Lubomir Kasakov

The use of the slowly degradable analog, α,β-methylene ATP, to produce desensitisation of the P2-purinoceptor: Effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder

alpha, beta-Methylene ATP has advantages over ATP in producing desensitisation of the P2-purinoceptor since it is degraded more slowly than ATP and does not initiate synthesis of prostaglandins. Following desensitisation of the excitatory P2-purinoceptors in the guinea-pig urinary bladder, the excitatory responses to non-adrenergic, non-cholinergic nerve stimulation were abolished, while those to acetylcholine and histamine were little affected. This result is consistent with the purinergic nerve hypothesis.

Purinergic innervation of the guinea-pig urinary bladder.

1 A number of criteria for considering adenosine 5′‐triphosphate (ATP) as a neurotransmitter in the guinea‐pig urinary bladder have been examined. In addition, the effect of tachyphylaxis to ATP on the response to non‐adrenergic, non‐cholinergic nerve stimulation has been re‐examined. 2 Quinacrine fluorescence histochemistry revealed a population of nerve fibres, ganglion cells, and nerve bundles in the bladder which were not seen in either the iris or vas deferens, where adrenergic and cholinergic nerves predominate. The distribution and morphology of the quinacrine‐positive nerves in the bladder were different from those observed with catecholamine fluorescence and cholinesterase histochemistry, and were unaffected by chemical sympathectomy. 3 Release of ATP from the bladder during stimulation of intramural excitatory nerves, in the presence of atropine and guanethidine increased to 3–12 times prestimulation levels. Tetrodotoxin abolished both the contractile response and the increase in ATP release resulting from intramural nerve stimulation. There was no increase in ATP release during contraction resulting from direct muscle stimulation following nerve paralysis with tetrodotoxin. 4 Sympathectomy with 6‐hydroxydopamine did not affect release of ATP in response to intramural nerve stimulation. 5 Release of ATP was dependent on the concentration of calcium ion in the medium. 6 Contractions in response to non‐adrenergic, non‐cholinergic intramural nerve stimulation were closely mimicked by ATP, but not by acetylcholine or histamine. 7 Adenosine and dipyridamole reduced the contractions to both ATP and non‐cholinergic nerve stimulation. 8 2–2′‐Pyridylisatogen was not a specific blocker of either ATP or intramural nerve stimulation in the guinea‐pig bladder. 2‐Substituted imidazolines initiated spontaneous activity making it impossible to assess any blocking action that they may have had. 9 Prostaglandins (E1 E2 and F2a) gave weak, slow contractions and an increase in spontaneous activity. Both the response to ATP and non‐adrenergic, non‐cholinergic nerve stimulation were greatly potentiated in the presence of prostaglandins. 10 In the presence of indomethacin the response to non‐adrenergic, non‐cholinergic nerve stimulation was virtually abolished following desensitization to ATP.

Direct evidence for ATP release from non-adrenergic, non-cholinergic ("purinergic") nerves in the guinea-pig taenia coli and bladder.

Demonstration of release of ATP from smooth muscle preparations during stimulation of purinergic nerves is complicated by the difficulty in showing whether it comes from nerve or muscle. ATP released during relaxation of the guinea-pig taenia coli and contraction of bladder strips in response to purinergic nerve stimulation was measured in the superfusate using the luciferin-luciferase ATP assay method. The amount of ATP increased 2-6 fold during isometric responses to purinergic nerve stimulation. This release was blocked by tetrodotoxin but not by adrenergic nerve destruction with 6-hydroxydopamine. No significant release of ATP was detected during comparable responses elicited by direct muscle stimulation. These results provide further support for the purinergic nerve hypothesis.

Inhibition of nitrergic relaxations by a selective inhibitor of the soluble guanylate cyclase

1 . The actions of 1H‐[1,2,4]oxadiazolo[4,3,‐a]quinoxalin‐1‐one (ODQ), a specific inhibitor of the soluble guanylate cyclase (SGC), were investigated in the rabbit anococcygeus muscle. 2 . ODQ (1 nM‐1 μm) inhibited in a concentration‐dependent manner the relaxations induced by electrical field stimulation (EFS; 50 V, 0.3 ms duration, 1 Hz, for 5 s, every 120 s). 3 . ODQ (1 μm) also inhibited the relaxations elicited by EFS (50 V, 0.3 ms duration, 1, 2.5, 5, 10 Hz, for 5 s) and sodium nitroprusside (SNP; 1 μm) without affecting those induced by isoprenaline (1 μm), atrial natriuretic peptide (ANP; 100 nM) or an analogue of cyclic GMP (8‐pCPT‐cyclic GMP; 500 μm). 4 . ODQ (1 μm) inhibited the elevations in the concentration of cyclic GMP induced by SNP or EFS, but not by ANP. ODQ did not affect the concentrations of cyclic AMP. 5 . Nitrergic relaxation in this tissue appears, therefore, to be mediated via activation of SGC.

Characterization of nitrergic neurotransmission during short‐and long‐term electrical stimulation of the rabbit anococcygeus muscle

1 Isolated preparations of rabbit anococcygeus muscle were exposed to electrical field stimulation (EFS; 50V, 0.3 ms duration, 0.08–40 Hz) for periods of 1–60 s (short‐term EFS) or 10 min‐2 h (long‐term EFS). 2 Both short‐ and long‐term EFS caused a contractile response which was enhanced by the nitric oxide (NO) synthase inhibitor, NG‐nitro‐L‐arginine (L‐NOARG), showing that it is modulated by endogenous NO. 3 In preparations treated with scopolamine and guanethidine and in which a constrictor tone was induced by histamine, both short‐ and long‐term EFS resulted in relaxation of the tissue. 4 Such relaxations were reversed by tetrodotoxin (TTX), ω‐conotoxin, inhibitors of NO synthase and the NO scavenger, oxyhaemoglobin, indicating that they are neuronal in origin and nitrergic in nature. 5 The relaxations to long‐term EFS persisted for the duration of the stimulation and were associated with sustained release of oxidation products of NO (NOx). The EFS‐induced release of NOx was decreased by N‐iminoethyl‐L‐ornithine (L‐NIO), an inhibitor of NO synthase, and by TTX. 6 Inhibitors of NO synthase, in addition, increased the basal tone of the tissue and reduced the basal output of NOx. The basal output of NOx was also reduced by TTX. 7 Long‐term EFS which induces ∼ 50% of the maximum relaxation could be enhanced by addition of L‐, but not D‐, arginine to the perfusion medium. 8 These data show that there is a continuous basal release of NO from nitrergic nerve terminals which maintains a relaxant tone in the rabbit anococcygeus muscle. 9 In addition, NO is released during short‐ and long‐term EFS which further relaxes the preparation and modulates sympathetic transmission. Activation of the L‐arginine: NO pathway for periods up to 2 h does not exhaust nitrergic transmission in any appreciable way.

Opioid Effects on 45Ca2+ Uptake and Glutamate Release in Rat Cerebral Cortex in Primary Culture

Abstract: Primary cultures of rat cortex, conveniently prepared from newborn animals, were used to study opioid effects on 45Ca2+ uptake and glutamate release. 45Ca2+ uptake, induced by treatment with glutamate or NMDA, was largely blocked by the NMDA antagonist MK‐801. K+ depolarization‐induced 45Ca2+ uptake was also reduced by MK‐801, indicating that the effect was mediated by glutamate release. Direct analysis verified that glutamate, and aspartate, were indeed released. Opioid peptides of the prodynorphin system were also released and these, or other peptides, were functionally active, because naloxone treatment increased glutamate release, as well as the 45Ca2+ uptake induced by depolarization. Opioid agonists, selective for μ‐, κ‐, and δ‐receptors, inhibited the 45Ca2+ uptake induced by K+ depolarization. The combination of low concentrations of MK‐801 and opioid agonists resulted in additive inhibition of K+‐ induced 45Ca2+ uptake. The results indicate that this system may be useful as an in vitro CNS model for studying modulation by opioids of glutamate release and Ca2+ uptake under acute, and perhaps also chronic, opiate treatment.

Noradrenergic-nitrergic interactions in the rat anococcygeus muscle: evidence for postjunctional modulation by nitric oxide.

1 The distribution of NADPH‐diaphorase positive and catecholamine‐containing nerve structures, and functional noradrenergic‐nitrergic interactions, were studied in the rat anococcygeus muscle. 2 The morphological findings demonstrated NADPH‐diaphorase positive neurones mostly as aggregates in intramural ganglia, nerve tracts and few single nerve fibres forming plexus‐like structures. 3 The nitric oxide synthase inhibitor NG‐nitro‐l‐arginine (l‐NOARG) inhibited concentration‐dependently the nitrergic relaxation, an effect reversed by l‐arginine. The drug had dual effects on noradrenergic contractile responses: at lower concentrations (0.1–10 μm) it decreased the amplitude of contractions and this was not affected by l‐arginine; higher concentrations (50–500 μm) potentiated the contractions, an effect that was prevented by l‐arginine. 4 The electron acceptor, nitro blue tetrazolium (NBT) produced a rapid inhibition of the noradrenergic contractile responses (EC50 0.178 ± 0.041 μm). The drug decreased the tone of the preparations. However, it potentiated concentration‐dependently the nitrergic relaxations. 5 NBT (1 μm) had no significant effect on the relaxations induced by exogenously applied nitric oxide (NO)‐donor sodium nitroprusside (SNP, 0.01–50 μm). However, the effect of NBT (0.1–10 μm) on the electrically induced relaxation was significantly decreased by l‐NOARG (10 and 50 μm). The inhibition was of a non‐competitive type. 6 Neither l‐NOARG (100 μm) nor NBT (1 μm) had any effect on the spontaneous or electrically‐induced release of 3H‐radioactivity from the tissues preincubated in [3H]‐noradrenaline. 7 It is concluded that l‐arginine‐NO pathway can modulate noradrenergic transmission in the rat anococcygeus muscle at postjunctional, but not prejunctional site(s).

A structure-activity study of nociceptin-(1-13)-peptide amide. Synthesis of analogues substituted in positions 0, 1, 3, 4 and 10.

A series of analogues of nociceptin, Noc(1-13)NH(2) (an agonist at the ORL1 receptor) was synthesized with following modifications: (1) N-terminal extension with Arg(0); (2) replacement of Gly(3) by basic or polar amino acids-Arg, Asn, Lys(For) or deletion; (3) exchange of Phe(1) or Phe(4) by Phe(NO(2)); (4) substitution of Ser(10) with D-Ser, Pro, D-Pro. The analogs were synthesized by solid-phase methodology using Fmoc-amino acid pentafluorophenyl esters. The affinity for the ORL1 and for the kappa, micro and delta-opioid receptors was investigated by radioligand binding assay and bioactivity by a mouse vas deferens (MVD) assay. The addition of the amino acid residue Arg to the N-terminal enhances the opioid receptor affinity of Noc(1-13)NH(2) while retaining ORL1 receptor affinity at a moderate level. The replacement of Gly in position 3 by the basic or polar amino acids-Arg, Asn, Lys(For) or its deletion led to inactive analogues. The replacement of Ser in position 10 by its D-isomer, Pro and D-Pro resulted in a series of analogues with the following order of activity: Ser(10)>D-Ser(10)>Pro(10)>D-Pro(10). In [D-Ser(10)]Noc(1-13)NH(2), introduction of an additional Phe(NO(2))(4) led to a >60-fold increase of ORL1 affinity, completely attenuating the loss of affinity brought about by Ser(10). In other analogues, introduction of Phe(NO(2))(4) did not change the magnitude of ORL1 binding significantly. Generally, while modifications in position 3 frequently led to a loss of most or all bioactivity, modifications in position 0 (Arg(0)) or 4 (Phe(NO(2))(4)) and 10 (D-Ser(10), Pro(10)) are tolerated.