Interaction of the synthetic immunomodulatory dipeptide bestim with murine macrophages and thymocytes

The tritium-labeled dipeptide bestim (gamma-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by high-temperature solid-state catalytic isotope exchange. It was found that [3H]bestim binds with a high affinity to murine peritoneal macrophages (Kd 2.1 +/- 0.1 nM) and thymocytes (Kd 3.1 +/- 0.2 nM), as well as with plasma membranes isolated from these cells (Kd 18.6 +/- 0.2 and 16.7 +/- 0.3 nM, respectively). The specific binding of [3H]bestim to macrophages and thymocytes was inhibited by the unlabeled dipeptide thymogen (L-Glu-L-Trp) (Ki 0.9 +/- 0.1 and 1.1 +/- 0.1 nM, respectively). After treatment with trypsin, macrophages and thymocytes lost the ability to bind [3H]bestim. Bestim in the concentration range of 10(-10) to 10(-6) M reduced the adenylate cyclase activity in the membranes of murine macrophages and thymocytes.     

Interaction of ACTH synthetic fragments with rat adrenal cortex membranes.

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.     

Synthetic peptide KKRR corresponding to the human ACTH fragment 15–18 is an antagonist of the ACTH receptor

Tritium-labeled synthetic fragments of human adrenocorticotropic hormone (ACTH) [³H]ACTH (11–24) and [³H]ACTH (15–18) with a specific activity of 22 and 26 Ci/mmol, respectively, were obtained. It was found that [³H]ACTH-(11–24) binds to membranes of the rat adrenal cortex with high affinity and high specificity (K _d 1.8 ± 0.1 nM). Twenty nine fragments of ACTH (11–24) were synthesized, and their ability to inhibit the specific binding of [³H]ACTH (11–24) to adrenocortical membranes was investigated. The shortest active peptide was found to be an ACTH fragment (15–18) (KKRR) (K _i 2.3 ± 0.2 nM), whose [³H] labeled derivative binds to rat adrenocortical membranes (K _d 2.1 ± 0.1 nM) with a high affinity. The specific binding of [³H]ACTH-(15–18) was inhibited by 100% by unlabeled ACTH (11–24) (K _i 2.0 ± 0.1 nM). ACTH (15–18) in the concentration range of 1–1000 nM did not affect the adenylate cyclase activity of adrenocortical membranes and, therefore, is an antagonist of the ACTH receptor.     

Buprenorphine‐elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ‐, μ‐opioid and nociceptin receptors

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ‐ (KOP), μ‐opioid (MOP) and nociceptin/opioid receptor‐like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium‐labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N‐linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U‐69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration‐dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP‐expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration‐dependent AC superactivation elicited by chronic buprenorphine exposure.     

Interaction between thymocytes and thymus-derived macrophages. II. Engulfment of thymocytes by macrophages

A high percentage (80-90%) of immature thymocytes were engulfed by syngeneic thymus-derived macrophages (TDM phi) following cocultivation for 3 days. Elimination occurred via internalization of thymocytes by the macrophages. We unequivocally demonstrated the presence of many live thymocytes inside the TDM phi by means of specific staining. Mature PNA- thymocytes were phagocytized to a lower degree than immature thymocytes, and T splenocytes were not eliminated at all. Bone marrow-derived macrophages internalized immature thymocytes to a degree similar to TDM phi. Since thymocyte survival was not at all affected by M phi culture supernatants alone, we conclude that cell to cell contact is necessary for thymocyte elimination. To identify the surface molecules which participate in internalization of thymocytes by the macrophages, both cell types were pretreated with a variety of agents. Treatment of thymocytes with tunicamycin (N-glycosylation inhibitor) and anti-Lyt-2 mAb decreased their elimination by M phi. Similarly, treatment of M phi with neuraminidase, trypsin, and anti-Ia mAb markedly suppressed their capacity to engulf thymocytes. On the other hand, thymocyte elimination was unaffected by (1) cell cultivation in syngeneic serum rather than heterologous serum; (2) use of allogeneic rather than syngeneic thymocytes; and (3) use of X-irradiated M phi and LPS-activated M phi rather than nontreated M phi.     

Stress-protective effect of the KKRR synthetic peptide corresponding to the 15–18 sequence of human adrenocorticotropic hormone

The activity of the KKRR synthetic peptide corresponding to the 15-18 sequence of human adrenocorticotropic hormone (ACTH) and its analogues: KKKK, RRRR, RRKK, kKRR, KkRR, KKrR, and KKRr (amino acid residues of the D configuration are designated by small letters), was studied in vivo on rats under cold and heat shock. Intranasal administration of the KKRR peptide at doses of 2–10 μg/animal 1 day before the shock was found to prevent a dramatic increase in the level of corticosterone in rat adrenal glands and blood plasma caused by the temperature effect. Amino acid substitutions in the KKRR peptide were shown to result in abrupt decrease in its activity. The peptide analogues exhibit a low stress-protective activity and had a low affinity for the ACTH receptor.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.     

Long-term treatment with fluoxetine induces desensitization of 5-HT4 receptor-dependent signalling and functionality in rat brain

The mode of action of antidepressant drugs may be related to mechanisms of monoamines receptor adaptation, including serotonin 5-HT₄ receptor subtypes. Here we investigated the effects of repeated treatment with the selective serotonin reuptake inhibitor fluoxetine for 21 days (5 and 10 mg/kg, p.o., once daily) on the sensitivity of 5-HT₄ receptors by using receptor autoradiography, adenylate cyclase assays and extracellular recording techniques in rat brain. Fluoxetine treatment decreased the density of 5-HT₄ receptor binding in the CA1 field of hippocampus as well as in several areas of the striatum over the doses of 5–10 mg/kg. In a similar way, we found a significant lower response to zacopride-stimulated adenylate cyclase activity in the fluoxetine 10 mg/kg/day treated group. Furthermore, post-synaptic 5-HT₄ receptor activity in hippocampus-measured as the excitatory action of zacopride in the pyramidal cells of CA1 evoked by Schaffer collateral stimulation was attenuated in rats treated with both doses of fluoxetine. Taken together, these results support the concept that a net decrease in the signalization pathway of 5-HT₄ receptors occurs after chronic selective serotonin reuptake inhibitor treatment: this effect may underlie the therapeutic efficacy of these drugs.     

Partial agonist actions of aripiprazole and the candidate antipsychotics S33592, bifeprunox, N-desmethylclozapine and preclamol at dopamine D(2L) receptors are modified by co-transfection of D(3) receptors: potential role of heterodimer formation

Aripiprazole and the candidate antipsychotics, S33592, bifeprunox, N-desmethylclozapine (NDMC) and preclamol, are partial agonists at D(2) receptors. Herein, we examined their actions at D(2L) and D(3) receptors expressed separately or together in COS-7 cells. In D(2L) receptor-expressing cells co-transfected with (D(3) receptor-insensitive) chimeric adenylate cyclase-V/VI, drugs reduced forskolin-stimulated cAMP production by approximately 20% versus quinpirole (48%). Further, quinpirole-induced inhibition was blunted by aripiprazole and S33592, confirming partial agonist properties. In cells co-transfected with equal amounts of D(2L)and D(3) receptors (1 : 1), efficacies of aripiprazole and S33592 were attenuated. Further, in cells co-transfected with D(2L) and an excess of D(3) receptors (1 : 3), aripiprazole and S33592 were completely inactive, and they abolished the actions of quinpirole. Likewise, bifeprunox, NDMC and preclamol lost agonist properties in cells co-transfected with D(2L)and D(3) receptors. Accordingly, at split D(2trunk)/D(3tail) and D(3trunk)/D(2tail) chimeras, agonist actions of quinpirole were blocked by aripiprazole and S33592 that, like bifeprunox, NDMC and preclamol, were inactive alone. Conversely, when a 12 amino acid sequence in the third intracellular loop of D(3) receptors was replaced by the homologous sequence of D(2L) receptors, aripiprazole, S33592, bifeprunox, NDMC and preclamol inhibited cAMP formation by approximately 20% versus quinpirole (42%). Moreover, at D(2L) receptor-expressing cells co-transfected with modified D(3i3(D2)) receptors, drugs behaved as partial agonists. To summarize, low efficacy agonist actions of aripiprazole, S33592, bifeprunox, NDMC and preclamol at D(2L) receptors are abrogated upon co-expression of D(3) receptors, probably due to physical association and weakened coupling efficacy. These findings have implications for the functional profiles of antipsychotics.     

Interferon-γ down-regulates membrane adenylate cyclase activity of a murine macrophage-like cell line (P388D1)

Effects of recombinant murine interferon-γ (rIFN-γ) on the membrane adenylate cyclase of a murine macrophage cell line (P388D₁) were investigated in order to explore the nature of a signal transmitted by IFN-γ receptor. Following the incubation of P388D₁ cells with 40 U/ml of rIFN-γ, the intracellular level of cAMP gradually increased about twofold over the control level within 60 min, and then began to gradually decline to about half the control level by 24 h incubation. The initial rise in cAMP level appeared to be due to the modest activation of adenylate cyclase and not due to the inhibition of cAMP-phosphodiesterase. Later decrease of intracellular cAMP may be due to quantitative down-regulation of the adenylate cyclase system. The basal enzymatic activity of the membrane prepared from P388D₁ cells exposed to IFN-γ for 24 h was found to be reduced to about 20% of that of the control membrane. However, the quality of the adenylate cyclase system appeared unchanged, because the relative degree of the response of the down-regulated membrane adenylate cyclase to prostaglandin PGE₂, NaF, guanylimidodiphosphate (GppNHp), cholera toxin (CT), or forskolin was found to remain unchanged. This quantitative down-regulation of adenylate cyclase must be due to the action of rIFN-γ, since the prior treatment of rIFN-γ with either acid (pH 2) or monoclonal anti-IFN-γ antibody inhibited the ability of IFN-γ to induce the down-regulation. The rIFN-γ-induced down-regulation is a reversible process, since the adenylate cyclase activity of the membrane was found to be restored when the rIFN-γ-exposed cells were cultured for 72 h in the absence of rIFN-γ. In addition, the 48 h-incubation of P388D₁ cells with rIFN-β or IFN-α was found not to significantly affect the membrane adenylate cyclase system.     

Signal transduction pathways of muscarinic receptors in circular smooth muscle from the rabbit caecum

The effects of muscarinic acetylcholine receptor stimulation on phosphoinositides breakdown and adenylate cyclase activity were examined in the circular smooth muscle of the rabbit caecum. InMyo-[³H]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [³H]inositol phosphates ([³H]IPs) accumulation (EC₅₀ of 3±1 M). The M₁-selective antagonist pirenzepine (PRZ), the M₂-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one) and the M₃-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [³]inositol phosphates accumulation with the following order of potency: p-F-HHSiD>PRZ>AF-DX 116. In saponin-permeabilized circular smooth muscle cells, carbachol and GTP[S] elicited a concentration-dependent increase in [³H]inositol phosphates accumulation. The concentration-response curve for GTP[S] was shifted to the left when cells were incubated with 1 M carbachol. The [³H]inositol phosphates accumulation elicited by simultaneous addition of 0.1 M GTP[S] and 1 M carbachol to permeabilized cells was significantly decreased (78.28±18.23% inhibition) when cells were preincubated for 5 min with 0.1 mM GDP[S]. In nonpermeabilized cells, pertussis toxin did not alter the carbachol-induced increase in [³H]inositol phosphates accumulation. On the other hand, the 0.1 mM carbachol-induced inhibition of forskolin-stimulated adenylate cyclase activity in circular smooth muscle homogenates was significantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 M final concentration). Moreover, the carbachol-induced inhibition of the cyclic AMP accumulation elicited by 10 M isoproterenol was abolished by pertussis toxin pretreatment of isolated circular smooth muscle cells. In conclusion, our data suggest that in circular smooth muscle of rabbit caecum, the muscarinic receptor stimulation of [³H]inositol phsophates accumulation is mediated by M₃ subtype receptors coupled to a pertussis toxin-insensitive G protein, whereas inhibition of adenylate cyclase activity is mediated by M₂ subtype receptors coupled to a pertussis toxin-sensitive GTP-binding protein G_i.     

The Interaction of Synthetic Decapeptide SLTCLVKGFY with Human T Lymphocytes

The synthetic peptide SLTCLVKGFY, corresponding to the 364–373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [¹²⁵I] -endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (K _i0.6 nM). The fragments 3–10, 4–10, 5–10, and 6–10 of Immunorphin also inhibited the binding (K _i2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and [Met]enkephaline and, therefore, are not opioid. The K _dvalues of the specific binding of ¹²⁵I-labeled Immunorphin and its 6–10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.     

Down-regulation of gonadotropin and beta-adrenergic receptors by hormones and cyclic AMP

Loss of gonadotropin receptors in murine Leydig tumor cells and of beta-adrenergic receptors in rat glioma C6 cells occurred following exposure of the cells to human chorionic gonadotropin and isoproterenol, respectively. Down-regulation of receptors was mimicked in part by other agents that elevated cyclic AMP levels in the cells such as cholera toxin and dibutyryl cyclic AMP. Whereas agonist-mediated receptor loss was rapid and almost total, down-regulation by cyclic AMP was slower and less extensive. Down-regulation of receptors did not appear to be accompanied by loss of the regulatory and catalytic components of adenylate cyclase. Hormone-mediated down-regulation was preceded by desensitization of hormone-stimulated adenylate cyclase. In contrast, there was no evidence that cyclic AMP caused desensitization. Finally, loss of receptors induced either by agonists or cyclic AMP required protein synthesis as cycloheximide inhibited down-regulation. We conclude that down-regulation of receptors in these cells is a complex process involving both cyclic AMP-independent and -dependent events.     

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [³H]bestim was found to bind with high affinity to mouse peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [³H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [³H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.     

Cerebellar astrocytes co-express several ADP receptors. Presence of functional P2Y<Subscript>13</Subscript>-like receptors

Astrocytes exhibit a form of excitability based on variations of intracellular Ca²⁺ concentration in response to various stimuli, including ADP, ATP, UTP and dinucleotides. Here, we investigate the presence of the recently cloned ADP-sensitive receptors, P2Y₁₂ and P2Y₁₃ subtypes, which are negatively coupled to adenylate cyclase, in cerebellar astrocytes. We checked the effect of specific agonists, 2-methylthioadenosine diphosphate (2MeSADP) and ADP, on adenylate cyclase stimulation induced by isoproterenol. Both agonists significantly reduced the cAMP accumulation induced by isoproterenol. The inhibitory effect was concentration-dependent with IC₅₀ values of 46 ± 13 and 23 ± 14 nM for 2MeSADP and ADP, respectively. The experiments were carried out in the presence of MRS-2179, a specific antagonist of P2Y₁ receptor, to avoid any contribution of this receptor. Using fura-2 microfluorimetry we also proved that astrocytes responded to 2MeSADP stimulations with calcium responses in the absence and also in the presence of MRS-2179. Both effects, inhibition of adenylate cyclase and intracellular calcium mobilization, were not modified by 2MeSAMP, an antagonist of P2Y₁₂ receptor, suggesting that were mediated by P2Y₁₃-like receptors.¹These authors equally contributed to this work.     

Stress-protective effect of the synthetic ACTH-like peptide leucocorticotropin

We found that the tritium-labeled synthetic ACTH-like octapeptide leucocorticotropin corresponding to the 81–88 sequence of the precursor of human interleukin-1α ([³H]GK VLKKRR) is bound by the ACTH receptor of rat adrenal cortex with a high affinity and specificity (K _d 2.2 ± 0.1 nM). This peptide was shown to exert no effect on the adenylate cyclase activity of the membranes of rat adrenal cortex in the concentration range from 1 to 1000 nM. Leucocorticotropin administration three times at doses of 10–20 μg/animal did not change the level of hydroxycorticosteroids (11-HOCS) in the rat adrenal glands in the absence of temperature action. At the same time, the peptide abolishes (at a dose of 20 μg/animal, three times) or significantly decreases (at a dose of 10 μg/animal, three times) the dramatic increase in the 11-HOCS content in the adrenal glands occurring in the case of cold or heat shock. Thus, leucocorticotropin normalizes the 11-HOCS level in the rat adrenal cortex during stress. The stress-protective effect of the peptide is mediated through the ACTH receptor.     

Expression of adenylate cyclase and phosphodiesterase in development of temperature-sensitive mutants with impaired metabolism of cAMP in drosophila melanogaster

The mode of the developmental expression of adenylate cyclase (AC) and phosphodiesterase (PDE) in D melanogaster indicates that PDE plays the major role in the maintenance of a certain level of cAMP in postembryonic development, while both enzymes function in concert in imago. The ts-mutants ts155 and ts622, characterized upon their isolation as having an increased cAMP content and normal PDE activity, manifest high levels of AC activity from the third day of imago life. The levels of PDE activity characteristic for adult mutants with altered enzyme activity (low in ts66 and ts980, high in ts398) are manifested in ts980 from larval instar II, and from the larval instar III in ts398 and ts66. Data on the dependence of PDE activity in adults upon temperature of incubation, being in agreement with the expectations for a ts-mutation in a gene coding for a form of PDE in case of ts66, suggest that ts398 affects not the enzyme-coding gene but rather one for an activator protein. The fact that in ts398 (the polyphasic ts-lethal mapping to 1-38.9) 1) AC activity is somewhat higher than normal at 22°C and is readily activated at 29°C, 2) activity of PDE-I assayed in heat-pretreated homogenates is higher than normal, 3) that boiled extracts of ts398 are potent activators of the wild type and of its own PDE-I indicates that it is a mutation affecting calmodulin, which is known to be stable at boiling and capable of activating both AC and PDE-I. Data on Ca²⁺ and EGTA effects suggest that the mutation presumably increases Ca²⁺-binding activity of calmodulin, ts980 and ts622, in which ts-lethality could be produced only by certain doses of haloperidol and triftazine, appear to be lethal in compounds with ts398, thus indicating that these mutations could affect the same calmodulin-controlling gene.     

Pharmacology of octopamine-, dopamine-, and 5-hydroxytryptamine-stimulated cyclic amp accumulation in the corpus cardiacum of the american cockroach,Periplaneta americana L

A variety of potential receptor agonists were incubated with isolated corpora cardiaca from the American cockroach Periplaneta americana to determine their effects on cyclic AMP production in this gland. Octopamine, dopamine, and 5-hydroxytryptamine elevated cyclic AMP levels in a dose-dependent manner with estimated K_a values of 15.8, 1.7, and 1.1 m?M, respectively, and their stimulation was found to be additive. Several vertebrate receptor antagonists were tested against the three amines and a preliminary pharmacological profile developed.     

The immunomodulatory effects of tachykinins and their receptors

Tachykinins (TKs) are a family of neuropeptides mainly expressed by neuronal and non-neuronal cell types, especially immune cells. Expression of TKs receptors on immune cell surfaces, their involvement in immune-related disorders, and therefore, understanding their immunomodulatory roles have become of particular interest to researchers. In fact, the precise understanding of TKs intervention in the immune system would help to design novel therapeutic approaches for patients suffering from immune disorders. The present review summarizes studies on TKs function as modulators of the immune system by reviewing their roles in generation, activation, development, and migration of immune cells. Also, it discusses TKs involvement in three main cellular mechanisms including inflammation, apoptosis, and proliferation.     

Role of cGMP- and cAMP-Dependent Systems in the Effects of Nitric Oxide on Transmitter Release and Potassium Currents in the Frog Neuromuscular Junction

We studied the molecular mechanisms responsible for nitric oxide (NO)-evoked modulation of the synaptic function in the frog neuromuscular junction using inhibitors of adenylate and guanylate cyclases and analogs of cyclic nucleotides. It was shown that application of an exogenous donor of NO, sodium nitroprusside, decreased transmitter release and increased the amplitude of voltage-dependent potassium current of the nerve endings. Our results indicate that NO regulates transmitter release and potassium current in the frog neuromuscular junction both via cAMP- and cGMP-dependent mechanisms.