Application Of Caffeine Research Articles

Progress in clinical application of caffeine in neonates

Caffeine is a methylxanthine alkaloid compound, which is not only effective, but also safe and needn′t monitor the plasma concentration for the prevention and treatment of apnea in preterm infants, compared with theophylline.In recent years, it has been reported that caffeine is beneficial to improve weaning extubation success rate, reduce the incidence of bronchopulmonary dysplasia in preterm infants, reduce the risk of surgical closure of patent ductus arteriosus, and improve the long-term neurological outcome in preterm infants. Key words: Caffeine; Apnea; Newborn

Risk assessment of synephrine in dietary supplements

Synephrine is asympathomimetic phenylethylamine derivative that occurs naturally in citrus fruits. It is often added to dietary supplements intended for weight loss and enhancement of sports performance, typically in the form of Citrus aurantium extracts and in many cases in combination with caffeine. The health risks of synephrine were evaluated on the basis of the available toxicological data and in accordance to the EFSA guidance on the safety assessment of botanicals and botanical preparations intended for use in food supplements. In animal studies, orally applied synephrine induced adrenergic effects on the cardiovascular system (increase of blood pressure, ventricular arrhythmias), which were enhanced by the concomitant application of caffeine as well as physical activity. Some human intervention studies investigating the acute effects of synephrine on blood pressure and heart rate of healthy, normotensive test persons indicate that synephrine can induce cardiovascular effects in humans. Aseries of published case reports of adverse cardiovascular effects (hypertension, cardiac arrhythmia, myocardial infarction) were associated with consumption of synephrine- and caffeine-containing dietary supplements. In conclusion, consumption of high amounts of synephrine, especially in combination with caffeine and physical exercise, is associated with an increased risk of adverse effects on the cardiovascular system. According to the assessment by the BfR (Bundesinstitut für Risikobewertung), daily intake of synephrine through dietary supplements should not exceed the median intake from conventional foods.

Protective effect of low dose caffeine on psychological stress and cognitive function

IntroductionCaffeine is an adrenergic antagonist that enhances neuronal activity. Psychological stress depresses cognitive function. AimTo investigate the effects of acute and chronic low dose caffeine on anxiety-like behavior and cognitive functions of acute or chronic psychological stressed rats. Material-methodAcute or chronic caffeine (3mg/kg) was administered to male Sprague Dawley rats (200–250g, n=42) before acute (cat odor) and chronic variable psychological stress (restraint overcrowding stress, elevated plus maze, cat odor, forced swimming) induction. Anxiety and cognitive functions were evaluated by hole-board and object recognition tests. The brain glutathione and malondialdehyde assays, myeloperoxidase, nitric oxide (NO), superoxide dismutase (SOD), luminol and lucigenin activity and histological examination were done. ANOVA and Student's t-test were used for statistical analysis. ResultsThe depressed cognitive function with chronic stress exposure and the increased anxiety-like behavior with both stress inductions were improved via both caffeine applications (p<0.05–0.001). Both caffeine pretreatments in chronic stressed rats, and chronic caffeine in acute stressed ones reduced the elevated myeloperoxidase activities (p<0.05–0.01). The increased malondialdehyde, lucigenin and NO levels with acute stress were inhibited with chronic caffeine (p<0.05–0.01), malondialdehyde and NO levels were declined by acute caffeine (p<0.001). Acute caffeine decreased SOD activity (p<0.01) and improved glutathione (p<0.01) and luminol levels (p<0.05). The induced histological damage with both stress exposures was ameliorated with chronic caffeine. ConclusionThe increased anxiety-like behavior and depleted cognitive functions under stress conditions were improved with both acute and predominantly chronic caffeine pretreatments by decreasing oxidative damage parameters.

Decreased Biosynthesis of Jasmonic Acid via Lipoxygenase Pathway Compromised Caffeine-Induced Resistance to Colletotrichum gloeosporioides Under Elevated CO2 in Tea Seedlings.

Caffeine, the major purine alkaloid in tea has long been known for its role in plant defense. However, its effect on Colletotrichum gloeosporioides that causes brown blight disease in tea is largely unknown especially under elevated CO2. Here we show that elevated CO2 reduced endogenous caffeine content in tea leaves, but sharply increased susceptibility of tea to C. gloeosporioides. The expression of C. gloeosporioides actin gene was gradually increased during the postinoculation period. In contrast, foliar application of caffeine decreased the C. gloeosporioides-induced necrotic lesions and the expression of C. gloeosporioides actin. Analysis of endogenous jasmonic acid (JA) content revealed that exogenous caffeine could induce JA content under both CO2 conditions in absence of fungal infection; however, in presence of fungal infection, caffeine increased JA content only under elevated CO2. Furthermore, exogenous caffeine enhanced lipoxygenase (LOX) activity and its biosynthetic gene expression under both CO2 conditions, indicating that increased JA biosynthesis via LOX pathway by caffeine might strengthen plant defense only under elevated CO2, while caffeine-induced defense under ambient CO2 might be associated with JA-independent LOX pathway in tea. These results provide novel insights into caffeine-induced plant defense mechanisms that might help to develop an eco-friendly approach for disease control.

Recent advances for clinical applications of caffeine in premature infants

Caffeine has been applied in clinic more than 30 years, nowadays, which is one of the safe and effective drugs for the treatment of neonatal disease.It has been proved that caffeine can significantly reduce the major acute neonatal morbidities include apnea of prematurity, bronchopulmonary dysplasia, severe retinopathy, patent ductus arteriosus with surgical ligation.Moreover, recent research also shown that caffeine can improve babies neurological outcome and lung function.This review summarized the latest progression in the pharmacological characteristics, clinical applications and untoward effect of caffeine. Key words: Caffeine; Aapnea of prematurity; Lung function; Nervous system

Electrophysiological basis of metabolic-syndrome-induced cardiac dysfunction.

Myocardial contractility is controlled by intracellular Ca2+ cycling with the contribution of sarcoplasmic reticulum (SR). In this study, we aimed to investigate the role of altered SR function in defective regulation of intracellular Ca2+ levels in rats with metabolic syndrome (MetS) induced by a 16-week high-sucrose drinking-water diet. Electric-field stimulated transient intracellular Ca2+ changes in MetS cardiomyocytes exhibited significantly reduced amplitude (∼30%) and prolonged time courses (2-fold), as well as depressed SR Ca2+ loading (∼55%) with increased basal Ca2+ level. Consistent with these data, altered ryanodine receptor (RyR2) function and SERCA2a activity were found in MetS cardiomyocytes through Ca2+ spark measurements and caffeine application assay in a state in which sodium calcium exchanger was inhibited. Furthermore, tetracaine application assay results and hyperphosphorylated level of RyR2 also support the "leaky RyR2" hypothesis. Moreover, altered phosphorylation levels of phospholamban (PLN) support the depressed SERCA2a-activity thesis and these alterations in the phosphorylation of Ca2+-handling proteins are correlated with altered protein kinase and phosphatase activity in MetS cardiomyocytes. In conclusion, MetS-rat heart exhibits altered Ca2+ signaling largely due to altered SR function via changes in RyR2 and SERCA2a activity. These results point to RyR2 and SERCA2a as potential pharmacological targets for restoring intracellular Ca2+ homeostasis and, thereby, combatting dysfunction in MetS-rat heart.

Interplay between non-NMDA and NMDA receptor activation during oscillatory wave propagation: Analyses of caffeine-induced oscillations in the visual cortex of rats

Generation and propagation of oscillatory activities in cortical networks are important features of the brain. However, many issues related to oscillatory phenomena are unclear. We previously reported neocortical oscillation following caffeine treatment of rat brain slices. Input to the primary visual cortex (Oc1) generates N-methyl-d-aspartate (NMDA) receptor-dependent oscillations, and we proposed that the oscillatory signals originate in the secondary visual cortex (Oc2). Because non-NMDA and NMDA receptors cooperate in synaptic transmission, non-NMDA receptors may also play an important role in oscillatory activities. Here we investigated how non-NMDA receptor activities contribute to NMDA receptor-dependent oscillations by using optical recording methods. After induction of stable oscillations with caffeine application, blockade of NMDA receptors abolished the late stable oscillatory phase, but elicited ‘hidden’ non-NMDA receptor-dependent oscillation during the early depolarizing phase. An interesting finding is that the origin of the non-NMDA receptor-dependent oscillation moved from the Oc1, during the early phase, toward the origin of the NMDA receptor-dependent oscillation that is fixed in the Oc2. In addition, the frequency of the non-NMDA receptor-dependent oscillation was higher than that of the NMDA receptor-dependent oscillation. Thus, in one course of spatiotemporal oscillatory activities, the relative balance in receptor activities between non-NMDA and NMDA receptors gradually changes, and this may be due to the different kinetics of the two receptor types. These results suggest that interplay between the two receptor types in the areas of Oc1 and Oc2 may play an important role in oscillatory signal communication.

Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes.

Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T. RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.

New Targeted Ca2+ Probes Reveal Mitochondrial Ca2+ Signaling Plays a Critical Role in Rat Sinoatrial (SA) Nodal Pacing

It remains uncertain to what extent cardiac pacing is governed by “membrane” or “Ca2+ clocks” and what in fact is the master oscillator of the heart? As we have already reported that rapid release of mitochondrial Ca2+ is critical in spontaneous beating of neonatal rat cardiomyocytes, we investigated whether a similar mechanism is at play in SA-nodal cells. Adult rat SA-nodal cells were enzymatically isolated and were cultured for 12 hours prior to viral infection with two genetically encoded fluorescent mitochondria-targeted Ca2+ probes GCaMP3, Kd 1 & 3µM. After 2-3 days in culture, the targeted GCaMP3-probes produced the typical punctate mitochondrial pattern in spontaneously beating myocytes. Such cells had robust expression of If, ICa, and INCX. Simultaneously recorded TIRF images of cytosolic (Cai, dialyzed K5Fura-2) and mitochondrial (Cam, GCaMP3 probes) Ca2+ distributions in blebistatin-treated cells showed differential patterns of rise and fall of Calcium. It was noted that only ∼50% of spontaneously beating SA-nodal cells expressed If when hyperpolarized negative to −70mV. Under current-clamp conditions, spontaneously triggered action potentials accompanied by in-phase Cai and Cam signals were recorded only in cells dialyzed with low concentrations of Ca2+ buffer (0.1 mM EGTA & Ca2+). Withdrawal of extracellular Na+ increased the rate of spontaneously generated Cai-oscillations at fixed holding potentials, but triggered diverse signals in different mitochondrial populations that ranged from continuous uptake of Ca2+ to spontaneously triggered oscillations to releasing of Ca2+. Similarly, application of caffeine during Na+ withdrawal while increasing Cai, showed Cam signals indicative of up-take of Ca2+ at some locations and release at others. We conclude that SR and mitochondrial Ca2+ signals and not If serve as the master oscillator of SA nodal cells. Supported by NIH Ro1HL-15162

Protein Phosphatase-1 Increases Calcium Spark Frequency in Murine Cardiomyocytes via Modulation of RyR2 Phosphorylation

Changes in cardiac ryanodine receptor (RyR2) phosphorylation are thought to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP1) activity has been observed in heart failure (HF), but the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP1 activity, we investigated the effect of the PP1 catalytic subunit on Ca2+ sparks in permeabilized cardiomyocytes. We used wild-type (WT) and transgenic mice in which the highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation. In WT myocytes, where cytosolic Ca2+ was clamped at 60nM, 2U/ml of PP1 initially increased Ca2+ spark frequency (CaSpF) by 2.2-fold, followed by a second phase during which CaSpF returned to control. Spark mass was decreased, but due to the high CaSpF, spark-mediated leak was increased by PP1. This was accompanied by depletion of the sarcoplasmic reticulum (SR) Ca2+ stores, as determined by application of caffeine. Changes in Ca2+ release and SR Ca2+ load were prevented by 5uM of okadaic acid, an inhibitor of PP1. S2808A mutant myocytes showed lower resting CaSpF compared to WT (1.18±0.23 vs 2.3±0.35 sparks/100um/s) and 2U/ml of PP1 failed to generate changes in CaSpF as well as in SR Ca2+ load. A higher concentration of PP1 (10U/ml) increased CaSpF 4-fold compared to control in WT, and 2.8-fold in S2808A cells, indicating a concentration-dependence. Our results suggest that increased intracellular PP1 activity stimulates RyR2-mediated SR Ca2+ release and that de-phosphorylation of RyR2-S2808 and at least one not yet identified phosphorylation site may be important in RyR2 modulation.

RNA synthesis and turnover in the molluscan nervous system studied by Click-iT method

RNA synthesis can be detected by means of the in vivo incorporation of 5-ethynyluridine (EU) in newly-synthesized RNA with the relatively simple Click-iT method. We used this method to study the RNA synthesis in the CNS tissue of adult and juvenile terrestrial snails Helix lucorum L. Temporally, first labeled neurons were detected in the adult CNS after 4-h of isolated CNS incubation in EU solution, while 12-h of incubation led to extensive labeling of most CNS neurons. The EU labeling was present as the nuclear and nucleolar staining. The cytoplasm staining was observed after 2–3 days of CNS washout following the EU exposure for 16h. In juvenile CNS, the first staining reaction was apparent as the staining of apical region in the procerebral lobe of cerebral ganglia after 1h of CNS incubation in EU, while the maximum pattern of staining was obtained after 4h of CNS incubation. Thus, age-related differences in RNA synthesis are present. Activation of neurons elicited by serotonin and caffeine applications noticeably increased the intensity of staining. EU readily penetrates into the bodies of juvenile snails immersed in the EU solution. When the intact juvenile animals were immersed in the EU solution for 1h, the procerebrum staining, similar to the one detected in the incubated juvenile CNS, was observed.

Altered Na/Ca exchange distribution in ventricular myocytes from failing hearts.

In mammalian cardiac ventricular myocytes, Ca efflux via Na/Ca exchange (NCX) occurs predominantly at T tubules. Heart failure is associated with disrupted t-tubular structure, but its effect on t-tubular function is less clear. We therefore investigated t-tubular NCX activity in ventricular myocytes isolated from rat hearts ∼18 wk after coronary artery ligation (CAL) or corresponding sham operation (Sham). NCX current (INCX) and l-type Ca current (ICa) were recorded using the whole cell, voltage-clamp technique in intact and detubulated (DT) myocytes; intracellular free Ca concentration ([Ca]i) was monitored simultaneously using fluo-4. INCX was activated and measured during application of caffeine to release Ca from sarcoplasmic reticulum (SR). Whole cell INCX was not significantly different in Sham and CAL myocytes and occurred predominantly in the T tubules in Sham myocytes. CAL was associated with redistribution of INCX and ICa away from the T tubules to the cell surface and an increase in t-tubular INCX/ICa density from 0.12 in Sham to 0.30 in CAL myocytes. The decrease in t-tubular INCX in CAL myocytes was accompanied by an increase in the fraction of Ca sequestered by SR. However, SR Ca content was not significantly different in Sham, Sham DT, and CAL myocytes but was significantly increased by DT of CAL myocytes. In Sham myocytes, there was hysteresis between INCX and [Ca]i, which was absent in DT Sham but present in CAL and DT CAL myocytes. These data suggest altered distribution of NCX in CAL myocytes.

Abstract 16089: Functional Coupling of SK Channels and L-type Calcium Channels in Cardiomyocytes

Background. Small conductance Ca 2+ -activated K + (SK) channels play significant roles in regulating the excitability of cardiomyocytes (CMs). SK channels are unique in that they are gated solely by intracellular Ca 2+ and hence, function to integrate intracellular Ca 2+ and membrane potentials on a beat-to-beat basis in the heart. Our previous studies revealed that cardiac SK2 channels coupled with L-type Ca 2+ channels (LTCCs) through a physical bridge, α-actinin2, suggesting that LTCCs may be functionally coupled with SK2 channels by providing local Ca 2+ domain to activate the SK channels. However, a recent study suggested that sarcoplasmic reticulum (SR) Ca 2+ release is necessary and sufficient for the activation of cardiac SK channels. The objective of the study is to examine the mechanisms of SK channel activation in native CMs. Methods and Results. By using a voltage-clamp protocol in rabbit CMs to activate LTCCs followed immediately by a test voltage to monitor the SK currents, we recorded apamin-sensitive SK currents in response to the Ca 2+ influx. By altering the duration of activation of LTCCs, the activation of SK currents as a function of the increasing Ca 2+ influx was quantified. EGTA or BAPTA were then used to test the intracellular Ca 2+ domain necessary for SK channel activation. BAPTA but not EGTA completely disengaged the coupling between LTCCs and SK channels. SK currents coupled with Ca 2+ influx via LTCCs continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in CMs. Super-resolution imaging of SK2, Ca v 1.2, and ryanodine receptor 2 (RyR2) using stimulated emission depletion (STED) microscopy was performed to directly quantify the distance among SK, LTCC and RyR2 in CMs. Conclusion. Ca 2+ influx through LTCCs provides the immediate and efficient Ca 2+ microdomain for the activation of SK channels. Furthermore, changes in SK channel activation kinetics following SR Ca 2+ depletion suggest both LTCC-mediated Ca 2+ influx and SR Ca 2+ release are necessary for the activation of cardiac SK channels. Funded by NIH (R01 HL085727 and R01 HL085844 to NC), VA (I01 BX000576 to NC) and AHA (14BGIA18870087 to XZ).

Abstract 19960: Human Cardiac Fibroblasts Increase SR-Dependency of Induced Pluripotent Stem Cell-Derived Cardiomyocyte Calcium Handling by Increasing SR Calcium Uptake and SERCA2a Expression via Direct Physical Contact

Introduction: Cardiac fibroblasts can influence cardiomyocyte structure and function through direct physical interaction and/or by the secretion of soluble factors. Here we assess the relative importance of these different modalities of interaction in terms of their effects on cardiomyocyte electrophysiology. Methods: Ventricular fibroblasts from the explanted hearts of dilated cardiomyopathy (DCM) patients (n=4) were seeded directly onto a confluent monolayer of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) at a ratio of 2:1 and cultured for 24 hours. iPS-CMs alone were used as control. Ca2+ transients and action potentials were recorded optically using fluo-4 AM and di-8-ANEPPS. Data are presented as percentage change from control where appropriate. Results: iPS-CM Ca2+ transient duration was significantly reduced by contact with fibroblasts (-8.4±2.2% n=12 p&lt;0.01). Sarcoplasmic reticulum (SR) Ca2+ uptake assessed by caffeine application was highly upregulated (2.07±0.12s-1 vs 0.63±0.07s-1 in control n=26, 23 p&lt;0.001), associated with a significant increase in SR Ca2+ content (F/F0: 3.74±0.11 vs 2.97±0.11 in control n=37, 32 p&lt;0.001) and SERCA2a protein expression (1.02±0.08 vs 0.55±0.01 n=3 p&lt;0.01). iPS-CMs in contact with fibroblasts displayed a greater dependence on SR uptake for Ca2+ extrusion than other mechanisms (SR/NCX/Slow: 60.7/31.9/7.4% vs 23.3/70.9/5.8% in control). Action potential duration (90%) was significantly prolonged in contact with fibroblasts (+18.8±2.5% n=22 p&lt;0.001 vs control). Screening of culture supernatants from the co-culture has identified the presence of a number of cytokines known to affect cardiomyocyte function including IL-6 (680pg/ml), MIF (1.16ng/ml), MCP-1 (1.25ng/ml) and TGF-β. (110pg/ml). Conclusions: DCM fibroblasts influence iPS-CM electrophysiology through different modalities; however physical contact with fibroblasts has effects not seen with soluble mediators alone. This contact-dependent shift towards greater SR-dependency represents a novel effect of fibroblasts on cardiomyocyte Ca2+ handling. Determining whether these effects are mediated through mechanical, electrical or paracrine signalling will be the focus of future work.

Caffeine-induced diuresis and natriuresis is independent of renal tubular NHE3.

Caffeine is one of the most widely consumed behavioral substances. We have previously shown that caffeine- and theophylline-induced inhibition of renal reabsorption causes diuresis and natriuresis, an effect that requires functional adenosine A1 receptors. In this study, we tested the hypothesis that blocking the Gi protein-coupled adenosine A1 receptor via the nonselective adenosine receptor antagonist caffeine changes Na(+)/H(+) exchanger isoform 3 (NHE3) localization and phosphorylation, resulting in diuresis and natriuresis. We generated tubulus-specific NHE3 knockout mice (Pax8-Cre), where NHE3 abundance in the S1, S2, and S3 segments of the proximal tubule was completely absent or severely reduced (>85%) in the thick ascending limb. Consumption of fluid and food, as well as glomerular filtration rate, were comparable in control or tubulus-specific NHE3 knockout mice under basal conditions, while urinary pH was significantly more alkaline without evidence for metabolic acidosis. Caffeine self-administration increased total fluid and food intake comparably between genotypes, without significant differences in consumption of caffeinated solution. Acute caffeine application via oral gavage elicited a diuresis and natriuresis that was comparable between control and tubulus-specific NHE3 knockout mice. The diuretic and natriuretic response was independent of changes in total NHE3 expression, phosphorylation of serine-552 and serine-605, or apical plasma membrane NHE3 localization. Although caffeine had no clear effect on localization of the basolateral Na(+)/bicarbonate cotransporter NBCe1, pretreatment with DIDS inhibited caffeine-induced diuresis and natriuresis. In summary, NHE3 is not required for caffeine-induced diuresis and natriuresis.

Renal Caffeine Effects are Independent of NHE3 Abundance, Trafficking or Phosphorylation

We have previously shown that the diuretic and natriuretic effect of caffeine is mediated by blockade of adenosine A1 receptors and both effects are unaffected in the absence of renal tubular NHE3. However, compensatory mechanisms in tubulus‐specific NHE3 knockout mice may mask caffeine effects on NHE3 abundance, trafficking or phosphorylation. Wild‐type mice were randomized to acute application of caffeine (45 mg/kg, n=6) or water (vehicle, n=6) via oral gavage (1% of bw) and kidneys harvested after 60 min. Renal NHE3 protein abundance (vehicle: 100±8, caffeine: 101±13 %), phosphorylation at serine 552 (vehicle: 100±14, caffeine: 86±10 %) or serine 605 (vehicle: 100±8, caffeine: 96±9 %) were comparable between groups. Membrane abundance analyzed via semi‐quantitative confocal laser scanning microscopy showed no significant difference between groups. We hypothesized that the Na+/HCO3‐ cotransporter NBC1 (NBCe1) may be responsible for mediating the caffeine effects. Mice were treated with vehicle (0.85% NaCl, n=12) or DIDS (NBC inhibitor, 40 mg/kg i.p., 2 µl/g bw, n=10) 30 min before application of caffeine (45 mg/kg, 3% of bw by oral gavage), and a 3 hour urine collection performed. DIDS+caffeine resulted in a significantly lower urinary flow rate vs vehicle+caffeine (191±31 vs 296±16 nl/min/g, P&lt;0.05) which was not different from vehicle alone (203±19 nl/min/g, n=12). Our data show that NHE3 is not required for caffeine‐induced renal effects but point towards a role of NBC1 in mediating caffeine‐induced diuresis.

Effect of caffeine on intrinsic mechanical properties of normal and malignant hyperthermia-susceptible muscle.

Malignant hyperthermia (MH) is a potentially lethal anesthesic complication. Pathological symptoms develop after exposure to triggering substances. It remains uncertain whether cellular alterations pre-exist. Mechanical properties of isolated muscle bundles were examined before and after exposure to a triggering substance. With prior written consent, muscle bundles of 12 MH-susceptible (MHS) and 56 MH-nonsusceptible (MHN) individuals were examined before and after exposure to incremental doses of caffeine. Mechanical properties (baseline tension, peak tension, time to peak tension, and relaxation time) were measured. Contraction and relaxation derivatives and contraction-relaxation coupling were calculated and analyzed. Mechanical properties were not different between the groups before caffeine application. Caffeine increased peak tension in both groups and baseline tension only in MHS muscle bundles; relaxation time/derivative and contraction-relaxation coupling were prolonged. Cellular changes seen in MH are not pre-existing. Exposure to triggering substance impairs relaxation in MHS muscle.

Effects of cannabidiol on contractions and calcium signaling in rat ventricular myocytes

Cannabidiol (CBD), a major nonpsychotropic cannabinoid found in Cannabis plant, has been shown to influence cardiovascular functions under various physiological and pathological conditions. In the present study, the effects of CBD on contractility and electrophysiological properties of rat ventricular myocytes were investigated. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the Ca2+ sensitive fluorescent indicator fura-2 AM. Whole-cell patch clamp was used to measure action potential and Ca2+ currents. Radioligand binding was employed to study pharmacological characteristics of CBD binding. CBD (1μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. CBD (1μM) significantly decreased the duration of APs. Further studies on L-type Ca2+ channels indicated that CBD inhibits these channels with IC50 of 0.1μM in a voltage-independent manner. Radioligand studies indicated that the specific binding of [3H]Isradipine, was not altered significantly by CBD. The results suggest that CBD depresses myocyte contractility by suppressing L-type Ca2+ channels at a site different than dihydropyridine binding site and inhibits excitation–contraction coupling in cardiomyocytes.

The perspective of caffeine and caffeine derived compounds in therapy.

Caffeine (1,3,7-trimethylxanthine) is a plant secondary metabolite with a significant impact on multiple processes and regulatory pathways in the body. Though major part of the population meets caffeine via coffee, tea or chocolate, it has also an important role in pharmacology and it is used as a supplementary substance in medicaments. Currently, the ability of caffeine to ameliorate some neurodegenerative disorders is proved in some studies. This review describes basic data about caffeine including toxicity, pharmacokinetics, biological mechanism of the action, and metabolism. Beside this, promising applications of caffeine, new medicaments and derivatives are discussed. Relevant papers and inventions are depicted in the manuscript. Caffeine is a pharmacologically promising substance that deserves big consideration in the current research and development. The compound has several reasons to be an object of scientific interest and to be used for pharmacology purposes. Despite an extensive research for a long time, no significantly negative effects on human health were proved hence caffeine can be considered as a completely safe compound. The recent data about amelioration of neurodegenerative and other disorders are promising and deserving more work on the issue. ARTICLE HIGHLIGHTS: Caffeine is a purine alkaloid from plants and it has a broad use in current pharmacology. Caffeine is a competitive antagonist of neurotransmitter adenosine on adenosine receptors. The substance is added as a supplementary to drugs and food.Besides interfering on adenosine receptors, caffeine interacts with acetylcholinesterase, monoamine oxidase, phosphodiesterase, ryanodine receptors and others.Current research is devoted to the role of caffeine in neurodegenerative diseases and immunity alteration. New chemical compounds based on caffeine moiety are prepared (Tab. 4, Fig. 6, Ref. 149).

Adenosine Receptor Blockade by Caffeine Inhibits Carotid Sinus Nerve Chemosensory Activity in Chronic Intermittent Hypoxic Animals.

Adenosine is a key excitatory neurotransmitter at the synapse between O(2)-sensing chemoreceptor cells-carotid sinus nerve (CSN) endings in the carotid body (CB). Herein, we have investigated the significance of adenosine, through the blockade of its receptors with caffeine, on the CB hypoxic sensitization induced by chronic intermittent hypoxia (CIH) in the rat. CIH animals were obtained by submitting rats during 15 days from 8:00 to 16:00 to 10 %O(2) for 40 s and 20 % O(2) for 80 s (i.e., 30 episodes/h). Caffeine (1 mM) was tested in spontaneous and 5 %O(2) evoked-CSN chemosensory activity in normoxic and CIH animals. CIH decreased basal spontaneous activity but increased significantly CSN activity evoked by acute hypoxia. Caffeine did not modify basal spontaneous activity in normoxic rats, but decreased significantly by 47.83 % basal activity in CIH animals. In addition, acute application of caffeine decreased 49.31 % and 56.01 % the acute hypoxic response in normoxic and CIH animals, respectively. We demonstrate that adenosine contributes to fix CSN basal activity during CIH, being also involved in hypoxic CB chemotransduction. It is concluded that adenosine participates in CB sensitization during CIH.