Regulation of adenosine concentration and cytoprotective effects of novel reversible adenosine deaminase inhibitors.

The physiological role of adenosine (Ado) is well known. Although a number of pharmacological attempts have been made to manipulate Ado concentrations in ischemic conditions in different tissues, none have been clinically accepted up to now, mostly due to insufficient elevation of Ado concentrations or unacceptable toxicity. In this study, we evaluated the biochemical and pharmacological actions of several novel erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) analogs to identify new reversible adenosine deaminase (ADA) inhibitors with potential clinical utility. In cell culture experiments, these compounds elevate cellular Ado concentrations under conditions of simulated ischemic stress but very little, if any, under normoxic conditions. Two compounds were selected for study: 9'-chloro-EHNA (CPC-405) and 9'-phthalimido-EHNA (CPC-406), which specifically inhibit ADA in cell-free preparations as well as in intact cells. CPC-405 and CPC-406 do not affect adenosine kinase activity, and they do not affect adenosine transport (influx). CPC-405 and CPC-406 are also more potent than EHNA in elevating adenosine release from human astrocytoma cells and bovine heart microvascular endothelial cells in 2-deoxyglucose-simulated ischemia or under anaerobic conditions. Inhibition of adenosine deaminase by CPC-405 or CPC-406, as well as the 2'-deoxyadenosine toxicity expressed in the presence of these ADA inhibitors, is reversed when the inhibitors are removed by washing the cells. In the isolated rat heart model of ischemia, these novel ADA inhibitors showed enhanced recovery of left ventricular end-diastolic pressure, left ventricular developed pressure, +dP/dtmax and -dP/dtmax. In the rat hippocampal slice model of hypoxia, these compounds also showed neuroprotective effects on CA1 hypoxic injury. In conclusion, these novel ADA inhibitors may represent clinically useful Ado elevating compounds that show cardioprotective, as well as neuroprotective, effects. Also, their potential for immunotoxicity, if any, appears to be transient in nature, representing an important clinical advantage compared with tight-binding ADA inhibitors such as deoxycoformycin.

Photodegradation of indo-1 and its effect on apparent Ca2+ concentrations.

BACKGROUND: Fluorescent indicators that show alterations in excitation and/or emission spectra in response to changes in [Ca2+] are widely used for quantitative cytosolic [Ca2+] measurements. There are several reports of changes in apparent [Ca2+] due only to illumination, however. These results have been attributed either to photodamage to the cells or to photodegradation of the indicator. Light-induced alteration in the behavior of the dye or cells would severely hamper the interpretation of experimental data. We examined this phenomenon in indo-1 loaded cells using confocal laser scanning microscopy. RESULTS: Illumination of indo-1 loaded GH3 cells leads to a decrease in apparent basal [Ca2+] and decreased peaks after depolarization with KCl. When cells were double loaded with indo-1 and fluo-3, the effect of UV illumination was noticed only with the former dye. UV irradiation of indo-1 in simple buffers caused overall photobleaching and conversion to a fluorescent but Ca2+-insensitive species. The latter effect cannot be canceled by ratiometric calibration and is due to loss of carboxymethyl groups from the anilino nitrogens. This photodegradation was inhibited by extracellular administration of 10 to 100 microM Trolox, a water-soluble vitamin E analog. CONCLUSIONS: Photodegradation processes like that observed for indo-1 are likely to be possible for all cation indicators that contain bis(carboxymethyl)anilino moieties, which include essentially all fluorescent indicators for Ca2+ and Mg2+ currently in biological use. If unrecognized, this photochemical dealkylation leads to an underestimation of the analyte concentrations depending on the intensity and duration of illumination. The problem can be avoided by including cell-permeant antioxidants such as Trolox in the bathing solution. The ultimate solution would be to redesign the indicators to minimize photodegradation in the absence of antioxidants.

Long-term depression in cerebellar Purkinje neurons results from coincidence of nitric oxide and depolarization-induced Ca2+ transients.

The role of nitric oxide (NO) in the induction of long-term depression (LTD) in the cerebellum was explored using a new, organic, membrane-impermeant form of caged NO. NO photolytically released inside Purkinje neurons mimicked parallel fiber (PF) activity in synergizing with brief postsynaptic depolarization to induce LTD. Such LTD required a delay of < 50 ms between the end of photolysis and the onset of depolarization, was prevented by intracellular Ca2+ chelation, and was mutually occlusive with LTD conventionally produced by PF activation plus depolarization. Bath application of NO synthase inhibitor or of myoglobin, a NO trap, prevent LTD induction via PF stimulation, but not that from intracellular uncaged NO, whereas intracellular myoglobin blocked both protocols. NO is therefore an anterograde transmitter in LTD induction. A biochemical requirement for simultaneous NO and elevation of intracellular free Ca2+ would explain why PF activity must coincide with postsynaptic action potentials.

Caged nitric oxide. Stable organic molecules from which nitric oxide can be photoreleased.

We report the synthesis and testing of a series of "caged" nitric oxide compounds that are stable indefinitely in oxygen-containing solutions until photolyzed by ultraviolet irradiation, whereupon they release nitric oxide (NO) with quantum yields of delta 8% for 3a (CNO-1) and delta 2% for compounds 3b-e (CNO2-5). After a flash, NO release is complete within 5 ms, so that precise temporal control of NO release is possible. NO donor 3d (CNO-4) includes two carboxylate negative charges at physiological pH, which reduce membrane permeability and enable photolytic generation of NO to be selectively confined to either extracellular or intracellular compartments. Esterification of these carboxyls with acetoxymethyl groups produces 3e (CNO-5), which is membrane-permeant and intracellularly hydrolyzable. Therefore, large populations of intact cells can be conveniently intracellularly loaded with "caged" NO donor 3d by incubation with 3e (CNO-5). The biological efficacy of these NO donors and their absolute dependence on UV-irradiation was demonstrated by inhibition of thrombin-stimulated platelet aggregation. Extracellular hemoglobin blocked the effects of NO generated outside but not inside platelets, verifying the sidedness of the NO donors and the limited spatial range of NO action. These molecules should permit precise spatial, temporal, and concentration control of NO release for investigation of its important biological functions.