μM Caffeine Research Articles

Molecular effects of taxol and caffeine on pancreatic cancer cells.

Pancreatic cancer is the fifth leading cause of cancer related deaths in the United States. Despite many recent advances in the treatment modalities, the mortality rate still remains very high. Paclitaxel (Taxol) and Caffeine have been used for the treatment of this disease, however the molecular mechanisms of these agents are not fully understood, which may be partly responsible for the failure of these agents in the treatment of pancreatic cancer. Human pancreatic adenocarcinoma cell lines, HPAC and PANC-1 containing wild-type and mutant p53 respectively, were used to investigate the effects of Taxol and Caffeine on cell growth, and their effects on the modulation of cell cycle and apoptosis related genes. Protein extracts from these cells treated with 100 nM of Taxol or 4 mM of Caffeine were subjected to Western blot analysis for this study. Drug treated cells were also analyzed to calculate the number of cells undergoing apoptosis. Dose and time dependent growth inhibition was observed in both PANC-1 and HPAC cells when treated with either Taxol or Caffeine. Western blot analysis showed an up-regulation of p21WAF1 in both cell lines treated with either Taxol or Caffeine. Furthermore, down-regulation of cyclin B and cdk1 was observed in Taxol and Caffeine treated HPAC cells. However, the results were drastically different in PANC-1 cells where cyclin B was down regulated only by Caffeine treatment and the level of cdk1 protein was undetectable in this cell line. Moreover, up-regulation of p53 and down-regulation of Bcl-2 was observed only in HPAC cells treated with Taxol. Apoptotic cell death analysis showed increasing number of cells undergoing apoptosis between 24 and 48 h of Caffeine treatment, however only Taxol showed greater than 50% cells under-going apoptosis only in HPAC cells. The up-regulation of p21WAF1 and down-regulation of cyclin B and cdk1 suggest their possible roles in G2/M cell cycle arrest caused by both Taxol and Caffeine as reported earlier. From these results we conclude that the differential molecular changes observed in this study may determine the cellular effects of these agents on pancreatic adenocarcinoma cells and that the effects of chemotherapeutic agents may be determined by the endogenous status of p53 mutation and, in turn, may determine the therapeutic effects of these agents in the treatment of pancreatic cancer.

Recovery as a function of the osmolality of the perfusion medium in microdialysis experiments

The effect of varying perfusion medium osmolality on the in vivo microdialysis recovery of caffeine and 5-chloro-2′,3′-dideoxy-3′-fluorouridine (FCdU) was investigated. The microdialysis probes were implanted in rats into the corpus striatum of the brain. Each probe was perfused with a solution containing 10 μM FCdU (in distilled water, Ringer's solution or in 20% sucrose in Ringer's solution) or with a solution containing 10 μM caffeine (in distilled water, Ringer's solution, in 20% sucrose in distilled water or in 20% sucrose in Ringer's solution). Recovery was calculated as the proportion of concentration loss over the microdialysis membrane to the perfusion medium (i.e. from 10 μM). The recovery was significantly dependent on the composition of the perfusion medium. The recoveries of FCdU were 0.097 (55%) for water solution, 0.176 (100%) for Ringer's solution and 0.292 (166%) for sucrose's solution. The recoveries of caffeine were 0.196 (73%) for water solution, 0.270 (100%) for Ringer's solution and 0.335 (124%) for sucrose's solution in water and 0.303 (112%) for sucrose's solution in Ringer. A linear correlation between the recovery over microdialysis membrane and osmolality of perfusion medium was demonstrated for FCdU. The model describing the change of the recovery of caffeine with osmolality of perfusion medium fitted a polynomial function of second order. It is concluded that the osmolality of the perfusion medium influences the recovery in microdialysis experiments. A possible mechanism for this phenomenon is an increase in the extracellular volume fraction with increasing osmolality, thereby increasing the effective diffusion in the tissue.

Recovery as a function of the osmolality of the perfusion medium in microdialysis experiments

The effect of varying perfusion medium osmolality on the in vivo microdialysis recovery of caffeine and 5-chloro-2′,3′-dideoxy-3′-fluorouridine (FCdU) was investigated. The microdialysis probes were implanted in rats into the corpus striatum of the brain. Each probe was perfused with a solution containing 10 μM FCdU (in distilled water, Ringer's solution or in 20% sucrose in Ringer's solution) or with a solution containing 10 μM caffeine (in distilled water, Ringer's solution, in 20% sucrose in distilled water or in 20% sucrose in Ringer's solution). Recovery was calculated as the proportion of concentration loss over the microdialysis membrane to the perfusion medium (i.e. from 10 μM). The recovery was significantly dependent on the composition of the perfusion medium. The recoveries of FCdU were 0.097 (55%) for water solution, 0.176 (100%) for Ringer's solution and 0.292 (166%) for sucrose's solution. The recoveries of caffeine were 0.196 (73%) for water solution, 0.270 (100%) for Ringer's solution and 0.335 (124%) for sucrose's solution in water and 0.303 (112%) for sucrose's solution in Ringer. A linear correlation between the recovery over microdialysis membrane and osmolality of perfusion medium was demonstrated for FCdU. The model describing the change of the recovery of caffeine with osmolality of perfusion medium fitted a polynomial function of second order. It is concluded that the osmolality of the perfusion medium influences the recovery in microdialysis experiments. A possible mechanism for this phenomenon is an increase in the extracellular volume fraction with increasing osmolality, thereby increasing the effective diffusion in the tissue.

RESPONSE TO NIMODIPINE IN CAFFEINE-INDUCED NEUROTOXICITY IN CEREBELLAR GRANULAR CELL CULTURE OF RAT PUPS

Methylxanthines (theophylline, theobromine and caffeine) are widely used as central nervous system stimulants and caffeine is used in the treatment of apnea in newborns. Plasma therapeutic concentration of caffeine is around 110 μm. Caffeine diffuses the blood brain barrier easily, increasing oxygen consumption in neurones and leading to cell death. In the present study, 4–7-day-old rats were used to obtain cerebellar granular cell cultures. Caffeine was used 50, 150, 250 and 350 μmconcentrations and the most toxic dose for it was found to be 350 μm. Death cell scores were 0.9±0.63 for control, 1.1±0.63 for 50 μm, 0.89±0.47 for 150 μm(P>0.05 for both), 3.84±0.8 for 250 μm(P=0.024) and 6.2±0.86 for 350 μm(P=0.001) caffeine concentrations. The role of voltage-dependent calcium channels in caffeine-induced neurotoxicity was tested with the doses of 100 and 200 μmnimodipine 45 min before or after the 350 μmcaffeine. Both doses of nimodipine after caffeine administration were found to be ineffective in blocking neurotoxicity. Doses administered 45 min prior to caffeine, reduced death cell score to 0.89±0.23 (P=0.000) for 100 μmnimodipine and 2.35±0.96 (P=0.000) for 200 μmnimodipine administration into the cultures. A dose-dependent manner of nimodipine in ischemic states is well-known. In the light of these results, nimodipine may be used in the treatment of newborn apneas together with caffeine to prevent neurotoxic side effects of high or repeated doses of it.

Fractional Ca2+Release from the Endoplasmic Reticulum Activates Ca2+Entry in Freshly Isolated Rabbit Aortic Endothelial Cells

The purpose of the present investigation was to examine the correlation between rates of endoplasmic reticulum (ER) depletion and activation of store operated channels (SOC) in freshly isolated rabbit aortic endothelial cells. We investigated the effects of 10μM cyclopiazonic acid (CPA), 10μM ryanodine, and 10mM caffeine on the rate of Ca2+depletion from the ER and on Ca2+and Mn2+influx using fura-2 fluorescence. We observed that the spontaneous loss of the ACh-sensitive pool is slow. Activation of ryanodine receptors (caffeine, ryanodine) or inhibition of the ER Ca2+pump (CPA) increased the rate of Ca2+loss from the ACh-sensitive pool. CPA stimulated Mn2+influx, while caffeine and ryanodine did not. Our results show non linear correlation between ER depletion and activation of divalent cation entry. In the case of CPA, less than 20% depletion of the ACh sensitive store was required for full activation of SOC, while caffeine and ryanodine deplete over 50% of the ACh sensitive store without activating any influx. These data suggest that only a small compartment of the ER is involved in regulation of Ca2+entry.

Caffeine-induced disturbances of early neurogenesis in whole mouse embryo cultures

In toto mouse embryos were cultivated at embryonic day 8.5 for 26 h with 105, 310 or 620 μM caffeine; 105–310 μM correspond to concentrations transferred by the placenta of heavy caffeine consumers. Failure of neural tube closure, excessive proliferation of neuroepithelial cells and premature evagination of telencephalic vesicles were present in 50% of treated embryos. When reaching the embryonic neural tube before neuronal migration, caffeine regionally modifies the schedule and/or rate of neural cell proliferation.

EFFECTS OF EPINEPHERINE-STIMULATED GLYCOGENOLYSIS ON FORCE AND CA2+ RELEASE IN MOUSE SINGLE SKELETAL MUSCLE FIBRES 1548

The purpose of this study was to determine whether reduced muscle glycogen content, independent of other fatigue-related factors, could account for the reduction in Ca2+ release observed during muscle fatigue. Intact single muscle fibres were dissected from the flexor brevis muscle of mice and injected with Indo-1 to measure intracellular free Ca2+ concentration([Ca2+]i). Force and [Ca2+]i were measured every 20 min at a range of frequencies in control fibres (CON; n=6) perfused with 5mM glucose and in epinepherine-treated fibres (EPI; n=4) perfused with no glucose + 10μM Epi to stimulate glycogenolysis. After 20 min EPI, force at 30Hz increased 2.3 -fold but by 60 min had returned to control levels. These changes in force were not due to changes in [Ca2+]i. Force and[Ca2+]i at all other frequencies (50, 70 and 100 Hz) and maximum Ca2+ activated force (100Hz + 5mM caffeine) were not altered. Performance in a final fatigue run, assessed by time to 30% initial force, was impaired after 60 min EPI (61 ± 7 s) compared to CON (222 ± 10 s) (p<0.05) indicating that glycogen concentration was reduced. We conclude that epinepherine-stimulated glycogenolysis does not impair Ca2+ release and that other fatigue-related factors in combination with glycogen depletion contribute to reductions in Ca2+ release during fatigue.

Transformation of sperm nuclei into metaphase chromosomes in maturing pig oocytes penetrated in vitro.

Cumulus-free pig oocytes at the germinal vesicle (GV) stage were incubated in modified Brackett & Oliphant's medium with 5% fetal calf serum and 5mM caffeine with or without cryopreserved, ejaculated spermatozoa. When oocytes were transferred into modified tissue culture medium (TCM-199B at pH 7.4) supplemented with 10 IU/ml eCG, 10 IU/ml hCG and 1 microg/ml oestradiol-17beta after 8 h of incubation with spermatozoa and cultured for 0-48 h, 86-99% of oocytes were penetrated. Most (95-100%) oocytes penetrated 0-16 h after transfer had decondensed sperm chromatin. However, 24 h after transfer 47% and 33% of penetrated oocytes contained recondensed sperm chromatin and sperm metaphase chromosomes, respectively. The proportion of penetrated oocytes containing sperm metaphase chromosomes increased after 36-48 h of transfer (51-65%). The transformation of sperm nuclei to metaphase chromosomes was obtained in 75% and 79% of anaphase I (AI) to telophase I (TI) and metaphase II (MII) oocytes, respectively, but only in 38% of metaphase I (MI) oocytes. Moreover, such transformation was observed only in 1 of 30 oocytes at the stages of GV breakdown to prometaphase I and none of 69 oocytes at the GV stage. The transformation of sperm nuclei into metaphase chromosomes was completely inhibited in oocytes penetrated by eight or more spermatozoa. Well-developed male and female pronuclei were observed in only 3 (4%) of 77 oocytes penetrated 48 h after transfer. The proportion of oocytes reaching MII was greatly inhibited by sperm penetration; only 18% of penetrated oocytes, but 87% of non-inseminated oocytes, reached MII by 48 h after transfer. None of the oocytes penetrated by seven or more spermatozoa reached MII. Most (75%) oocytes were inhibited from the transition from MI to MII even though they were cultured for 48 h. The present results indicate that: (1) the cytoplasm of maturing oocytes possesses an activity for transforming sperm nuclei into metaphase chromosomes, (2) immature pig oocytes penetrated by spermatozoa can undergo meiotic maturation to MI, and (3) the transition of such oocytes from MI to MII is inhibited, suggesting that an activity of mitogen-activated protein kinase may be retarded.

Dissociation of subsarcolemmal from global cytosolic [Ca2+] in myocytes from guinea-pig coronary artery.

1. Changes in cytosolic Ca2+ concentration (delta[Ca2+]c) were measured by global indo-1 fluorescence and compared with changes in subsarcolemmal Ca2+ concentration (delta[Ca2+]sl) indicated by Ca(2+)-activated K+ currents (IK(Ca)). 2. At -50 mV holding potential, 10mM caffeine increased both IK(Ca) and [Ca2+]c without measurable delay. While IK(Ca) peaked within 0.3 +/- 0.16 s (mean +/- S.D.) and decayed to 50% within 0.4 +/- 0.2 s, delta[Ca2+]c peaked within 1.5 +/- 0.5 s and decayed to 50% within 5.2 +/- 1.0 s. The different time courses support the idea that [Ca2+]sl and [Ca2+]c deviate. 3. When 10 mM caffeine was applied 20 s after an initial 2 s caffeine application, IK(Ca) was suppressed to 22 +/- 5% and delta [Ca2+]c to 40 +/- 4%. During the following 1 min caffeine-free period, IK(Ca) recovered to 61 +/- 7% while delta [Ca2+]c remained at 40 +/- 3%. The differences between IK(Ca) and delta[Ca2+]c suggest that Ca2+ deprivation and Ca2+ refilling is faster in peripheral than in central sarcoplasmic reticulum (SR). 4. During the loading period of indo-1, a spontaneous delta[Ca2+]c of 30-80 nM appeared both at -50 mV and at more positive potentials. The amplitude of spontaneous delta[Ca2+]c increased with the amplitude, the frequency or the fusion of spontaneous transient outward currents (STOCs). 5. Block of sarcolemmal Ca2+ fluxes by 1 mM La3+ increased [Ca2+]c by 250 +/- 100 nM and suppressed the spontaneous delta[Ca2+]c. However, La3+ did not significantly retard the rate of decay of STOCs which may therefore be limited by Ca2+ diffusion into the cytosol and not by Ca2+ extrusion. 6. The dissociation of IK(Ca) (or STOCs) and delta[Ca2+]c may indicate a Ca2+ concentration gradient during Ca2+ release directed from the sarcolemma towards the centre of the cell, which later reverses direction.

Uptake and release of Ca2+ in chemically skinned aortic strips from spontaneously hypertensive (SHR) and normotensive (WKY) rats.

The uptake and release of Ca2+ were studied in EGTA-skinned aortic strips from spontaneously hypertensive rats (SHR strain: SAP = 191 +/- 5 mmHg, n = 27) and normotensive control rats (WKY strain: SAP = 131 +/- 2 mmHg, n = 25). 45Ca uptake was measured as a function of time (0.5 to 30 min.), at pCa 6.6, in the presence of 10 mM of K oxalate. Skinned aortic strips of SHRs accumulated more Ca2+ after 30 min of uptake than those of WKY rats (0.66 +/- 0.05 vs 0.52 +/- 0.03 nmole.mg-1 wet tissue; p < 0.05). A lower activity of the transport system in the hypertensive group was evidenced by the fraction of these maximal uptake values accumulated after 2 minutes of uptake, 56% compared with 98% in the normotensive group. 45Ca release was assayed in skinned aortic strips preloaded for 30 minutes with 45Ca in the absence of K oxalate and desaturated with washing solutions containing 3 nM free Ca2+. 30 mM of caffeine, 5 microM of norepinephrine or 10 microM of IP3 resulted in greater increases in the rates of Ca2+ efflux in WKY than in SHR aortic strips. Net effluxes of Ca2+ upon stimulation with all these drugs were statistically significant only in the hypertensive group due to its slightly but consistently higher Ca2+ content. Changes in both rate of efflux and net efflux induced by 30 mM of caffeine could be blocked by 0.6 mM of ryanodine. The sarcoplasmic reticulum is characterized in the genetically hypertensive rats by a low transport activity of its Ca(2+)-ATPase, a high Ca2+ content and a Ca2+ release mechanism with low responsiveness to stimulation by caffeine, norepinephrine and IP3.

Forskolin stimulation of cyclic AMP accumulation in rat brain cortex slices is markedly enhanced by endogenous adenosine.

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.

In-vitro penetration of pig oocytes matured in culture by frozen-thawed ejaculated spermatozoa

Pig oocytes matured in culture were inseminated with frozen-thawed ejaculated spermatozoa without preincubation in modified tissue culture medium (TCM) 199. High penetration rates (85-89%) and increased incidence of polyspermy were obtained at 25-100 x 10(6) spermatozoa/ml. Wide variation in penetration rates (16-89%) was observed in oocytes inseminated in medium containing 5mM caffeine and at 25-50 x 10(6) spermatozoa/ml obtained from 6 boars, regardless of sperm motility. At 25-50 x 10(6) spermatozoa/ml, penetration rates of oocytes were dependent upon the concentration of caffeine in the medium: there was no penetration without caffeine, but penetration was highest (89%) with 5mM caffeine. None of the oocytes was penetrated in the medium supplemented with heparin at 5-40 micrograms/ml. When heparin was included in the medium with 5mM caffeine, it inhibited the efficacy of caffeine to promote sperm penetration of oocytes.

The Nuclear-Cytopiasmic [Ca2+] Gradient in Single Mammalian Vascular Smooth Muscle Cells

We studied the nuclear-cytoplasmic [Ca2+] gradient in single, freshly dispersed smooth muscle cells from the ferret portal vein, by means of fluorescence ratio imaging microscopy. Cells were loaded with the cell-permeant calcium indicator fura-2 AM. Caffeine and platelet-derived growth factor (PDGF) were used in order to mobilize Ca2+ from the sarcoplasmic reticulum. Basal levels of nuclear and cytoplasmic [Ca2+] were 299 +/- 51 nM and 141 +/- 23 nM respectively (n = 10, p less than 0.001). At 3 min after 10mM caffeine, nuclear [Ca2+] decreased to 117 +/- 13 nM (n = 10, p less than 0.001), cytoplasmic [Ca2+] also decreased to 75 +/- 11 nM (n = 10, p less than 0.001) and the nuclear-cytoplasmic difference was no longer significant (p greater than 0.05). Similarly, 300pM of PDGF also abolished the nuclear-cytoplasmic [Ca2+] gradient (n = 12, p greater than 0.1). We conclude that in single muscle cells from the ferret portal vein a perinuclear sarcoplasmic reticulum could be a factor that contributes to the high apparent nuclear Ca2+ levels either through a dynamic Ca2+ influx, or by a fura-2 signal in the sarcoplasmic reticulum masking the true nuclear [Ca2+].

Caffeine inhibits forskolin-stimulated cyclic AMP accumulation in rat brain

Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex. The IC 50 for this inhibition was 21 ± 2.9 μM, and 200 μM caffeine almost completely blocked the forskolin response. Theophylline which is a behavioural stimulant mimicked the effect of caffeine. However, there was only a slight inhibition of the forskolin response by theobromine and IBMX (3-isobutyl-1-methylxanthine), both of which lack behavioral stimulant effects.

Interaction of caffeine with the GABA A receptor complex: alterations in receptor function but not ligand binding

Behavioral and neurochemical evidence indicates interactions between caffeine and other adenosine receptor ligands and the γ-aminobutyric acid (GABA)-benzodiazepine system. To assess the effects of caffeine on binding and function at the GABA A receptor, we studied the effects of behaviorally-active doses of caffeine on benzodiazepine and Cl − channel binding and on overall function of the GABA A receptor as measured by Cl − uptake. There was no effect of caffeine on benzodiazepine receptor binding in cortical synaptosomal membranes at concentrations of 1–100 μM. No effects on benzodiazepine binding were found ex vivo in mice treated with caffeine, 20 and 40 mg/kg. At the putative Cl − channel site labeled by t-butylbicyclophosphorothionate (TBPS), binding was unchanged in vitro after caffeine treatment (1 and 10 μM) in washed and unwashed membranes. However, in ex vivo studies caffeine (20 and 40 mg/kg) increased numbers of TBPS sites in unwashed but not washed membranes. Muscimol-stimulated Cl − uptake into cortical synaptoneurosomes was decreased in mice treated with caffeine, 20 and 40 mg/kg. Similar results were observed in in vitro preparations treated with 50 μM but not 100 μM caffeine. These results indicate that caffeine administration significantly alters the Cl − transport function of the GABA A receptor complex.

Modulation of divalent cation‐activated chloride ion currents

1. Voltage-sensitive calcium channel currents carried by Ca2+ (ICa) or Ba2+ (IBa) were followed by tail currents carried by Cl- ions in approximately 45% of cultured dorsal root ganglion neurones. 2. Extracellular application of (-)-baclofen (100 microM) inhibited IBa and ICl(Ba). Bay K 8644 (5 microM) potentiated both currents. 3. Intracellular GTP-gamma-S increased the proportion of neurones in which ICl(Ba) was recorded. In addition, the activation by GTP-gamma-S of a pertussis toxin-sensitive GTP binding (G)-protein resulted in a steady increase in the Cl- tail current with time, despite a concurrent reduction in IBa. 4. Extracellular application of 10mM caffeine selectively reduced ICl(Ba) without significant change in IBa. When Ca2+ was the charge carrier, caffeine had little effect on ICl(Ca), and increased the inactivation of ICa. 5. We conclude that, in addition to being regulated by divalent cation entry through Ca2+ channels, the Cl- current is also regulated by G-protein activation. The mechanism of activation of ICl(Ba) may involve Ca2+ release from intracellular stores.

Regulation of the dephosphorylation of phosphorylase A by glucose, AMP and polyamines

1. 1. The effect of glucose, caffeine, AMP and polyamines was investigated on the dephosphorylation of phosphorylase a by the catalytic subunits of protein phosphatase-1 and -2A. 2. 2. Caffeine at 1–20 μM inhibited the dephosphorylation of the dimeric phosphorylase a at 37°C using skeletal muscle enzymes; 0.1–10 mM of caffeine enhanced the rate of dephosphorylation greatly at 13°C and slightly at 37°C. 3. 3. α- d-Glucose was more effective in accelerating both the dephosphorylation and the tryptic digestion of phosphorylase a than the β-anomer. 4. 4. Polyamines were found to moderate the inhibitory effect of AMP at concentrations which may occur in the tissues. In the presence of 5 mM glucose polyamines could cancel the AMP inhibition of the dephosphorylation of liver phosphorylase a by hepatic protein phosphatase-1 and −2A.

Effects of external sodium removal on the contracture of frog skeletal muscle.

Effects of removal of the external sodium on the contractility of the frog's skeletal muscle were studied. Sodium-free solution, replaced by choline, strongly augmented the twitch tension before it abolished the action potential. A similar marked potentiation was also observed in the sustained contracture induced by a small depolarization under the current-clamped condition where generation of conductive action potential was blocked. On the other hand, the maximum contracture induced either by electrical stimulation or by high potassium solution was not modified by lowering the external sodium concentration. Furthermore, contraction threshold measured on the electrically induced contracture was shifted to more negative value of the membrane potential. Caffeine contracture, examined by a short-term application of 5 mM caffeine, was first inhibited by sodium-free solution but afterwards restored or even augmented during the sodium-free perfusion. From these observations it is concluded that a removal of external sodium lowered the mechanical threshold of the muscle by affecting some step of the coupling process at the T-SR junction without affecting the contractile machinery.

Caffeine inhibits post-tetanic potentiation but does not alter long-term potentiation in the rat hippocampal slice

The effects of caffeine were investigated on the extracellular excitatory postsynaptic potentials (EPSPs) recorded in the stratum radiatum of CA 1 of the rat hippocampal slice in response to stimulation of the Schaffer collaterals. Caffeine in concentrations from 500 μM to 10 mM caused an increase in the amplitude of the EPSP, which reached a peak after 5–10 min perfusion. This increase was antagonized by pretreatment with 20 μM trifluoperazine. Paired-pulse facilitation, augmentation and potentiation were strongly inhibited by caffeine. Augmentation was most sensitive to caffeine, being abolished by 500 μM caffeine. Long-term potentiation (LTP) induced by high-frequency stimulation was not significantly inhibited by caffeine either by 5–10 min or by 60 min perfusion with 10 mM caffeine. Moreover, the caffeine-induced increase in the low-frequency EPSP could be reversed by 30 min washout, demonstrating that it was not an LTP type increase.

Evidence for negative control of growth by adenosine in the mammalian embryo: Induction of Hmx/+ mutant limb outgrowth by adenosine deaminase

We investigated the growth-regulatory actions of adenosine and adenosine deaminase (ADA) during embryonic limb development in the mouse. Polydactylous outgrowth, an expression of the Hemimelia-extra toe (Hm x) mutant phenotype, was experimentally regulated in hind-limb buds explanted into a serum-free in vitro system at stage 18 of gestation. Its expression was promoted by exposure to 0.1 or 0.2 IU/ml exogenous ADA and suppressed by co-exposure to 10 nM (—)-N 6-(R-phenylisopropy1)-adenosine (N 6-PIA). Evidence that N 6-PIA acted as a high-affinity agonist against the external adenosine receptor was provided by experiments in which 100 μM caffeine, a known antagonist, competitively blocked its effect. The endogenous adenosine content was analyzed by reversed-phase high-performance liquid chromatography with fluorometric detection following its conversion to the 1,N 6ethenoadenosine derivative. At stage 18. the adenosine levels were 0.5 pmol/μg DNA in whole embryos and 0.08 pmol/pg DNA in hindlimb buds. At the same stage, partially purified extracts of the embryonal plasma enriched fraction contained high levels of ADA activity (0.04-0.06 IU/embryo, or 0.7-1.0 IU/mg protein). In contrast, blood cells contained 0.0001 IU/embryo (or 0.01 IU/mg protein). This enzyme occurred as a single kinetic form with a molecular weight of 4500047000 daltons and an apparent K m of 36-38 μM. Its presence in the embryonal plasma argues against an endocrine mechanism of adenosine secretion in favor of autocrine (self-regulatory) or paracrine (proximate-regulatory) mechanisms. Taken together, our results suggest that the in vitro outgrowth of the prospective polydactylous region is induced upon escape from the local growth-inhibitory influence of extracellular adenosine. It is concluded that growth during early limb development is under systemic as well as local control.