Structure activity relations for inhibition of neurotransmission in rat vas deferens by adenosine

Adenosine inhibits the isometric contractions of the rat vas deferens in response to field stimulation in vitro by presynaptic inhibition of transmitter release. In the present study the structure activity relations for the inhibition of neurotramsmission in the rat vas deferens by adenosine were examined. Adenosine and adenosine-N¹-oxide were the most potent inhibitors studied. 6-Methylaminopurine riboside, 6-hydroxylaminopurine riboside, 5′-deoxyadenosine and 5′-nitroadenosine were slightly less potent inhibitors. The common structural requirements for activity include a primary or secondary amine function at C₆ of the purine ring with little tolerance for major steric changes or substitutions on the sugar moiety. None of the analogues studied prevented the presynaptic inhibitory action of adenosine.     

Co-transmission in the rat vas deferens: postjunctional synergism of noradrenaline and adenosine 5'-triphosphate

In the isolated prostatic half of the rat vas deferens, joint application of noradrenaline (NA) and adenosine 5'-triphosphate (ATP) produced a contractile response whose magnitude was greatly larger than the addition of the tension generated by the application of each agent alone. The effect of ATP was mimicked by two non-hydrolyzable ATP analogs, but not by GTP, AMP or adenosine. In sympathectomized rats, ATP potentiated NA effects, increasing both the peak tension and the duration of the vas deferens contractile response. The synergism was concentration related. Prazosin antagonized the NA synergism but not the ATP response. Likewise, desensitization of the P2-purinoceptor blocked the ATP synergism without modifying the NA-induced contraction.     

Adenosine-induced depression of synaptic transmission in the isolated olfactory cortex: receptor identification

We have investigated the type of purine receptor in the guinea-pig olfactory cortex, using pial surfaces slices maintained in vitro. Adenosine (0.1 to 100 mumol/l) bath applied in the presence of the uptake inhibitor nitrobenzylthioinosine, depressed the evoked potentials in a dose related fashion. Synthetic and uptake resistant adenosine analogues had the same effect as adenosine and the order of potency of these was: 5'-N-ethylcarboxamide adenosine greater than L-N6-phenylisopropyl adenosine (L-PIA) = N6-cyclohexyladenosine = 2-chloroadenosine greater than adenosine greater than D-N6-phenylisopropyladenosine (D-PIA). The D-stereoisomer of PIA was 45 times less potent than L-PIA. The methylxanthine compounds 8-phenyltheophylline (3 mumol/l) and 3-isobutyl-1-methylxanthine (50 mumol/l) antagonised the depression produced by L-PIA. Rolipram, a phosphodiesterase inhibitor, in concentrations up to 100 mumol/l had no effect on the evoked potentials or on adenosine action. Forskolin, a cAMP stimulant, slightly increased the amplitude of the evoked potential, and partly reversed the depressant effect of adenosine. Noradrenaline had no effect either alone or in the presence of adenosine. The results of these experiments indicate the existence of A1 subtype adenosine receptors in the guinea pig olfactory cortex probably linked to a depression of intracellular cAMP.     

Dual effects of adenosine and adenosine analogues on motor activity of the human fallopian tube

Effects of adenosine and adenosine analogues on spontaneous contractility of the human fallopian tube during different phases of the menstrual cycle were studied. In isthmic preparations, a stimulatory effect by L-N6-phenylisopropyladenosine (L-PIA), with preference for adenosine A1-receptors, was seen mainly during the proliferative phase. In ampullary preparations, stimulation by L-PIA was seen both in the secretory and proliferative phases. Adenosine and 2-chloroadenosine exerted similar stimulatory effects. 5'-N-ethylcarboxamide-adenosine (NECA), with selectivity for adenosine A2-receptors, or D-PIA never showed a stimulatory effect. At concentrations above those needed for stimulation, adenosine, 2-chloroadenosine and L-PIA inhibited spontaneous contractions, in common with NECA and D-PIA. Here, NECA was more potent than L-PIA. The D-PIA and L-PIA were equipotent. The inhibition was seen during the whole menstrual cycle. The competitive adenosine antagonist 8-p-sulphophenyltheophylline (PS?T) reversibly antagonized the stimulatory and inhibitory effects elicited by adenosine and the analogues. The PS?T alone could exert a stimulatory or an inhibitory action on spontaneous contractility. We suggest that adenosine can modulate contractile activity in the human fallopian tube via stimulatory A1-and inhibitory A2-receptors. These receptors are located on the smooth muscle cells, and might act via cAMP. The relative receptor dominance may be influenced by cyclic hormonal changes.     

A comparison of the electrical properties and morphological characteristics of the smooth muscle of the rat and guinea-pig vas deferens

Summary Microelectrodes were used to compare a variety of electrophysiological parameters of the rat and guinea-pig vas deferens. In comparison to the guinea pig, spontaneous junction potentials in the rat tissue were of shorter duration and occurred with greater frequency and amplitude. Action potentials induced by nerve stimulation could be observed in the smooth muscle of both species. However, in the rat tissue the majority of action potentials were generated in the impaled cell while 60% of the action potentials in the guinea-pig vas deferens were propagated. When current was intracellularly applied, spike potentials could be induced in approximately 90% of the cells of the rat vas deferens but in less than 10% of the cells of the guinea-pig vas deferens, The space constant was 1.48 mm for the guinea-pig vas deferens, but less than 0.5 mm for the rat vas deferens. Electronmicroscopic examination of the homologous tissues indicates that the differences in electrical properties can be accounted for in part by differences in morphology. The incidence and intimacy of neuromuscular contacts was greater in the rat vas deferens while the incidence of nexuses between smooth muscle cells was greater in the guinea-pig tissue.Supported by grants from the National Institute of Neurological Diseases and Stroke (NS 08300) and the West Virginia University Medical Corporation     

Species differences in histamine receptors in the vas deferens

Histamine, specific H₁-and H₂-receptor agonists in conjunction with specific H₁-and H₂-receptor antagonists and other types of classical antagonists were used to characterize histamine receptors in the vasa deferentia of mice, rats and guinea pigs. The H₁-receptor mediates contraction while the H₂-receptor produces inhibition. There were marked qualitative and quantitative differences in the distribution of the two types of histamine receptors in the vas deferens of different species. Results indicate that mouse and rat vas deferens contain an inhibitory H₂-receptor, but virtually no excitatory H₁-receptor. In contrast, guinea pig vas deferens contained an excitatory H₁-receptor but was essentially devoid of an inhibitory H₂-receptor. The rank order of relative potencies of various agonists as well as the calculated pA ₂ values of cimetidine in the mouse and rat vas deferens suggest that the two species probably have the same H₂-receptor. High concentrations of histamine and 2-methyl histamine have a stimulant action in the mouse and rat vas deferens which was secondary to release of endogenous noradrenaline rather than to the stimulation of an excitatory H₁-receptor.     

Reserpine-induced potentiation of the inhibitory action of neuropeptide Y on the rat vas deferens neurotransmission

The inhibitory action of neuropeptide Y (NPY) on the muscular activity of the prostatic end of the rat vas deferens elicited by transmural electrical stimulation was examined in control and in reserpinized rats. Pretreatment with 1 mg/kg reserpine for 48 h induced a 6-fold increase in NPY potency. Likewise, the potency of clonidine to inhibit the electrically induced muscular activity or noradrenaline to contract the ductus musculature was also potentiated. It is hypothesized that reserpine via a denervation super-sensitivity-like process increases the density of the NPY receptors. The functional significance of NPY in the motor activity of the vas deferens is discussed.     

An inhibition of post-ganglionic motor transmission in the mammalian vas deferens by D-lysergic acid diethylamide

1. Under certain conditions D-lysergic acid diethylamide (LSD), 10(-9)-10(-6) g/ml., exerted an immediate, prolonged and slowly reversible inhibitory effect upon the post-ganglionic motor transmission in desheathed guinea-pig vas deferens preparations.2. The most critical factor influencing this action of LSD appeared to be the train length. With short trains of less than 4 or 5 pulses the twitch inhibition produced by LSD was often total. With longer trains (5-20 pulses), the degree of inhibition declined with increase in train length. These results suggest the existence of two components in the motor response to post-ganglionic stimulation, distinguished by their susceptibility to LSD.3. The inhibition of the LSD-susceptible component was related to the dose of LSD in the range 10(-9)-10(-6) g/ml., reaching a maximum at 0.5-1 x 10(-6) g/ml. The response remnants elicited by trains of more than 5 pulses under these conditions could not be reduced further by a ten- to twenty-fold increase in LSD concentration to 10(-5) g/ml. and were in fact slightly potentiated.4. The inhibition of post-ganglionic motor transmission by LSD was not explicable on the basis of an alpha-adrenoceptor blockade because it was not associated with any reduction in motor responses to noradrenaline.5. The use of propranolol excluded mediation of the LSD-inhibition by beta-adrenoceptors.6. The LSD effect was not due to a non-specific smooth muscle depression because it was not associated with any reduction in motor responses to acetylcholine, ATP or bradykinin.7. The inhibitory effect of LSD on post-ganglionic transmission resembled that of noradrenaline in that it was antagonized by phentolamine; another alpha-adrenoceptor blocking agent, phenoxybenzamine, was less effective than phentolamine in this respect.8. The LSD-inhibition was obtained in preparations taken from reserpinized guinea-pigs.9. The inhibition of motor transmission in the vas deferens by LSD was confirmed in rats, Meriones shawii and rabbits.10. The inhibition of post-ganglionic transmission by LSD was unrelated to its ability to antagonize 5-hydroxytryptamine (5-HT), to which the longitudinal muscle of the guinea-pig vas deferens is insensitive. The more potent 5-HT antagonists, methysergide and BOL 148 were either virtually inactive or considerably weaker than LSD.     

Release of 3H-noradrenaline from the rat vas deferens under various in vitro conditions.

The release of 3H-(-)-noradrenaline (NA) from rat vas deferens in vitro was examined under various experimental conditions. It was found that in normal and reserpinized vas deferens the release of NA evoked by (+)-amphetamine (5 X 10(-6) M) or low external Na+ (26 mM) was antagonized by imipramine methiodide and desipramine, inhibitors of the NA uptake, but was not dependent on the presence of Ca2+ in the medium and was not antagonized by the potent local anaesthetic agent bethoxycaine. The release evoked by veratridine in reserpinized tissue was antagonized by the uptake inhibitors but was in normal tissue only partially inhibited in presence of Ca2+ but almost completely in absence of Ca2+. The release by high K+ (117 mM)+low Na+ (26 mM) in normal tissue was dependent on the presence of Ca2+ and was antagonized by the muscarinic agonists carbacholine and metacholine and by high concentrations of desipramine. In the reserpinized vasa the corresponding release was not dependent on Ca2+ and was not antagonized by the muscarinic agents but was inhibited by high concentrations of desipramine.     

2-Hydroxy-saclofen: an improved antagonist at central and peripheral GABAB receptors

2-hydroxy-saclofen (2-OH-S), a sulphonic analogue of baclofen, slightly increased the twitch height and reversibly antagonised the GABA- and baclofen-induced depression of twitch contractions in the guinea pig vas deferens and isolated ileum, causing a parallel dextral shift in the baclofen dose-response curve in a competitive manner (pA2 = 5.0) in the latter tissue. 2-OH-S (10-50 microM) reversibly elevated the spike height and antagonised the baclofen (8-20 microM)-induced suppression of ictal discharges in rat cortical slices superfused in Mg2+-free Krebs solution, the spike height declining to control level within 15 min of washout. The antagonism by 2-OH-S on GABAB receptor-mediated actions is selective, as 2-OH-S did not affect depressive responses to adenosine or morphine, or contractile responses to GABA (GABAA receptor-mediated), acetylcholine and carbachol in the ileum. Compared to phaclofen, 2-OH-S is a more potent competitive antagonist of GABAB receptor-mediated actions in the central and peripheral nervous system.     

Binding of adenosine and receptor-specific analogues to lymphocytes from control subjects and patients with common variable immunodeficiency.

Studies were performed on the binding of tritiated adenosine and its analogues, 5'-N-ethylcarboxamide adenosine (NECA) and N6-phenylisopropyladenosine (PIA), to human peripheral blood lymphocytes. These revealed binding only of adenosine (Kd, 1-10 microM, 14,000 binding sites/cell), which was abolished by dipyridamole, a specific adenosine transport inhibitor, suggesting that the binding is to the nucleoside transporter. The absence of high affinity (Kd less than or equal to 1 microM) binding of adenosine or of the two analogues. NECA and PIA suggests that the previously reported effects of adenosine on cAMP formation are not mediated by cell surface specific nucleoside receptors. Binding of adenosine to the carrier in lymphocytes from patients with common variable immunodeficiency was similar to those from control subjects.     

Sympathetic denervation of the smooth muscle of the vas deferens

1. The post-ganglionic nerve fibres to the vas deferens of the guinea-pig and rat were interrupted in vivo by stripping one vas deferens of its serous coat; the other vas deferens was left intact as a control.2. Four to eight days later the stripped vas deferens did not contract in response to electrical transmural stimulation in vitro at 0.1 msec pulse duration. Pulses of 1.0 msec duration produced small contractions which were not abolished by local anaesthetic or adrenergic neurone-blocking drugs.3. Log dose-response curves to noradrenaline were, for stripped vasa deferentia, to the left of those for control vasa. The increase in sensitivity to noradrenaline at 8 days was about sixteenfold for rat vasa and about tenfold for guinea-pig vasa. Tyramine did not contract stripped vasa from guinea-pigs or rats.4. The noradrenaline and adrenaline content of guinea-pig and rat vasa was greatly reduced or abolished 8 days after the stripping operation.5. Fluorescent nerve terminals were usually absent when transverse sections of stripped vasa were examined by fluorescence microscopy after treatment by the formaldehyde condensation method for demonstrating catecholamines.6. It is concluded that post-ganglionic sympathetic denervation is achieved by stripping the vas deferens in vivo of its serous coat and mesenteric attachments.     

Pyrazolo [3,4-d] pyrimidines, a new class of adenosine antagonists

A variety of nitrogen heterocycles structurally related to caffeine and theophylline have been tested for activity as adenosine receptor antagonists. Preliminary screening, utilizing displacement of [3H]N6-phenylisopropyladenosine binding to rat brain membrane A1-adenosine receptors, identified several pyrazolo[3,4-d]pyrimidines with potential antagonist activity. These were then tested for their ability to antagonize the adenosine-stimulated adenylate cyclase system of guinea-pig brain slices. One of these, 4,6-bis-alpha-carbamoylethylthio-1-phenylpyrazolo[3,4-d]pyrimidine (DJB-KK), was over an order of magnitude more potent than theophylline in blocking adenosine-stimulated increases in cyclic AMP levels.     

Adenosine modulation of neuroeffector transmission in guinea-pig uterine smooth muscle.

Effects of adenosine and adenosine analogues on neuroeffector transmission in separate layers of the guinea-pig uterine smooth muscle during different phases of the oestrus cycle were studied. Adenosine (ADO), N6-[(R)-1-methyl-2-phenylethyl]- adenosine (R-PIA) and 5'-N-ethylcarboxamideadenosine (NECA) enhanced spontaneous contractile activity as well as contractile responses to nerve stimulation or direct muscle stimulation in the longitudinal and circular muscle layers both at the time of ovulation and implantation. The agonist potency order was R-PIA greater than or equal to NECA greater than ADO. Furthermore, in the circular muscle layer inhibitory effects were seen at time of implantation and the agonist potency for this inhibitory effect was NECA greater than R-PIA greater than or equal to ADO. 8-p-sulphophenyltheophylline (8-PSOT) antagonized both the stimulatory and inhibitory effects of the purines and 8-p-sulphophenyltheophylline applied to untreated preparations inhibited nerve-induced contractile activity. NECA inhibited stimulation-induced release of 3H in preparations incubated with [3H]noradrenaline. We suggest that adenosine and its analogues can modulate adrenergic neuroeffector transmission in guinea-pig uterine smooth muscle via action at postjunctional receptors stimulating contractile activity and prejunctional receptors inhibiting transmitter release. In the circular muscle layer postjunctional receptors inhibiting contractile activity were evident at time of implantation. Furthermore, endogenous purines exerting a stimulatory action on the neuroeffector transmission were likely formed during the present experimental conditions.     

Post- and prejunctional consequences of ecto-ATPase inhibition: electrical and contractile studies in guinea-pig vas deferens

At sites of purinergic neurotransmission, synaptic ecto-ATPase is believed to limit the actions of ATP following its neural release. However, details of the modulation by this enzyme of the ATP-mediated conductance change and the possible mechanisms mediating this modulation remain unelucidated. We have addressed these issues by studying the effect of ARL 67156, a selective ecto-ATPase inhibitor, on ATP-mediated electrical and contractile activity in the sympathetically innervated guinea-pig vas deferens. ARL 67156 at 100 μ m significantly potentiated the amplitude of spontaneous excitatory junction potentials (SEJPs) by 81.1% ( P < 0.01) and prolonged their time courses (rise time by 49.7%, decay time constant by 38.2%; P < 0.01). Moreover, the frequency of occurrence of SEJPs was strikingly increased (from 0.28 ± 0.13 to 0.90 ± 0.26 Hz; P < 0.01), indicating an additional, primarily presynaptic, effect of ecto-ATPase inhibition. The frequency of occurrence of discrete events (DEs), which represent nerve stimulation-evoked quantal release of neurotransmitter, was also increased (∼6-fold; P < 0.01), along with the appearance of DEs at previously 'silent' latencies. Purinergic contractions of the vas deferens were potentiated significantly ( P < 0.01) by ARL 67156; these potentiated contractions were suppressed by the A1 agonist adenosine ( P < 0.01) but left unaffected by the A1 antagonist 8-phenyltheophylline (8-PT). Our results indicate (i) that ecto-ATPase activity, in addition to modulating the ATP-mediated postjunctional conductance change, may regulate transmitter release prejunctionally under physiological conditions, and (ii) that the prejunctional regulation may be mediated primarily via presynaptic P2X, rather than A1, receptors.     

Release of 3H-noradrenaline from the rat vas deferens under various in vitro conditions

The release of ³H-(-)-noradrenaline (NA) from rat vas deferens in vitro was examined under various experimental conditions. It was found that in normal and reserpinized vas deferens the release of NA evoked by (+)-amphetamine (5 times 10^?6 M) or low external Na⁺ (26 mM) was antagonized by imipramine methiodide and desipramine, inhibitors of the NA uptake, but was not dependent on the presence of Ca²⁺ in the medium and was not antagonized by the potent local anaesthetic agent bethoxycaine. The release evoked by veratridine in reserpinized tissue was antagonized by the uptake inhibitors but was in normal tissue only partially inhibited in presence of Ca²⁺ but almost completely in absence of Ca²⁺. The release by high K⁺ (117 mM) + low Na⁺ (26 mM) in normal tissue was dependent on the presence of Ca²⁺ and was antagonized by the muscarinic agonists carbacholine and metacholine and by high concentrations of desipramine. In the reserpinized vasa the corresponding release was not dependent on Ca²⁺ and was not antagonized by the muscarinic agents but was inhibited by high concentrations of desipramine.     

The storage of endogenous noradrenaline in sympathetic nerve terminals

1. The subcellular distribution of noradrenaline in sympathetic nerve terminals of rat vas deferens and cat spleen has been studied by cell fractionation methods combined with fluorescence and electronmicroscopic histochemical methods for noradrenaline.2. Pinched-off axon varicosities (synaptosomes) were isolated and identified by fluorescence and electronmicroscopy in the mitochondrial pellet.3. The proportion of large to small dense-cored vesicles in electronmicrographs of sympathetic nerve terminals varies in different organs. In rat vas deferens 4% and in cat spleen 20% are large vesicles.4. Density gradients of rat vas deferens have a single low density peak of noradrenaline at 0.6 M sucrose, whereas those of cat spleen have an additional peak of noradrenaline at 1.1 M sucrose.5. Small dense-cored vesicles were identified electronmicroscopically in the low density fractions and large dense-cored vesicles in the high density fractions from density gradients.6. We conclude that both small and large dense-cored vesicles store noradrenaline.     

Isolation of a polypeptide from the venom of Bungarus multicinctus that binds to ganglia and blocks the ganglionic transmission in mammals

A 15,000 dalton polypeptide purified from Bungarus multicinctus venom (which normally copurifies with alpha-bungarotoxin) was characterized biochemically and its biological effects were studied. This polypeptide, P15, had an aminoacid composition and molecular weight different from those of both alpha- and beta-bungarotoxin. It inhibited the ganglionic transmission in the guinea-pig hypogastric nerve-vas deferens preparation and did not block, even at very high concentrations, the neuromuscular transmission in the rat phrenic nerve-diaphragm preparation. In the same preparations alpha-bungarotoxin was unable to block the response at the ganglionic synapse while it was fully active in blocking the neuromuscular transmission. However, a pretreatment of the vas deferens preparation with alpha-bungarotoxin prevented the inhibitory effect of P15. 125I-Labeled P15 showed a specific and saturable binding to rat superior cervical ganglia homogenate and to a Torpedo postsynaptic membrane fraction. The binding of P15 to ganglia was inhibited by curare. The binding was Ca2+ dependent. The density of binding sites was of 300 fmol/mg of protein in the ganglion and 500 fmol/mg of protein in Torpedo membranes. The amount of P15-binding sites in ganglia was not modified by denervation, indicating that P15 binds to postsynaptic receptors. The binding of 125I-labeled P15, both in ganglia and Torpedo membranes, was inhibited by alpha-bungarotoxin. P15 had a Ca2+-dependent phospholipase A2 activity. Lowering Ca2+ concentration in incubation media affected the phospholipase A2 activity more than binding properties and inhibition of phospholipase activity with p-bromophenacyl bromide did not affect the activity of P15 on vas deferens preparation, suggesting that the phospholipase activity is not necessary for the activity of P15 on nicotinic receptors. Our results suggest that P15 toxin may be a specific and valuable probe for studying the ganglionic nicotinic receptor.     

Evidence that adenosine mediates the depression of spinal dorsal horn neurons induced by peripheral vibration in the cat

Nociceptive neurons in the dorsal horn of the cat spinal cord are depressed by vibration applied to the ipsilateral hind limb. The present study investigated the pharmacological properties of this depression because of the possibility that it represents the neural basis at the spinal level for the analgesic effects of vibration in humans. Experiments were done in cats anesthetized with sodium pentobarbital and acutely spinalized at the first lumbar level. Extracellular recordings were made from nociceptive neurons in the lower lumbar segments. The depression of these neurons induced by vibration to the hindlimb was attenuated by administration of the P1-purinergic (adenosine) receptor antagonist, caffeine (20-60 mg/kg i.v.); the maximum attenuation was 100%. Effects of caffeine began within 2 min after the start of injection (1-3 min injection period), were greatest in the 10 min period after the end of injection and lasted for up to 2 hr. Importantly, another P1-purinergic receptor antagonist, which does not cross the blood-brain barrier, 8-sulphophenyltheophylline (8-16 mg/kg), had no effect on the depression when given intravenously (n = 5); however, when administered by iontophoresis 8-sulphophenyltheophylline blocked the depression in 2 of 6 units. Dipyridamole (1.0-2.0 mg/kg i.v.), an inhibitor of adenosine uptake, potentiated the depression in 2 of 5 cases. These results prompt us to suggest that depression induced by vibration may be mediated by adenosine via the activation of P1-purinergic receptors. On the other hand, the GABAA antagonist, bicuculline, failed to attenuate vibration-induced depression when administered either intravenously (0.2-0.4 mg/kg; n = 5) or by iontophoresis (n = 10) and the glycine antagonist, strychnine (0.2-0.6 mg/kg; n = 3) and the opiate antagonist, naloxone (0.1-0.4 mg/kg; n = 4) were similarly ineffective. These findings suggest that vibration-induced depression of these units occurs without involvement of bicuculline-sensitive GABA receptors, strychnine-sensitive glycine receptors and naloxone-sensitive opiate receptors. In view of the fact that vibration-induced depression is evoked synaptically, this study is the first to demonstrate in the central nervous system a synaptic response which is mediated by adenosine. In addition, we suggest that the analgesic effects of vibration in humans may be mediated at the spinal level by activation of P1-purinergic receptors.