TRPV4 Channel-Regulated ATP Release Contributes to γ-Irradiation-Induced Production of IL-6 and IL-8 in Epidermal Keratinocytes.

External stimuli, such as radiation, induce inflammatory cytokine and chemokine production in skin, but the mechanisms involved are not completely understood. We previously showed that the P2Y11 nucleotide receptor, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) all participate in interleukin (IL)-6 production induced by γ-irradiation. Here, we focused on the transient receptor potential vanilloid 4 (TRPV4) channel, which is expressed in skin keratinocytes and has been reported to play a role in inflammation. We found that irradiation of human epidermal keratinocytes HaCaT cells with 5 Gy of γ-rays (137Cs: 0.75 Gy/min) induced IL-6 and IL-8 production. HaCaT cells treated with TRPV4 channel agonist GSK1016790A also showed increased IL-6 and IL-8 production. In both cases, IL-6/IL-8 production was not increased at 24 h after stimulation, but was increased at 48 h. ATP was released from cells exposed to γ-irradiation or TRPV4 channel agonist, and the release was suppressed by TRPV4 channel inhibitors. The γ-irradiation-induced increase in IL-6 and IL-8 production was suppressed by apyrase (ecto-nucleotidase), NF157 (selective P2Y11 receptor antagonist) and SB203580 (p38 MAPK inhibitor). GSK1016790A-induced inhibitor of kappa B-alpha (IκBα) decomposition, which causes NF-κB activation was suppressed by NF157 and SB203580, and γ-irradiation-induced IκBα decomposition was suppressed by TRPV4 channel inhibitors. Our results suggest that γ-irradiation of keratinocytes induces ATP release via activation of the TRPV4 channel, and then ATP activates P2Y11 receptor and p38 MAPK-NF-κB signaling, resulting in IL-6/IL-8 production.

Stoichiometry of ATP hydrolysis and chlorophyllide formation of dark-operative protochlorophyllide oxidoreductase from Rhodobacter capsulatus.

Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing a reduction of the C17 = C18 double bond of Pchlide to form chlorophyllide a (Chlide) in bacteriochlorophyll biosynthesis. DPOR consists of an ATP-dependent reductase component, L-protein (a BchL dimer), and a catalytic component, NB-protein (a BchN-BchB heterotetramer). The L-protein transfers electrons to the NB-protein to reduce Pchlide, which is coupled with ATP hydrolysis. Here we determined the stoichiometry of ATP hydrolysis and the Chlide formation of DPOR. The minimal ratio of ATP to Chlide (ATP/2e(-)) was 4, which coincides with that of nitrogenase. The ratio increases with increasing molar ratio of L-protein to NB-protein. This profile differs from that of nitrogenase. These results suggest that DPOR has a specific intrinsic property, while retaining the common features shared with nitrogenase.

Microwave irradiation decreases ATP, increases free [Mg²âº], and alters in vivo intracellular reactions in rat brain.

Rapid inactivation of metabolism is essential for accurately determining the concentrations of metabolic intermediates in the in vivo state. We compared a broad spectrum of energetic intermediate metabolites and neurotransmitters in brains obtained by microwave irradiation to those obtained by freeze blowing, the most rapid method of extracting and freezing rat brain. The concentrations of many intermediates, cytosolic free NAD(P)(+) /NAD(P)H ratios, as well as neurotransmitters were not affected by the microwave procedure. However, the brain concentrations of ATP were about 30% lower, whereas those of ADP, AMP, and GDP were higher in the microwave-irradiated compared with the freeze-blown brains. In addition, the hydrolysis of approximately 1 µmol/g of ATP, a major in vivo Mg(2+) -binding site, was related to approximately five-fold increase in free [Mg(2+) ] (0.53 ± 0.07 mM in freeze blown vs. 2.91 mM ± 0.48 mM in microwaved brains), as determined from the ratio [citrate]/[isocitrate]. Consequently, many intracellular properties, such as the phosphorylation potential and the ∆G' of ATP hydrolysis were significantly altered in microwaved tissue. The determinations of some glycolytic and TCA cycle metabolites, the phosphorylation potential, and the ∆G' of ATP hydrolysis do not represent the in vivo state when using microwave-fixed brain tissue.

Influence of irradiation on in vitro red-blood-cell (RBC) storage variables of leucoreduced RBCs in additive solution PAGGS-M.

BACKGROUND: The effect of gamma irradiation on leucoreduced red-blood-cells (RBCs) stored in an additive solution (AS) containing phosphate, adenine, glucose, guanosine, saline and mannitol (PAGGS-M) has not yet been studied, and there are different recommendations about storage time of leucoreduced RBCs after irradiation. STUDY DESIGN AND METHODS: We studied 63 leucoreduced RBC units. All RBCs were stored in AS PAGGS-M and leucoreduced on the collection day. Twenty-one components were irradiated on Day +14 with 30 Gy and 22 served as non-irradiated controls. Samples were drawn and analysed from these 43 units on Day +7, +14, +21, +28, +35, +42 and +49 from the collection day. From 20 units, no samples were taken earlier than on Day +49. Of these, 10 components had been irradiated on Day +14 with 30 Gy and 10 served as non-irradiated controls. RESULTS: Gamma irradiation induced an enhanced in vitro haemolysis rate in the irradiated components. One of the irradiated units showed a haemolysis rate over the recommended limit of 0·8% on Day +42 and four on Day +49. The leakage of potassium ions from irradiated RBCs started to increase faster than that of unirradiated RBCs from the day of irradiation. Lactate dehydrogenase levels increased faster in irradiated units 3 weeks after irradiation. We showed that taking samples weekly does not affect the final result. CONCLUSIONS: Our findings show that the European recommendations should not be changed in regard to the limitation of the storageability after irradiation of leucoreduced RBCs. The damage after irradiation and storage cannot be prevented by using the high-quality AS PAGGS-M.

In vitro effects on platelets irradiated with short-wave ultraviolet light without any additional photoactive reagent using the THERAFLEX UV-Platelets method.

BACKGROUND: A novel short-wave ultraviolet light (UVC) pathogen reduction technology (THERAFLEX UV-Platelets; MacoPharma, Mouvaux, France) without the need of any additional photoactive reagent has recently been evaluated for various bacteria and virus infectivity assays. The use of UVC alone has on the one hand been shown to reduce pathogens but may, on the other hand, have some impact on the platelet (PLT) quality. The purpose of this study was to determine the potential effects on PLT quality of pathogen inactivation treatment using the novel UVC method for PLT concentrates. STUDY DESIGN AND METHODS: Buffy-coat-derived PLTs suspended in SSP+ were irradiated with UVC light in plastic bags (MacoPharma) made of ethyl vinyl acetate, considered to be highly permeable to UVC light. The UVC-treated (test, n=8) as well as the untreated (reference, n=8) PLT units were stored in PLT storage bags composed of n-butyryl, tri n-hexyl citrate-plasticized polyvinyl chloride (MacoPharma) on a flat bed agitator for in vitro testing during 7 days of storage. RESULTS: No significant difference in PLT counts and lactate dehydrogenase between the groups was detected. During storage, glucose decreased more and lactate increased more in the test units. Statistically significant differences were found for glucose (P<0·01) and lactate (P<0·05) on day 7. ATP levels were higher (P<0·01 from day 5) in the reference units. With exception of day 7 (P<0·01 reference vs. test), hypotonic shock response reactivity was not different between groups. Extent of shape change was lower (P<0·01), and CD62P (P<0·05 day 5) was higher in the test units. CD42b and CD41/61 showed similar trends throughout storage, without any significant difference between the units. pH was maintained at >6·8 (day 7) and swirling remained at the highest level (score = 2) for all units throughout storage. CONCLUSION: Our results suggest that irradiation with UVC light has a slight impact on PLT in vitro quality and appears to be insignificant with regard to current in vitro standards.

Phenylephrine Alleviates 131I Radiation Damage in Submandibular Gland Through Maintaining Mitochondrial Homeostasis.

PURPOSE: The impairment of the salivary glands is a permanent side effect of 131I ablation therapy for patients with differentiated thyroid cancer. Effective and safe treatments for protecting the salivary glands against 131I are currently not available. Mitochondria are susceptible to ionizing radiation, but alterations after 131I exposure are unknown. Here, we investigated the mechanisms of 131I damage in submandibular glands (SMGs) and evaluated the cytoprotective effect of phenylephrine (PE) against mitochondrial radiation damage. METHODS AND MATERIALS: Rats were randomly divided into 4 groups: control, PE alone, 131I alone, and 131I with PE pretreatment. The mitochondrial structure of SMGs was observed under transmission electron microscopy. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cytochrome c, cleaved-caspase 3, SIRT1, NAMPT, and PGC-1α protein levels were determined with Western blot and immunohistochemistry. Levels of mitochondrial membrane potential, nicotinamide adenine dinucleotide (NAD), and adenosine triphosphate (ATP) were measured with relevant kits. RESULTS: After exposing rat SMGs to 131I, the mitochondrial membrane structures were destroyed, the mitochondrial membrane potential decreased, the release of cytochrome c increased, and cleaved-caspase 3 and cell apoptosis were activated. Moreover, the expression of SIRT1, NAMPT, and PGC-1α was downregulated, and the levels of NAD and ATP decreased. In contrast, PE alleviated the 131I-induced mitochondrial damages and upregulated the expression of SIRT1/NAMPT/PGC-1α and the levels of NAD and ATP. CONCLUSIONS: These findings demonstrate that 131I impairs the salivary glands via the downregulation of SIRT1/NAMPT/PGC-1α signal pathways, which disturbs mitochondrial homeostasis. PE alleviated the 131I damage in SMGs at the mitochondrial level, suggesting that PE could be used as a potential radioprotector for patients with differentiated thyroid cancer with radiation sialadenitis.

Aptamer-based fluorescence sensor for rapid detection of potassium ions in urine.

We unveil a new homogeneous assay-using OliGreen and an ATP-binding aptamer-for the highly selective and sensitive detection of potassium ions.

Low level lasers in dentistry.

Low level laser therapy (LLLT) uses light energy, in the form of adenosine triphosphate (ATP), to elicit biological responses in the body. The increased cellular energy and changes in the cell membrane permeability result in pain relief, wound healing, muscle relaxation, immune system modulation, and nerve regeneration. This article investigates the clinical effects of LLLT and explains how it can be applied in the dental field.

Ga-As (808 nm) laser irradiation enhances ATP production in human neuronal cells in culture.

OBJECTIVE: The aim of the present study was to investigate whether Ga-As laser irradiation can enhance adenosine triphosphate (ATP) production in normal human neural progenitor (NHNP) cells in culture. METHODS: NHNP were grown in tissue culture and were treated by Ga-As laser (808 nm, 50 mW/cm(2), 0.05 J/cm(2)), and ATP was determined at 10 min after laser application. RESULTS: The quantity of ATP in laser-treated cells was 7513 +/- 970 units, which was significantly higher (p < 0.05) than the non-treated cells, which comprised 3808 +/- 539 ATP units. CONCLUSION: Laser application to NHNP cells significantly increases ATP production in these cells. These findings may explain the beneficial effects of low-level laser therapy (LLLT) in stroked rats. Tissue culture of NHNP cells might offer a good model to study the mechanisms associated with promotion of ATP production in the nervous system by LLLT.

Effects of proton beam irradiation on mitochondrial biogenesis in a human colorectal adenocarcinoma cell line.

Proton beam therapy has recently been used to improve local control of tumor growth and reduce side-effects by decreasing the global dose to normal tissue. However, the regulatory mechanisms underlying the physiological role of proton beam radiation are not well understood, and many studies are still being conducted regarding these mechanisms. To determine the effects of proton beams on mitochondrial biogenesis, we investigated: mitochondrial DNA (mtDNA) mass; the gene expression of mitochondrial transcription factors, functional regulators, and dynamic-related regulators; and the phosphorylation of the signaling molecules that participate in mitochondrial biogenesis. Both the mtDNA/nuclear DNA (nDNA) ratio and the mitochondria staining assays showed that proton beam irradiation increases mitochondrial biogenesis in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced aggressive HT-29 cells. Simultaneously, proton beam irradiation increases the gene expression of the mitochondrial transcription factors PGC-1α, NRF1, ERRα, and mtTFA, the dynamic regulators DRP1, OPA1, TIMM44, and TOM40, and the functional regulators CytC, ATP5B and CPT1-α. Furthermore, proton beam irradiation increases the phosphorylation of AMPK, an important molecule involved in mitochondrial biogenesis that is an energy sensor and is regulated by the AMP/ATP ratio. Based on these findings, we suggest that proton beam irradiation inhibits metastatic potential by increasing mitochondrial biogenesis and function in TPA-induced aggressive HT-29 cells.

Intermolecular cross-linking of a novel rice Kinesin k16 motor domain with a photoreactive ATP derivative.

A fluorescent photoreactive ATP derivative, 2'(3')-O-(4-benzoylbenzoyl)-1,N(6)-etheno-ATP (Bz(2)-epsilonATP), was synthesized and reacted with the rice kinesin K16 motor domain (K16MD). In the presence of ADP or ATP, UV irradiation of the K16MD solution containing Bz(2)-epsilonATP resulted in a new 100 kDa band, which was an intermolecular cross-linked product of motor domains. In contrast, no cross-linking was observed in the absence of nucleotides. For the motor domain of mouse brain kinesin and skeletal muscle myosin subfragment-1, no such intermolecular photo cross-linking by Bz(2)-epsilonATP was observed. Our results indicate that Bz(2)-epsilonATP acts unusually as a photoreactive crosslinker to detect conformational changes in K16MD induced by nucleotide binding resulting in the formation of dimers.

Photoinduced electron transfer between Fe(III) and adenosine triphosphate-BODIPY conjugates: Application to alkaline-phosphatase-linked immunoassay.

Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes.

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP.

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F1) as the triphosphate, either in the absence or presence of Mg2+, label covalently bound is not hydrolysed. 2. In the presence of Mg2+ the nitreno-ATP is bound to both the alpha and beta subunits, mainly (63%) to the alpha subunits. 3. After successive photolabelling of F1 with 8-azido-ATP (no Mg2+) and 8-azido-ADP (with Mg2+) 4 mol label is bound to F1, 2 mol to the alpha and 2 mol to the beta subunits. 4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F1 previously labelled with 8-nitreno-ADP. It is concluded that binding to the alpha-subunits hinders binding to the beta subunits. 5. F1 that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F1. 6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F1.

Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.

The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A.

Modification of the intrinsic fluorescence and the biochemical behavior of ATP after irradiation with visible and near-infrared laser light.

In this work, the effects of visible (655 nm) and near-infrared (830 nm) light on ATP in solution were examined. The addition of irradiated ATP to the hexokinase reaction caused significant differences in the reaction rates and in the Michaelis-Menten kinetic parameters, k(m) and v(max). Irradiated ATP cleavage by hexokinase occurred in less time. Changes were wavelength and dose dependent. Excitation of ATP with a 260 nm wavelength ultraviolet light induced a fluorescence emission that was decreased when Mg2+ was added due to ion binding of the phosphates, which are the structures that modify the fluorescence produced by the adenine dipoles. The irradiation of this ATP.Mg2+ solution using 655 and 830 nm light increased the fluorescence by a possible displacement of Mg2+ from the phosphates. In conclusion, visible and near-infrared light modifies the biochemical behavior of ATP in the hexokinase reaction and the fluorescence intensity of the molecule thus altering the Mg2+ binding strength to the oxygen atoms in the phosphate group.

Photo-infrared pulsed bio-modulation (PIPBM): a novel mechanism for the enhancement of physiologically reparative responses.

OBJECTIVE: The present manuscript describes the non-invasive, long-range, energy transport of a singular infrared pulsed laser device (IPLD) and the upstream components of the original action mechanism, designated photo-infrared pulsed bio-modulation (PIPBM). BACKGROUND DATA: Major strides have been taken in recent years towards scientifically acceptable clinical applications of low-energy lasers. Nevertheless, challenges still abound. For instance, the range of potential target tissues for laser therapy in medicine has been, until now, limited by the optical penetration of the beam or to sites accessible by fiberoptics. In addition, much needs to be learned about the action mechanisms of pulsed lasers, which can induce unique biological effects. METHODS: We present a review of the IPLD laser technology and the PIPBM mechanism. RESULTS: The studies reviewed suggest that the PIPBM enhances physiologically reparative processes in a non-toxic and selective manner through the activation and modulation of chaotic dynamics in water. These, in turn, lead not only to local, but also long-distance (systemic) effects. CONCLUSIONS: Though additional studies are necessary to fully explore the biological effects of the PIPBM induced by the IPLD, this mechanism may have multiple potential applications in medicine that are the subject of active current and future investigations.

Nucleotide binding by the HIV-1 integrase protein in vitro.

Recombinant human immunodeficiency virus type 1 (HIV-1) integrase was shown to bind ATP and other nucleoside triphosphates and nucleotide analogs in vitro. Cross-linking of ATP and the photoaffinity analog 8-azido-ATP to integrase occurred in a UV dose-dependent manner. Covalent binding of ATP to integrase was also achieved without UV irradiation when the nucleotide was oxidized to the 2',3'-dialdehyde derivative (oxidized ATP) prior to incubation with the protein, indicating the presence of a reactive lysine residue in the nucleotide binding region of the protein. A number of experimental observations indicate that nucleotides and DNA substrates bind at the same or overlapping site(s) on the integrase protein. For example, the binding of nucleotides or nucleotide analogs to integrase was blocked by prior incubation with DNA substrates, and the covalent cross-linking of 8-azido-ATP to integrase inhibited the DNA binding and oligonucleotide cleavage activities of the protein. Oxidized ATP inhibited the oligonucleotide cleavage activity of integrase at concentrations that had no effect on DNA binding, suggesting that oxidized nucleotides may specifically target the catalytic center of the enzyme. These studies indicate that nucleotide analogs may serve as probes for the DNA binding and catalytic sites of the enzyme and may serve as models for the design of active site inhibitors of retroviral integrase.

Fourier transform infrared photolysis studies of caged compounds.

Time-resolved FTIR difference spectroscopy is a powerful tool for investigating molecular reaction mechanisms of proteins. In order to detect, beyond the large background absorbance of the protein and the water, absorbance bands of protein groups that undergo reactions, difference spectra have to be performed between a ground state and an activated state of the sample. Because the absorbance changes are small, the reaction has to be started in situ, in the apparatus, and in thin protein films. The use of caged compounds offers an elegant approach to initiate protein reactions with a nanosecond UV laser flash. Here, time-resolved FTIR and FT-Raman photolysis studies of the commonly used caged compounds, caged Pi, caged ATP, caged GTP, and caged calcium are presented. The use of specific isotopic labels allows us to assign the IR bands to specific groups. Because metal ions play an important role in many biological systems, their influence on FTIR spectra of caged compounds is discussed. The results presented should provide a good basis for further FTIR studies on molecular reaction mechanisms of energy or signal transducing proteins. As an example of such investigations, the time-resolved FTIR studies on the GTPase reaction of H-ras p21 using caged GTP is presented.

Quantitative changes of metabolic and bioenergetic parameters in experimental tumors during fractionated irradiation.

PURPOSE: Previous studies with rat rhabdomyosarcomas indicate that during fractionated irradiation profound alterations of the tumor microvasculature and the oxygenation status occur when the total dose exceeds 45 Gy. At this dose a destruction which included all structures of the vessels and a significant worsening in tumor oxygenation were found. The aim of the present study was to analyze whether these effects of fractionated irradiation on the microvasculature and on tumor oxygenation also induce changes in the bioenergetic and metabolic status in the tumors during radiation treatment. METHODS AND MATERIALS: R1H rhabdomyosarcomas of the rat implanted into the flank were irradiated with 60Co-gamma-rays using 5 fractions of 3 Gy per week over 5 weeks. During this irradiation schedule, tumors were investigated each week for the microregional distributions of glucose, lactate, and ATP concentrations. For this, tumors were rapidly excised, shock-frozen and quantitative bioluminescence measurements were performed on tumor tissue sections. RESULTS: ATP concentrations remained unchanged during fractionated irradiation up to a total dose of 45 Gy. Above this dose, a significant decrease in ATP levels was observed. Lactate concentrations changed only slightly during irradiation whereas glucose levels increased continuously over the whole irradiation period. CONCLUSIONS: During fractionated irradiation of R1H tumors with a total dose of 75 Gy, the bioenergetic and metabolic status of the tumors changed considerably. This became most obvious once a dose of 45 Gy had been achieved. The severe energy depletion and worsening of tumor oxygenation might be the result of destruction of tumor blood vessels as has been described previously in the same tumor model. The modification of the tumor micromilieu appears to be an important parameter in the responsiveness of tumor cells to radiation and for local tumor control.

Positive chronotropic responses of rabbit sino-atrial node cells to flash photolysis of caged isoproterenol and cyclic AMP.

The kinetics of onset and the intracellular biochemical signalling mechanisms which are responsible for the positive chronotropic effect of sympathetic stimulation in rabbit cardiac pacemaker cells were examined by using flash photolysis of caged isoproterenol (ISO) and cyclic AMP (cAMP). When caged ISO (10 microM) was present in the superfusate, a single ultraviolet flash caused gradual increases in the spontaneous beating frequency and action potential height of S-A node cells. Both these effects developed after an initial latency of approximately 5 s. Photorelease of ISO also increased the L-type Ca2+ current (ICa-L) with a time-course similar to that of the changes in action potential waveform and heart rate. All of these ISO-induced effects were blocked completely by 1 microM propranolol, demonstrating that they were beta-adrenergic responses. Flash photolysis of caged cAMP (50 microM) also resulted in increased firing frequency and ICa-L. However, these responses to cAMP developed with little or no latency. Intracellular dialysis with a selective inhibitor of the cAMP-dependent protein kinase, Rp-cAMPS, completely abolished the increase in ICa-L demonstrating that it is mediated exclusively via cAMP-dependent activation of protein kinase A, as opposed to a direct G-protein mediated mechanism.