diaphragm contractility: cellular levels of 45Ca and cAMP.

The relationship between variations in diaphragmatic contractility and corresponding changes in total tissue levels of 45Ca and adenosine 3',5'-cyclic monophosphate (cAMP) was examined. The contractile performance of perfused contracting rat diaphragms was manipulated with theophylline (10(-4) M), induced fatigue, or both. The increased contractility associated with theophylline was related to significant increases in 45Ca levels without changes in cAMP levels. Fatigue-diminished contractility was associated with increases in both 45Ca and cAMP levels. The increased 45Ca and cAMP levels associated with fatigue persisted, even in the presence of theophylline. Calcium channel blockade with 10(-4) M verapamil blocked the positive inotropic influence of theophylline as well as the theophylline-associated increase in 45Ca levels. Verapamil had no effect on either the fatigue-associated decreases in contractility or the fatigue-enhanced 45Ca uptake. The results of this study strongly suggest that the enhanced contractility associated with theophylline is related to its influence on cellular calcium metabolism. The elevated level of isotopic calcium measured in fatigued muscle probably represents calcium sequestered in the sarcoplasmic reticulum, the result of cAMP-enhanced Ca-adenosine triphosphatase activity.

Effects of purine derivatives on calcitriol metabolism in rats

The effects of theophylline and sodium urate on metabolic production (PR) and clearance rate (MCR) of calcitriol were determined by the constant isotope infusion method in normal rats. Calcitriol PR was significantly reduced after infusion for 20 h of either theophylline (1 mg/h, PR = 22.3 +/- 1.6 ng.kg-1.day-1, P less than 0.001, n = 5) or sodium urate (0.5 mg/h, PR = 18.6 +/- 1.2 ng.kg-1.day-1, P less than 0.001, n = 5) compared with control rats infused with saline (PR = 32.0 +/- 1.5 ng.kg-1.day-1, n = 5). Renal 1 alpha-hydroxylase activity of kidney homogenate was significantly inhibited in rats infused with theophylline or urate. Suppression of 1 alpha-hydroxylase activity was also observed when the kidney homogenate was preincubated for 3 h with various concentrations of xanthine (0.11-3.0 mg/dl). In addition, the MCR of calcitriol was decreased in rats infused with either theophylline (MCR = 21.0 +/- 0.88 microliter.min-1.100 g-1, P less than 0.005) or urate (MCR = 22.9 +/- 0.91 microliter.min-1.100 g-1, P less than 0.05) compared with saline-infused control rats (MCR = 25.2 +/- 0.41 microliter.min-1.100 g-1). Because calcitriol degradation is a receptor-mediated process that requires binding of the receptor-hormone complex to chromatin, we studied the binding affinity of labeled calcitriol receptor for DNA-cellulose in the presence of theophylline or urate. Both theophylline and urate inhibited receptor binding affinity for DNA-cellulose. We conclude that these purine derivatives suppress calcitriol synthesis and inhibit receptor binding affinity for DNA. The altered receptor binding affinity could explain the decreased MCR of calcitriol.

Theophilline Is an Antagonist of Adenosine in Human Forearm Arterioles

The pharmacodynamics of theophylline in humans is attributed to the inhibition of phosphodiesterase activity, a concept based on in vitro studies using drug concentrations much higher than those achieved by therapeutic doses. Recent in vitro studies suggest that theophylline might act as an antagonist of adenosine. This hypothesis was tested in hypertensives by assessing the effect of theophylline on adenosine mediated vasodilation. Drugs were infused into the brachial artery while forearm blood flow (venous plethysmography) was continuously monitored. Adenosine, infused cumulatively (5, 15, and 50 micrograms/min for 3 min each; n = 5), caused a dose-dependent vasodilation that was clearly blunted in the presence of theophylline in a dose not affecting basal arterial flow (1000 micrograms/min for 20 min). On the contrary, papaverine, a selective phosphodiesterase activity inhibitor, infused at two increasing doses (100 and 300 micrograms/min for 20 min, n = 5), did not affect adenosine mediated vasodilation. Our data show that theophylline acts as a specific antagonist of adenosine in humans, an effect which might be relevant for its pharmacological action.

Modulation of transferrin-receptor activity and recycling after induced differentiation of BeWo choriocarcinoma cells

BeWo human choriocarcinoma cells normally grow as cytotrophoblast cells. However, in the presence of 100 microM-forskolin or 5 mM-theophylline, these cells form syncytia similar to morphologically well differentiated syncytiotrophoblasts. We have examined the effect of syncytia formation on transferrin-receptor activity and recycling. Although cellular proliferation stops upon growth in the presence of forskolin or theophylline, the number of cell-surface transferrin-receptors unexpectedly increased 2-fold, whereas the total cellular number increased at most 15%. The rate of biosynthesis of the transferrin receptor as well as class I MHC glycoprotein did not change measurably during syncytium formation. The biosynthesis of human chorionic gonadotropin increased 35-fold after 30 h of growth in the presence of theophylline. The redistribution of the transferrin receptor in syncytia is maintained by a decreased rate constant of endocytosis (0.141 min-1 compared with 0.231 min-1 for control cells) and an increased rate constant of externalization (0.122 min-1 compared with 0.060 min-1 for control cells). These altered rates of endocytosis and externalization resulted in an increased rate of iron accumulation in the syncytia. Furthermore, the recycling time of the transferrin receptor decreased in cells grown in the presence of theophylline (14.6 min compared with 21.2 min in control cells).

Involvement of Dihydropyridine‐Sensitive Ca2+ Channels in Adenosine‐Evoked Inhibition of Acetylcholine Release from Guinea Pig Ileal Preparation

The effects of adenosine and nifedipine on endogenous acetylcholine (ACh) release evoked by electrical stimulation from guinea pig ileal longitudinal muscle preparations exposed to physostigmine were evaluated using an HPLC with electrochemical detection (ECD) system. Resting ACh release, which was sensitive to tetrodotoxin (0.3 microM), was enhanced by Bay K 8644 (0.5 microM; a Ca2+ antagonist) or 4-aminopyridine (30 microM; a K+ channel blocker) but not by theophylline (100 microM; a P1 purinoceptor antagonist) or atropine (0.3 microM). The enhancement of the resting ACh release by Bay K 8644 was virtually unaffected by atropine. Electrically evoked ACh release was enhanced by around two- to fourfold in the presence of theophylline, atropine, Bay K 8644, 4-aminopyridine, or atropine. On the other hand, the evoked ACh release was reduced by adenosine (10-30 microM), nifedipine (0.1-0.3 microM; a dihydropyridine Ca2+ channel antagonist), or bethanechol (1-3 microM) in a concentration-related fashion. The reduction induced by adenosine or nifedipine was almost abolished by either theophylline or Bay K 8644, whereas that induced by bethanechol was virtually unaffected by these drugs. The inhibition by adenosine of ACh release was not influenced in the presence of 4-aminopyridine or atropine. However, this inhibition by adenosine was considerably enhanced by halving the Ca2+ concentration in the Krebs solution and was diminished by doubling the Ca2+ concentration. These findings suggest that adenosine produces a cholinergic neuromodulation presumably via modifying dihydropyridine-sensitive Ca2+ channel activities in the cholinergic neurons, and thus L-type Ca2+ channels may exist on the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased insulin binding to porcine adipocytes in vitro by beta-adrenergic agonists.

The present study was conducted to determine the influence of dibutyryl-cAMP (dbcAMP), epinephrine, ractopamine and clenbuterol on insulin binding to porcine adipocytes. Dibutyryl-cAMP decreased insulin binding to swine adipocytes by 40 and 20% at 1.8 and 25.8 ng insulin/ml, respectively. Ractopamine and clenbuterol directly reduced insulin binding at the low insulin concentration and decreased binding at high insulin concentrations in the presence of adenosine deaminase. Scatchard analysis suggested that the reduction of insulin binding was due to a decrease in receptor number. Epinephrine alone did not influence insulin binding. In the presence of theophylline, epinephrine decreased binding at both low and high insulin concentrations; however, ractopamine plus theophylline decreased binding only at the low insulin concentration. Clenbuterol did not affect insulin binding in the presence of theophylline. Propranolol blocked the inhibitory effect of epinephrine on insulin binding. These beta-adrenergic agonists can inhibit insulin binding and, thus, antagonize insulin action in swine adipocytes.