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We have previously identified the suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) as a low nanomolar potency antagonist of recombinant P2X1receptors. Here, we characterize, by two-electrode voltage-clamp electrophysiology, three isomeric suramin analogs designated para-4,4',4'',4''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetrakis-benzenesulfonic acid (NF110), meta-(3,3',3'',3''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF448), and ortho-(2,2',2'',2''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (MK3) with respect to their potency in antagonizing rat P2X receptor-mediated inward currents in Xenopus laevis oocytes. Meta, para, and ortho refer to the position of the single sulfonic acid group relative to the amide bond linking the four symmetrically oriented benzenesulfonic acid moieties to the central, invariant suramin core. NF448, NF110, and MK3 were >200-fold less potent in blocking P2X1receptors than NF449, from which they differ structurally only by having one instead of two sulfonic acid residues per benzene ring. Although the meta- and ortho-isomers retained P2X1receptor selectivity, the para-isomer NF110 exhibited a significantly increased activity at P2X3receptors (Ki∼ 36 nM) and displayed the following unique selectivity profile among suramin derivatives: P2X2+3= P2X3> P2X1> P2X2>> P2X4> P2X7. The usefulness of NF110 as a P2X3receptor antagonist in native tissues could be demonstrated by showing that NF110 blocks αβ-methylene-ATP-induced currents in rat dorsal root ganglia neurons with similar potency as recombinant rat P2X3receptors. Together, these data highlight the importance of both the number and exact location of negatively charged groups for P2X subtype potency and selectivity.

Whole‐cell patch clamp experiments were used to investigate the transduction mechanism of adenosine A2Areceptors in modulating N‐methyl‐D‐aspartate (NMDA)‐induced currents in rat striatal brain slices. The A2Areceptor agonist 2‐p‐(2‐carboxyethyl)phenethylamino‐5′‐N‐ethylcarboxamidoadenosine (CGS 21680) inhibited the NMDA, but not the (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) current in a subset of striatal neurons.Lucifer yellow‐filled pipettes in combination with immunostaining of A2Areceptors were used to identify CGS 21680‐sensitive cells as typical medium spiny striatal neurons.Dibutyryl cyclic AMP and the protein kinase A activator Sp‐cyclic AMPs, but not the protein kinase A inhibitors Rp‐cyclic AMPS or PKI(14–24)amide abolished the inhibitory effect of CGS 21680. The phospholipase C inhibitor U‐73122, but not the inactive structural analogue U‐73343 also interfered with CGS 21680. The activation of protein kinase C by phorbol 12‐myristate 13‐acetate or the blockade of this enzyme by staurosporine did not alter the effect of CGS 21680. Heparin, an antagonist of inositol 1,4,5‐trisphosphate (InsP3) and a more efficient buffering of intracellular Ca2+by BAPTA instead of EGTA in the pipette solution, abolished the CGS 21680‐induced inhibition.The calmodulin antagonist W‐7 and cytochalasin B which enhances actin depolymerization also prevented the effect of CGS 21680; the calmodulin kinase II inhibitors CaM kinase II(281–309) and KN‐93 but not the inactive structural analogue KN‐92 were also effective. The calcineurin inhibitor deltamethrin did not interfere with CGS 21680.It is suggested that the transduction mechanism of A2Areceptors to inhibit NMDA receptor channels is the phospholipase C/InsP3/calmodulin and calmodulin kinase II pathway. The adenylate cyclase/protein kinase A and phospholipase C/protein kinase C pathways do not appear to be involved.