The effect of P2 receptor antagonists and ATPase inhibition on sympathetic purinergic neurotransmission in the guinea-pig isolated vas deferens

1. Intracellular microelectrodes were used to record the transmembrane potential and excitatory junction potentials (e.j.p.s) produced by sympathetic nerve stimulation (1 Hz) in smooth muscle cells of the guinea-pig isolated vas deferens. 2. The symmetrical 3′-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid (NF023) produced a concentration-dependent inhibition of e.j.p. magnitude (IC(50)=4.8×10(−6) M), but had no effect on the resting membrane potential of the smooth muscle cells. 3. Pyridoxal-5-phosphate (P-5-P) also depressed e.j.p. magnitude in a concentration-dependent manner, but was less potent than NF023 (IC(50)=2.2×10(−5) M). At 10(−4) M and above P-5-P significantly depolarized the smooth muscle cells. 4. The nucleoside triphosphatase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156) (5×10(−5) M) significantly increased e.j.p. amplitude. ARL 67156 (10(−4) M) further increased e.j.p. amplitude such that they often reached threshold for initiation of action potentials, causing muscle contraction and expulsion of the recording electrode. 5. After reduction of e.j.p.s by NF023 or P-5-P (both 10(−5) M), subsequent co-addition of ARL 67156 (10(−4) M) significantly increased their magnitude. 6. The overflow of endogenous ATP evoked by field stimulation of sympathetic nerves (8 Hz, 1 min) was measured by HPLC and flurometric detection. ARL 67156 (10(−4) M) enhanced ATP overflow by almost 700% compared to control. 7. We conclude that for electrophysiological studies NF023 is preferable to other P2X receptor antagonists such as pyridoxalphosphate -6-azophenyl-2′,4′-disulphonic acid (PPADS), suramin or P-5-P. Furthermore, breakdown of endogenous ATP by nucleoside triphosphatases is an important modulator of purinergic neurotransmission in the guinea-pig vas deferens

Release of a soluble ATPase from the rabbit isolated vas deferens during nerve stimulation

1. The properties of the ATPase released during electrical field stimulation (EFS) (8 Hz, 25 s) of the sympathetic nerves of the superfused rabbit isolated vas deferens were investigated. 2. Superfusate collected during EFS rapidly metabolised exogenous ATP (100 μM) and 50% was broken down in 5.67±0.65 min. The main metabolite was ADP, virtually no AMP was produced and adenosine was absent. No enzyme activity was seen in samples collected in the absence of EFS. 3. Lineweaver-Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 40 μM and V(max) of 20.3 nmol ATP metabolized min(−1) ml(−1) superfusate. ATPase activity was unaffected by storage at room temperature for 24 h, but was abolished at pH4 or by heating at 80°C for 10 min. 4. ARL 67156 inhibited ATP breakdown in a concentration-dependent manner (IC(50)=25 μM (95% confidence limits=22–27 μM), Hill slope=−1.06±0.04). 5. When EFS was applied three times at 30 min intervals, ATP metabolism was 20–30% less in superfusate collected during the second and third stimulation periods compared with the first. ATPase activity was released in a frequency-dependent manner, with significantly greater activity seen after stimulation at 4 and 8 Hz than at 2 Hz. 6. In conclusion, EFS of the sympathetic nerves in the rabbit vas deferens causes release of substantial ATPase, but little ADPase activity into the extracellular space. This contrasts with the guinea-pig vas deferens, which releases enzymes that degrade ATP to adenosine. Thus, the complement of enzymes released by nerve stimulation is species-dependent

Characterization of the ATPase released during sympathetic nerve stimulation of the guinea-pig isolated vas deferens

1. The release of ATPase activity evoked by electrical field stimulation (EFS) (8 Hz, 25 s) was investigated in several tissues in which adenosine 5′-triphosphate (ATP) acts as a neurotransmitter. 2. Superfusate collected during EFS of sympathetic nerves of the guinea-pig, rat and mouse isolated vas deferens and parasympathetic nerves of the guinea-pig isolated urinary bladder contained ATPase activity. ATP breakdown was fastest in superfusate collected from the guinea-pig isolated vas deferens. However, EFS of the enteric nerves of the guinea-pig isolated taenia coli did not release any detectable ATPase. 3. The ATPase released from the guinea-pig isolated vas deferens metabolized ATP at similar rates at incubation temperatures of 37°C and 20°C. Lineweaver–Burke analysis of the initial rates of ATP hydrolysis gave a K(M) of 39 μM and a V(max) of 1039 pmol ATP metabolized min(−1) ml(−1) superfusate. 4. 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156), pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and pyridoxal-5′-phosphate (P-5-P) all inhibited the ATPase activity in a concentration-dependent manner with a potency order of ARL 67156=PPADS>P-5-P. 5. In conclusion, EFS of several tissues in which ATP is a neurotransmitter causes the release of an ATPase and activity is greatest in the guinea-pig vas deferens. The enzyme has pharmacological and kinetic characteristics that are similar to ectonucleoside triphosphate diphosphohydrolases

Physiological level of norepinephrine increases adenine nucleotides hydrolysis in rat blood serum

Extracellular adenosine 5′-triphosphate (ATP) and its breakdown products, adenosine 5′-diphosphate (ADP) and adenosine, have significant effects on a variety of biological processes. NTPDase enzymes, responsible for adenine nucleotides hydrolysis, are considered the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATP and ADP hydrolysis in rat blood serum are higher during the dark (activity) phase compared to the light (rest) phase. In nocturnal animals (e.g., rats), important physiological changes occur during the dark phase, such as increased circulating levels of melatonin, corticosterone, and norepinephrine (NE). This study investigated the physiological effects, in vivo and in vitro, of melatonin, dexamethasone, and NE upon nucleotides hydrolysis in rat blood serum. For in vivo experiments, the animals received a single injection of saline (control), melatonin (0.05 mg/kg), dexamethasone (0.1 mg/kg), or NE (0.03 mg/kg). For in vitro experiments, melatonin (1.0 nM), dexamethasone (1.0 μM), or NE (1.0 nM) was added directly to the reaction medium with blood serum before starting the enzyme assay. The results demonstrated that ATP and ADP hydrolysis in both in vitro and in vivo experiments were significantly higher with NE treatment compared to control (in vitro: ATP = 36.63%, ADP = 22.43%, P < 0.05; in vivo: ATP = 44.1%, ADP = 37.28%, P < 0.001). No significant differences in adenine nucleotides hydrolysis were observed with melatonin and dexamethasone treatments. This study suggests a modulatory role of NE in the nucleotidases pathway, decreasing extracellular ATP and ADP, and suggests that NE might modulate its own release by increasing the activities of soluble nucleotidases

Second Black Sea Symposium For Young Scientists In Biomedicine March 27-30, 2014, Varna, Bulgaria

Варненски медицински форум (Varna Medical Forum) V. 3, Suppl 1(2014

Abstracts Of The Proceedings And The Posters From The Third Scientific Session Of The Medical College Of Varna

October 2-3, 201