Cyclic adenosine monophosphate (cAMP)-dependent protein kinase, labeled with fluorescein and rhodamine on the catalytic and regulatory subunits, respectively, was injected into Aplysia sensory neurons either in culture or in intact cell clusters. Energy transfer between the subunits, a measure of cytosolic cAMP concentration ([cAMP]), and compartmentation of the dissociated subunits were monitored by confocal fluorescence microscopy. Bath application of serotonin produced a much greater elevation of [cAMP] in the processes than in the central bodies of the neurons. The resulting gradients must drive a sizable centripetal flux of cAMP because direct microinjection of cAMP showed that it diffused readily. Perinuclear increases in [cAMP] slowly caused the translocation of the freed catalytic subunit into the nucleus to an extent proportional to the percentage of its dissociation from the regulatory subunit.

We have investigated the purinoceptor subtypes responsible for calcium signaling in human platelets, which previous studies have shown to involve both Ca 2؉ influx via receptor-operated cation channels and release of Ca 2؉ from intracellular stores. Fura-2 measurements of [Ca 2؉ ] i in stirred platelet suspensions showed that both ADP (40 M) and the non-hydrolyzable ATP analogue ␣␤-meATP (␣,␤-methyleneadenosine 5-triphosphate, 10 M) activated a rapid Ca 2؉ influx whereas only ADP mobilized Ca 2؉ from internal stores. In "nystatin" whole-cell patch clamp recordings, ATP, ADP, and the non-hydrolyzable ATP analogues, ␣,␤-meATP and ATP␥S (adenosine 5-O-(3-thiotriphosphate), all activated a cation channel permeable to both monovalent and divalent cations with a single-channel conductance of 11 picosiemens in NaCl saline. The current response to ATP (40 M) was activated within 20 ms and desensitized with a time constant of 47-107 ms in the continued presence of agonist, which are characteristics of P 2X1 receptors in other tissues. We conclude that human platelets possess a P 2X1 purinoceptor, which mediates a rapid phase of ADP-or ATP-evoked Ca 2؉ entry via a cation channel, whereas one or more separate ADP-selective P 2 purinoceptors evoke release of calcium from intracellular stores.In many cell types, extracellular ATP and ADP interact with a family of P 2 purinoceptors (1). Two subgroups have been recently cloned: ionotrophic (P 2X1 , P 2X2 , P 2X3 , P 2X4 ) and Gprotein-coupled (P 2Y , P 2U ) purinoceptors (2-8). Data suggest putative P 2D and P 2Z purinoceptors (9, 10) also exist. Human platelets are reported to possess a unique ADP-selective purinoceptor, termed P 2T , which mediates shape change and aggregation (11). Major actions of ADP in platelets include mobilization of intracellular calcium stores (13) and activation of a non-selective cation channel (12). In cell-attached patch clamp recordings (14), ADP evoked single channel activity if included in the pipette but not when added to the bath saline, demonstrating that this channel is activated by a direct receptoroperated or G-protein-linked mechanism rather than via a diffusible second messenger. Whether the actions of ADP in Ca 2ϩ signal generation in human platelets are mediated by one or multiple purinoceptors is, however, uncertain. In the present study we have used both whole-cell patch clamp recordings and fura-2 intracellular calcium measurements to investigate the purinoceptor subtypes involved in human platelet calcium signaling. MATERIALS AND METHODSSolutions and Reagents-Unless otherwise stated, standard platelet saline contained (mM) 150 NaCl, 10 Hepes, 1 MgCl 2 , 1 EGTA at pH 7.35 (with NaOH). BaCl 2 saline contained (mM) 110 BaCl 2 , 10 Hepes, 1 MgCl 2 at pH 7.35 (with N-methyl D-glucamine base). The pipettes were filled with a solution containing (mM) 50 KCl, 70 K 2 SO 4 , 10 Hepes, 5 MgCl 2 , 0.1 EGTA, pH 7.2 (KOH). 50 -100 M nystatin was added to the internal pipette saline from a 50 mM stock, made in dimethyl sulfoxide, immediately ...

] o respectively. Endocardial APD 90 s correspondingly increased from 51.6 AE 1.9 ms (n ¼ 7) to 62.8 AE 2.8 ms (n ¼ 7) and 62.9 AE 5.9 ms (n ¼ 11) giving reductions in endocardial-epicardial differences, DAPD 90 , from 14.4 AE 2.6 to 4.4 AE 5.0 and )3.4 AE 6.0 ms respectively. Early afterdepolarizations (EADs) occurred in epicardia in three of seven spontaneously beating hearts at 4 mm [K + ] o with triggered beats followed by episodes of non-sustained VT in nine of 11 preparations at 3 mm. Programmed electrical stimulation never induced arrhythmic events in preparations perfused with normokalemic solutions yet induced VT in two of seven and nine of 11 preparations at 4 and 3 mm