Neuroscience Letters, 151 (1993) 21-24 0 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$06.00 21 NSL 09302 Facilitation of [3H]-ACh release by forskolin depends on A,-adenosine receptor activation Paulo Correia-de23 and Joaquim Alexandre Ribeiro Laboratory of Pharmacology, ICBAS, University of Oporto, and Laboratory of Pharmacology, Gulbenkian Institute of Science, Oeiras (Portugal) (Received 31 August 1992; Revised version received 2 November 1992; Accepted 27 November 1992) Key wor& Presynaptic A, adenosine receptor; Forskolin; [‘HI-Acetylcholine release; Phrenic nerve ending The effect of forskolin (FSK) on [‘HI-acetylcholine release ([‘HI-ACh) from the phrenic motor nerve terminals, and its modification by adenosine deaminase (ADA), by the A,-adenosine receptor agonist 2-[p-(2-carboxyethyl)phenethylamino]-5’-N-ethylcarbox~ide adenosine (CGS 2168OC), by the A,-adenosine receptor agonist R-N 6-phenylisopropyl adenosine (R-PIA), by the AZ-antagonist N-(2-(dimethylamino)ethyl)-N-methyl-4-(2,3,6,7- tetrahydro-2,6-dioxo-l,3dipropyl-lH-purine-8-yl)-benzene sulphonamide (PD 115,199), and by the A,-antagonist 1,3-dipropyl-8-cyclopentylxan- thine (DPCPX) were studied on the rat phrenic-hemidiaphragm preparation. It is concluded that the excitatory effect of FSK on evoked [‘I-II-ACh release depends on tonic A,-adenosine receptor activation. Phrenic motor nerve terminals possess both, A,-inhib- itory and AZ-excitatory, xanthine sensitive adenosine re- ceptors modulating evoked [3H]-acetylcholine ([3H]- ACh) release [3]. The effect of the endogenous agonist, adenosine, is inhibitory [7], and inhibition of transmis- sion occurs when the inactivation pathways of adenosine are blocked [12], which indicates that endogenous ex- tracellular adenosine tonically inhibits transmission at the phrenic diaphragm. It thus appears that the inhib- itory adenosine receptors have a preponderant role in the modulation of neuromuscular transmission. The physio- logical meaning of the excitatory AZ-receptors, which ap- pear to be positively coupled to adenylate cyclase/cyclic AMP [4] (for a review see ref. 5), is unknown. In the present work we studied the potential physiological role of the A,-adenosine receptors, by investigating if the ex- citatory effect of an activator of adenylate cyclase, fors- kolin (FSK) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA [l 11, on [3H]-ACh release was dependent on A,-adenosine receptor activation. The experiments were carried out on rat phrenic nerve-hemidiaphragm preparations (8 mm width) from Wistar rats of either sexes of about 200 g in weight. The procedures used for labelling the preparations and meas- uring evoked [3H]-ACh release were previously described [3] with minor modifications. Briefly, the preparations were superfused (3 ml/min’) in 3 ml organ baths at 37°C Correspondence: J.A. Ribeiro, Laboratory of Pharmacology, Gulben- kian Institute of Science, 2781 Geiras, Portugal. Fax: (351) 14431631. with Tyrode solution continuously gassed with 95% O2 and 5% CO*, containing (mM): NaCl 137, KC1 2.7, CaCl, 1.8, MgCl, 1, NaH,PO, 0.4, NaHCO, 11.9, glu- cose 11.2 and choline 0.001. After a 30 min equilibration period, the perfusion was stopped and the nerve endings were labelled during 40 min with 1 ,LM [3H]-choline (spe- cific activity 2.5 @i/nmol) under electrical stimulation at 1 Hz with supramaximal rectangular pulses of 40 ,DS du- ration. After the end of the labelling period, the prepara- tions were again superfused (15 ml/min) and the nerve stimulation stopped. From this time onwards hemi- cholinium-3 (10 PM) was present to prevent uptake of choline. After a 60 min period of washout, the perfusion was stopped, and 3 ml bath samples were collected every 3 min by emptying and refilling again the organ bath with the solution in use. Tritium content of the samples was measured by liquid scintillation spectrometry. [3H]- ACh release was evoked by nerve stimulation at 5 Hz during 3 min with supramaximal rectangular pulses of 40 ,us duration. Two stimulation periods (S, and S,) sepa- rated by an interval of 24 min were used. Electrical stim- ulation of the phrenic nerve increases only the release of [3H]-ACh, while the output of [3H]-choline remains un- changed [14]. Therefore, evoked [3H]-ACh release was calculated by subtracting the basal tritium outflow from the total tritium outflow during the stimulation period (cf. ref. 3). Test drugs were added 15 min before S, and were present up to the end of the experiments. Their ef- fects were expressed by the ratios S,/S,, i.e., the ratio