P2-purinoceptor-mediated autoinhibition of sympathetic transmitter release in mouse and rat vas deferens

Effects of drugs acting at P₂-purinoceptors on the release of newly taken up [³H]-noradrenaline were studied in slices of mouse and rat vas deferens. The slices were superfused and stimulated electrically, in most experiments by trains of 60 pulses/8 Hz.In mouse vas deferens, the P₂-purinoceptor antagonists reactive blue 2 (1.8–100 M) and brilliant blue G (10–300 M) increased the stimulation-evoked overflow of tritium in a concentration-dependent manner as shown previously for suramin. Reactive blue 2, which preferentially blocks the P_2Y-subtype, was the most potent compound and the compound with highest maximal effect, an increase by 104%. Pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS), in contrast, caused a small increase only at a single concentration (30 M). The effects of reactive blue 2, brilliant blue G and suramin were not additive. The P₂ agonist adenosine 5-O-(3-thio)-triphosphate (ATPS) reduced the evoked overflow of tritium. As shown previously for suramin, reactive blue 2 30 M and brilliant blue G 100 M antagonized the effect of ATPS. From the shift of the ATPS concentration-response curve to the right, an apparent pK_B value of 5.3 was estimated for reactive blue 2 and an apparent pKB of 4.5 for brilliant blue G. In rat vas deferens, reactive blue 2 (3–30 M), brilliant blue G (10 M) and suramin (30–300 M) also increased the evoked overflow of tritium. As in the mouse, reactive blue 2 was the most potent compound and the compound with highest maximal effect, an increase by 9001o. As previously demonstrated in the mouse, suramin (300 M) increased the evoked overflow of tritium only when rat vas deferens slices were stimulated by trains of 60 pulses at 1 or 8 Hz, but not when they were stimulated by trains of 6 pulses/100 Hz.The results confirm the operation of a P₂-purinoceptor-mediated prejunctional negative feedback controlling the release of noradrenaline in mouse vas deferens and demonstrate the same mechanism in rat vas deferens. The prejunctional P₂-purinoceptors are P_2Y-like in both species. They are a novel kind of autoreceptors, operating in parallel to prejunctional ₂-autoreceptors. Correspondence to: I. von Kügelgen at the above address     

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by α,β-methylene ATP (α,β-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by α,β-MeATP (3 μM), with IC₅₀-values ranging from 1.1 to 100 μM. Pyridoxalphosphate-6-azophenyl-2’,4’-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4’-diisothiocyanatostilbene-2,2’-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8’-[Carbonylbis(imino-3,1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate) (NF023), sura- min, pyridoxalphosphate-6-azophenyl-2’,5’-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34–43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6’-(1,1’-biphenyl-4,4’-diylbisazo)-bis-4-amino-5-hydroxy-naphtha-lene-1,3-disulphonate (NH01) and Evans blue even enhan- ced the response to ATP. For antagonists causing full or partial inhibition, the IC₅₀-values against ATP were close to those against α,β-MeATP. All antagonists attenuated the removal of ATP, with IC_25%-values ranging from 0.8 μM to >320 μM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X₁). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered. Received: 23 October 1998 / Accepted: 11 January 1999     

The mechanisms of enhancement and inhibition of field stimulation responses of guinea-pig vas deferens by prostacyclin analogues

1. In the guinea-pig isolated vas deferens preparation bathed in Tyrode''s solution, the prostacyclin analogues, cicaprost, TEI-9063, iloprost, taprostene and benzodioxane-prostacyclin, enhanced twitch responses to submaximal electrical field stimulation (20%-EFS). The high potency of cicaprost (EC₁₅₀=1.3 nM) and the relative potencies of the analogues (equi-effective molar ratios=1.0, 0.85, 1.6, 17 and 82, respectively) suggest the involvement of a prostacyclin (IP-) receptor.
2. Maximum enhancement induced by cicaprost in 2.5 mM K⁺ Krebs-Henseleit solution was similar to that in Tyrode solution (2.7 mM K⁺), but was progressively reduced as the K⁺ concentration was increased to 3.9, 5.9 and 11.9 mM. There was also a greater tendency for the other prostacyclin analogues to inhibit EFS responses in 5.9 mM standard K⁺ Krebs-Henseleit solution; this may be attributed to their agonist actions on presynaptic EP₃-receptors resulting in inhibition of transmitter release.
3. The EFS enhancing action of cicaprost was not affected by the α₁-adrenoceptor antagonist prazosin (100 and 1000 nM). Cicaprost (20 and 200 nM) did not affect contractile responses of the vas deferens to either ATP (5 μM) or α,β-methylene ATP (1 μM) in the presence of tetrodotoxin (TTX, 100 nM). In addition, enhancement by cicaprost of responses to higher concentrations of ATP (30 and 300 μM) in the absence of TTX, as shown previously by others, was not seen. Prostaglandin E₂ (PGE₂, 10 nM) and another prostacyclin analogue TEI-3356 (20 nM) enhanced purinoceptor agonist responses. Unexpectedly, TTX (0.1 and 1 μM) partially inhibited contractions elicited by 10–1000 μM ATP; contractions elicited by 1–3 μM ATP were unaffected. Further studies are required to establish whether a pre- or post-synaptic mechanism is involved.
4. In a separate series of experiments, cicaprost (5–250 nM), TEI-9063 (3–300 nM), 4-aminopyridine (10–100 μM) and tetraethylammonium (100–1000 μM) enhanced both 20%-EFS responses and the accompanying overflow of noradrenaline to a similar extent. In further experiments with the EP₁-receptor antagonist AH 6809, TEI-3356 (1.0–100 nM) and the EP₃-receptor agonist, sulprostone (0.1–1.0 nM) inhibited both maximal EFS responses and noradrenaline overflow, thus confirming previous reports of the high activity of TEI-3356 at the EP₃-receptor. Cicaprost had no significant effect on noradrenaline overflow at 10 and 100 nM, but produced a modest inhibition at 640 nM.
5. In conclusion, our studies show that prostacyclin analogues (particularly TEI-3356) can inhibit EFS responses of the guinea-pig vas deferens by acting as agonists at presynaptic EP₃-receptors. Prostacyclin analogues (particularly cicaprost and TEI-9063) can also enhance EFS responses through activation of IP-receptors. The mechanism of the enhancement has not been rigorously established but from our results we favour a presynaptic action to increase transmitter release.

     

Cholinergic innervation of the guinea-pig isolated vas deferens

Recently, a cholinergic neurogenic component of contraction has been characterised in the aganglionic mouse vas deferens. In this paper, a cholinergic component of contraction in the guinea-pig vas deferens is characterised pharmacologically. A residual, tetrodotoxin-sensitive (TTX, 0.3 μM), neurogenic contraction was revealed after prolonged exposure (5 h) to the adrenergic neurone blocker bretylium (20 μM) or in the presence of prazosin (100 nM) and α,β-methylene ATP (1 μM), a purinergic agonist which desensitizes P2X receptors. The bretylium-resistant component was potentiated by the acetylcholinesterase (AChE) inhibitor neostigmine (10 μM) and inhibited by the muscarinic-receptor (mAChR) antagonist cyclopentolate (1 μM). Nicotine (30 μM) enhanced the bretylium-resistant component. Neostigmine increased the second component of contraction in the presence of prazosin and α,β-methylene ATP, whilst yohimbine (1 μM), an α₂ adrenergic receptor antagonist, enhanced both the first and second components of the electrically evoked contraction. These enhanced contractions were blocked by cyclopentolate in both cases. Nicotine enhanced the cholinergic component of contraction revealed by neostigmine but failed to have any detectable effects in the presence of cyclopentolate. Neostigmine alone increased the slow component of contraction which was reversed by cyclopentolate to control levels. The M₃ receptor-antagonist 4-DAMP (10 nM) markedly inhibited the cholinergic component of contraction to a level comparable with cyclopentolate. Laser microscopy has shown that neostigmine also increased the frequency of spontaneous Ca²⁺ transients remaining in smooth muscle cells after perfusion with prazosin and α,β-methylene ATP, an effect blocked by 4-DAMP. These experimental data show that there is a functional cholinergic innervation in the guinea-pig vas deferens whose action is limited by acetylcholinesterase, blocked by cyclopentolate and mediated through M3 receptors. Moreover, by blocking the cholinesterase, the increased amount of ACh generates spontaneous Ca²⁺ transients in smooth muscle cells. An erratum to this article is available at .     

P1-purinoceptor-mediated modulation of neural noradrenaline and ATP release in guinea-pig vas deferens

The effect of P₁-purinoceptor activation on contractions, release of noradrenaline and release of ATP elicited by electrical field stimulation (210 pulses, 7 Hz) was studied in the superfused vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of total tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferinluciferase technique.Electrical stimulation elicited reproducible contraction, tritium overflow and ATP overflow. In the absence of other drugs, adenosine (10–100 M) did not change evoked contractions but reduced the evoked overflow of tritium and ATP. In subsequent experiments ₁-adrenoceptors were blocked by prazosin, P₂-purinoceptors by suramin and ₂-adrenoceptors by rauwolscine. No or almost no contraction remained under these conditions. The evoked overflow of tritium was 505% and the evoked overflow of ATP 34% of that observed in the absence of prazosin, suramin and rauwolscine. Adenosine (1–100 M) again reduced the evoked overflow of tritium and ATP, and so did the A₁-selective agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA; 0.032–0.32 M). Adenosine and CCPA decreased the evoked overflow of ATP to a greater extent than the evoked overflow of tritrium.It is concluded that neural release of both postganglionic sympathetic cotransmitters, noradrenaline and ATP, is decreased upon activation of prejunctional P₁- (A₁-) purinoceptors in guinea-pig vas deferens. The A₁-receptor-mediated inhibition of the release of ATP is more marked than the inhibition of the release of noradrenaline, a pattern opposite to the inhibition produced by activation of prejunctional ₂-autoreceptors. Correspondence to: B. Driessen at the above address     

A1 adenosine receptor modulation of electrically-evoked contractions in the bisected vas deferens and cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon both electrically-evoked and phenylephrine-induced contractile responses were investigated in the bisected vas deferens and the cauda epididymis of the guinea-pig. Electrical field-stimulation (10 s trains of pulses at 9 Hz, 0.1 ms duration, supramaximal voltage) elicited biphasic and monophasic contractile responses from preparations of bisected vas deferens and cauda epididymis, respectively; these responses were abolished by tetrodotoxin (300 nM).
2. In the prostatic half of the vas deferens the A₁ selective adenosine receptor agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) and the non-selective A₁/A₂ adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) inhibited electrically-evoked contractions (pIC₅₀±s.e.mean values 6.15±0.24, 5.99±0.26 and 5.51±0.24, respectively). The responses to CPA were blocked by the A₁ adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine, DPCPX (100 nM).
3. In the epididymal half of the vas deferens NECA potentiated (at ⩽100 nM) and inhibited (at ⩾1 μM) electrically-evoked contractions. In the presence of the non-selective α-adrenoceptor antagonist phentolamine (3 μM), the α₁-adrenoceptor antagonist, prazosin (100 nM), or at a reduced train length (3 s) NECA inhibited electrically-evoked contractions (pIC₅₀ values 6.05±0.25, 5.97±0.29 and 5.71±0.27, respectively). CPA (at 10 μM) also inhibited electrically-evoked contractions in this half of the vas deferens. In the presence of prazosin (100 nM), CPA also inhibited electrically-evoked contractions (pIC₅₀ 6.14±0.67); this effect was antagonized by DPCPX (30 nM, apparent pK_B 8.26±0.88). In the presence of the P2 purinoceptor antagonist, suramin (300 μM), CPA (up to 1 μM) potentiated electrically-evoked contractions.
4. NECA, CPA and APNEA potentiated electrically-evoked contractions in preparations of cauda epididymis (pEC₅₀ values 7.49±0.62, 7.65±0.74 and 5.84±0.86, respectively), the response to CPA was competitively antagonized by DPCPX (100 nM) with an apparent pK_B value of 7.64±0.64.
5. The α₁-adrenoceptor agonist phenylephrine elicited concentration-dependent contractile responses from preparations of bisected vas deferens and cauda epididymis. NECA (1 μM) potentiated responses to phenylephrine (⩽1 μM) in the epididymal, but not in the prostatic half of the vas deferens. In preparations of epididymis NECA (1 μM) shifted phenylephrine concentration response curves to the left (4.6 fold). In the presence of a fixed concentration of phenylephrine (1 μM), NECA elicited concentration-dependent contractions of preparations of the epididymal half of the vas deferens and of the epididymis (pEC₅₀ values 7.57±0.54 and 8.08±0.18, respectively). NECA did not potentiate responses to ATP in either the epididymal half of the vas deferens or the epididymis.
6. These studies are consistent with the action of stable adenosine analogues at prejunctional A₁ and postjunctional A₁-like adenosine receptors. The prejunctional A₁ adenosine receptors only inhibit the electrically-evoked contractions of purinergic origin (an effect predominant in the prostatic half of the vas deferens). At the epididymis, where electrically-evoked contractions are entirely adrenergic, the predominant adenosine receptor agonist effect is a potentiation of α₁-adrenoceptor-, but not of ATP-induced contractility.

     

Inhibition by nucleotides acting at presynaptic P2-receptors of sympathetic neuro-effector transmission in the mouse isolated vas deferens

Summary Effects of nucleotides and nucleosides on smooth muscle tension and the release of previously stored [³H]-noradrenaline were studied in the mouse isolated vas deferens. The tissue was stimulated twice by 20 electrical field pulses delivered at 2 Hz (S₁, S₂)., \-Methylene-ATP, ATPS, ATP and UTP elicited contraction, with potency decreasing in that order; there was no contractile response to adenosine (up to 100 mol/1) and uridine (up to 1 mmol/1). The electrically evoked overflow of tritium was reduced by the drugs in the following order of potency: ATPS > ATP = adenosine > UTP; ,\-meth-ylene-ATP (up to 10 µmol/l) and uridine (up to 1 mmol/1) did not significantly change the evoked overflow. 8-(p-Sulphophenyl)theophylline did not alter the contractile responses to the nucleotides; it prevented the overflow-inhibiting effect of adenosine and reduced that of UTP; the overflow-inhibiting effects of ATP and ATPS were not significantly attenuated. After prolonged exposure to ,-methylene-ATP, all contractile nucleotide effects were abolished; in contrast, the depression by adenosine and the nucleotides of the evoked overflow of tritium persisted. None of the effects was changed by indometacin, yohimbine or reactive blue 2.It is concluded that ATP, ATPS, ,\-methylene-ATP and UTP produce contraction of the vas deferens by activation of P_2x-receptors. Moreover, the nucleotides inhibit per se the release of [3H]-noradrenaline (and presumably the co-transmitter mixture of noradrenaline and ATP); the effect of ATP is not, or only to a small extent, due to breakdown to adenosine. The presynaptic site of action of the purine nucleotides is a P₂-receptor which differs from the P_2X-receptor and may be a reactive blue 2-resistant P_2y-like receptor.     

Inhibition by ethanol of contractions of rat vas deferens: no evidence for selective blockade of P2X-purinoceptors

Summary Some ligand-gated ion channels are important sites of action of ethanol. The aim of the study was to find out whether the P_2X-purinoceptors mediating contraction of the rat isolated vas deferens also are selectively sensitive to ethanol. Contractions were elicited by ATP (1 mmol/1), ,ß-methylene ATP (0.3 mol/1), noradrenaline (3 mol/1), high K⁺ (20 mmol/1) or electrical (neural) stimulation by pairs of pulses 3 s apart. In electrical stimulation experiments, purinergic and adrenergic response components were isolated by prazosin and suramin, respectively. Concentration-effect curves were determined for ethanol and, for comparison, nifedipine. Tritium outflow from tissues preincubated with ³H-noradrenaline was also examined.Ethanol at relatively low concentrations reduced contractions elicited by high K⁺ (IC₃₀ 145 mmol/1), ATP (IC₃₀ 211 mmol/1) and ,ß-methylene ATP(IC₃₀ 215 mmol/1) as well the purinergic component of neurogenic twitches (IC₃₀ 110-126 mmol/1; a significant effect at 10–32 mmol/1) and the adrenergic component of twitch 2 of the twitch pairs (IC₃₀ 63 mmol/1). These contractions also were very sensitive to nifedipine. Higher concentrations of ethanol were needed to reduce contractions elicited by noradrenaline (IC₃₀ 365 mmol/1) and the adrenergic component of twitch 1 of the twitch pairs (IC₃₀ 382 mmol/1), contractions that also were less sensitive to nifedipine. Ethanol 1 mol/l abolished all contractions. In contrast, concentration-effect curves for the inhibition by nifedipine of contractions evoked by ATP, ,ß-methylene ATP and noradrenaline (rapid phase) levelled off at 60–70% inhibition. The contractions that remained when these agonists were administered in the presence of nifedipine 10 mol/l were depressed by ethanol (IC₃₀ 242–387 mmol/1). Ethanol 320 mmol/1 did not change the electrically evoked overflow of tritium from vasa deferentia preincubated with ³H-noradrenaline.It is concluded that the P_2X-purinoceptors of rat vas deferens smooth muscle, although ligand-gated ion channels, are not selectively sensitive to ethanol. The reduction of contractions can be explained by, first, an inhibition of L-type voltage-dependent Ca²⁺ channels for which relatively low concentrations of ethanol are needed, and second, a non-specific depressant effect at an unknown site or at unknown sites which requires relatively high concentrations. Correspondence to R. Bultmann at the above address     

The fade of the purinergic neurogenic contraction of the guinea-pig vas deferens: analysis of possible mechanisms

The purinergic response of the guinea-pig vas deferens to long trains of pulses at high frequency consists of an initial twitch followed by a much lower plateau. Mechanical, neurochemical and electrophysiological techniques were used to examine the reason for the fade. Mechanical measurements. In tissues stimulated by trains of 180 pulses/10 Hz and treated with prazosin to suppress the noradrenergic contraction component, the response to ,-methylene ATP and to exogenous ATP was as high during the secondary plateau of the purinergic neurogenic contraction as it was outside electrical stimulation periods; the response to 50 pulses/100 Hz was also unchanged during the low plateau. The plateau was not increased by reactive blue 2, 8-(p-sulphophenyl)theophylline, propranolol or capsaicin. Neurochemical measurements. In tissues preincubated with [³H]-noradrenaline, electrical stimulation elicited an overflow of tritium and of ATP. In the absence of drugs as well as in the presence of prazosin and suramin to suppress contractions, the overflow of tritium per pulse decreased slightly in the course of trains of 90 pulses/10 Hz; the overflow of ATP per pulse decreased to a greater extent on average, but the decrease was not statistically significant. In the presence of prazosin and nifedipine, also to suppress contractions, the overflow of tritium per pulse again decreased slightly in the course of trains of 105 pulses/10 Hz, but the overflow of ATP per pulse if anything tended to increase. Electrophysiological measurements. Extracellular recording in the presence of prazosin showed that electrical stimulation by 180 pulses/10 Hz elicited excitatory junction currents (EJCs) which facilitated and summated to reach threshold for the initiation of action potentials in the smooth muscle cells. In most tissues, smooth muscle action potentials ceased after a few seconds although EJCs continued. Intracellular recording in the presence of prazosin and nifedipine showed that excitatory junction potentials (EJPs) elicited by 180 pulses/10 Hz facilitated and summated to a plateau after about 10 stimuli. The EJPs continued unchanged, and the plateau depolarization was maintained, throughout the train.It is concluded that the fade of the purinergic neurogenic contraction is not due to P_2Y-purinoceptor desensitization. It also is not due to a secondary relaxation mediated by P_2Y- or P₁-purinoceptors, -adrenoceptors or a compound originating from primary afferent axons. Moreover, a fade of the release of ATP in the course of the pulse train is not responsible for the contraction fade. Rather, the reason is a failure of the process by which the smooth muscle cell depolarization triggers action potentials. Inactivation of l-type Ca²⁺ channels that are under the control of released ATP may be the underlying mechanism. Correspondence to: B. Driessen at the above address     

Prostaglandin-versus alpha-adrenoceptor-mediated control of sympathetic neurotransmitter secretion in guinea-pig isolated vas deferens

Noradrenaline (NA) secretion of isolated superfused guinea-pig vas deferenswas studied by determination of total field stimulation-induced efflux of tritium, after preincubation with ³H-l-NA. The medium contained optimal concentrations of desmethylimipramine and normetanephrine to block local rebinding of free NA. Further addition of the two chemically different inhibitors of prostaglandin synthetase, 5,811,14 eicosatetraynoic acid or indomethacin, consistently enhanced the nerve stimulation-induced output of tritium at a frequency of 5/sec, but not at 10/sec. The α-adrenoceptor blocking agent, phenoxybenzamine further strongly elevated nerve stimulation-induced output of tritium. This rise was abolished by low concentrations of exogenous prostaglandin E₂. The results show that sympathetic neurotransmitter secretion in the guinea-pig vas deferens, during low frequency stimulation, is restricted by local formation of prostaglandin E. However and in addition, neurotransmitter secretion appears to be restricted by an α-adrenoceptor-mediated mechanism, which does not appear to depend on endogenous prostaglandin E as a chemical mediator.     

Characterization of the prejunctional muscarinic receptors mediating inhibition of evoked release of endogenous noradrenaline in rabbit isolated vas deferens

The aim of the present study was to characterize the prejunctional modulation of evoked release of endogenous noradrenaline in rabbit vas deferens by the use of muscarinic receptor agonists and subtype-prefering antagonists.Vasa deferentia of the rabbit were stimulated electrically by trains of 120 pulses delivered at 4 Hz or trains of 30 pulses at 1 Hz. The inhibition by muscarinic agonists of the stimulation-evoked overflow of endogenous noradrenaline in the absence and presence of antagonists was used to determine affinity constants for antagonists. These values were compared with those observed at putative M₁ receptors inhibiting neurogenic twitch contractions in the rabbit vas deferens and with affinity data obtained at M₁(m₁)-M₄(m₅) receptors in functional studies and binding experiments.The evoked overflow of noradrenaline from sympathetic nerves was enhanced by the Al receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the P₂ purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2,4-disulfonic acid (PPADS) and indomethacin, indicating a tonic inhibition by endogenous A₁ and P₂ purinoceptor agonists and prostanoids, respectively. The stimulation-evoked overflow at 4 Hz was not sensitive to inhibition by the muscarinic agonists methacholine or 4-(4-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium iodide (4-Cl-McN-A-343). In contrast, at a stimulation frequency of 1 Hz the evoked noradrenaline release was decreased by muscarinic agonists (EC₅₀): arecaidine propargyl ester (0.062 M), 4-Cl-McN-A-343 (0.32 M), 4-(4-fluorophenylcarbamoyloxy)-2-butynylN-methyl-pyrrolidinium tosylate (4-F-PyMcN⁺; 0.48 M) and methacholine (0.86 M). The affinity constants of most of the muscarinic antagonists [atropine: pK_B = 9.47; (R)-trihexyphenidyl: pK_B = 9.18; pirenzepine: pA₂ = 7.68; methoctramine: pK_B = 6.90] are consistent with estimates of these antagonists at M₁(m₁) receptors determined in various functional and binding studies. The high antagonistic potency of pirenzepine and (R)-trihexyphenidyl and the agonistic activity of 4-F-PyMcN⁺ argue for the involvement of M₁, and against that of M₂ and M₃ receptors in the inhibition of evoked noradrenaline overflow. However, the high apparent pK_B of 8.30 for himbacine is not in accordance with an M₁ receptor; by contrast, it would be compatible with the presence of M₂ or M₄ receptors. The potencies of the tested muscarinic agonists and antagonists largely agree with those obtained for the inhibition of neurogenic twitch responses (0.05 Hz) in the rabbit vas deferens. In conclusion, the rabbit vas deferens is endowed with prejunctional muscarinic receptors mediating heteroinhibition of noradrenaline release that are probably of the same subtype as the putative M₁ receptors inhibiting neurogenic twitch contractions, and are not of the M₂, M₃ or m₅ subtype. Correspondence to: U. Grimm at the above address     

Ouabain-induced potentiation on the contractions of the guinea-pig vas deferens

This contractile responses of the guinea-pig vas deferens to norepinephrine, isoproterenol, tyramine, ACh, carbachol, histamine and 5-HT were non-specifically potentiated by ouabain (10^?6?10^?5 g/ml); potentiations of tyramine and 5-HT were particularly marked. Ouabain enhanced the responses to norepinephrine, 5-HT and angiotensin on the denervated preparation.The response of the tissue to 5-HT consisted of a fast and a slow component. The latter was augmented by methysergide. The marked potentiation of the slow phase by ouabain was prevented by pretreatment with reserpine, cocaine or phentolamine. Denervation abolished the slow phase abolished but increased the fast phase. The latter was inhibited by phenoxybenzamine, but not by phentolamine. Therefore, the slow and fast phases may be due to actions of 5-HT at pre- and post-synaptic sites, respectively.From these results, it is concluded that ouabain potentiation is due to increased catecholamine release and to excitation of the post-synaptic membrane.     

Modulation of neural noradrenaline and ATP release by angiotensin II and prostaglandin E2 in guinea-pig vas deferens

Effects of angiotensin II and prostaglandin E₂ on contractions, release of noradrenaline and release of ATP elicited by electrical stimulation (210 pulses, 7 Hz) were studied in the isolated vas deferens of the guinea pig. Release of noradrenaline was assessed as overflow of tritium after preincubation with [³H]-noradrenaline. ATP was measured by means of the luciferin-luciferase technique. In some experiments postsynaptic a ₁-adrenoceptors and P_2X-purinoceptors were blocked by prazosin and suramin, respectively, to isolate the neural fraction of the overflow of ATP.Electrical stimulation elicited an overflow of tritium and ATP and, in the absence of prazosin and suramin, contraction. In the absence of prazosin and suramin, angiotensin II (1–100 nM) enhanced contractions as well as the evoked overflow of tritium and ATP. All parameters were increased by about the same percentage for a given concentration of angiotensin 11. The effect of prostaglandin E₂ (1–100 nM) was complex. Contractions were mainly enhanced, the evoked overflow of tritium was reduced, whereas the evoked overflow of ATP was predominantly increased. No or almost no contraction remained in the presence of prazosin and suramin, and the evoked overflow of ATP was decreased to about 16%. Angiotensin II (1–100 nM) again enhanced the evoked overflow of tritium and ATP. Both were increased by about the same percentage for a given concentration of angiotensin II and also were increased by about the same percentage as obtained in the absence of prazosin and suramin. Prostaglandin E₂ (1–100 nM) decreased the evoked overflow of tritium and ATP in the presence of prazosin and suramin, both by about the same percentage at a given prostaglandin E₂ concentration.It is concluded that neural release of ATP, like the release of noradrenaline, is presynaptically facilitated by angiotensin II and depressed by prostaglandin E₂. In the case of angiotensin II, increases in neural and postsynaptic ATP release contribute to the increase in ATP over flow observed in the absence of prazosin and suramin. In the case of prostaglandin E₂, an increase in postsynaptic ATP release can override the reduction in neural ATP release and give rise to an increase in ATP overflow in the absence of prazosin and suramin. No evidence for a differential modulation of neural noradrenaline versus ATP release was found. Correspondence to: B. Driessen at the above address     

Evans blue blocks P2X-purinoceptors in rat vas deferens

Summary In rat vas deferens, Evans blue 100 M increased contractions elicited by high K⁺ and by noradrenaline but markedly reduced contractions elicited by the P_2X-purinoceptor-selective agonist ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was shifted to the right by Evans blue 30 M and the maximal contraction was increased. In tissues incubated with nifedipine 10 M, Evans blue 100 M tended to increase the residual contraction elicited by noradrenaline and abolished the residual response to ,-methylene ATP (3 M). The concentration-response curve of ,-methylene ATP was progressively shifted to the right by increasing concentrations of Evans blue in the presence of nifedipine; maximal contractions were increased by Evans blue 10 and 30 but not 100 M. From the shifts to the right caused by Evans blue 30 M, apparent pK_B values of 5.9 (no nifedipine) and 6.0 (nifedipine present) were calculated. It is concluded that Evans blue blocks P_2X-purinoceptors in rat vas deferens and in addition causes a non-receptor-specific enhancement of contractions.Correspondence to: R. Bültmann at the above address     

Effects of T-type calcium channel blockers and thalidomide on contractions of rat vas deferens

Background and purpose:

In rat vas deferens, nerve mediated-contractions to a single electrical stimulus consist of an early purinergic and a later adrenergic component with differing sensitivities to L-type calcium channel blockers. We have investigated the effects of the T-type calcium channel blockers mibefradil and (1S, 2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylic dihydrochloride (NNC 55-0396) against contractions in rat vas deferens. In addition, the actions of thalidomide were examined.

Experimental approach:

Prostatic and epididymal portions of rat vas deferens were stimulated with a single electrical stimulus every 5 min, and mouse whole vas deferens was stimulated with 40 pulses at 10 Hz every 5 min.

Key results:

Both mibefradil and NNC 55-0396 (100 µM) produced inhibition of contractions of epididymal portions (42 ± 13%, n= 7, and 43 ± 4%, n= 15, of control respectively). However, both agents produced small inhibitions of responses in prostatic portions, presumably by L-type calcium channel block. Thalidomide (100 µM) inhibited contractions in epididymal (55 ± 4% of control, n= 17) but not in prostatic portions of rat vas deferens. Thalidomide (10–100 µM) also inhibited contractions in mouse vas deferens.

Conclusions and implications:

The T-type calcium channel blockers mibefradil and NNC 55-0396 block particularly the adrenoceptor-mediated, nifedipine-resistant response to nerve stimulation in rat vas deferens, and this may suggest that this component involves T-type calcium channels. In addition, thalidomide has actions that resemble those of the T-type calcium channel blockers, in that it blocks nifedipine-resistant contractions in epididymal portions.     

P2-purinoceptor antagonists: I. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by small aromatic isothiocyanato-sulphonates

Effects of eight small aromatic isothiocyanatosulphonates, of the aliphatic 2-isothiocyanatoethene-1-sulphonate (IES), and of the parent amines were studied on contractions of the rat vas deferens elicited by ,-methy-lene ATP (,-MeATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue.The aromatic isothiocyanato-sulphonates all reduced contractions of the rat vas deferens elicited by ,-methy-lene ATP. The antagonism was non-competitive, with depression of the maximum of the concentration-response curve of ,-MeATP and incomplete reversibility. The IC₅₀ values were between 11 and 54 M. In the guineapig taenia coli, the aromatic compounds shifted the concentration-response curve of ADPS to the right in a surmountable manner (one exception), and where three concentrations were tested, the Arunlakshana-Schild regression was linear and its slope did not differ from 1. The apparent K _d values were between 10 and 214 M. The removal of ATP from the medium by vas deferens tissue was decreased by the aromatic isothiocyanates with IC_25% values between 25 and 464 M. IES and the parent amines were inactive or almost inactive (parent amines not tested on ATP breakdown).The results indicate that the isothiocyanato residue as well as the aromatic core are essential for P₂-purinoceptor blockade. At the P_2X-purinoceptor, potency increases with the size of the molecules but is independent of the position of the isothiocyanato and sulphonate substituents. No simple structure-activity relationship for the P_2Y-purinoceptor and the ATP-degrading ecto-nucleotidases can be derived beyond the apparent lack of a major influence of the position of the substituents. 2-Isothiocyanatonaphthalene1-sulphonate (-INS) seems to be interesting because of relatively high P_2X-selectivity versus both the P_2Y-purinoceptor and ecto-nucleotidases.     

P2-purinoceptor antagonists: III. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to suramin

Effects of suramin and five analogs or fragments of suramin were studied on contractions of the rat vas deferens elicited by ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue. One compound, NF023, differed from suramin by removal of two p-methylbenzamido groups, whereas another, BSt101, differed from NF023 by additional removal of the three sulphonate residues from one of the terminal naphthalene rings.The compounds all shifted the concentration-response curve of ,-MeATP in the rat vas deferens to the right and simultaneously increased the maximum of the curve. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent K _d values were between 1 and 3672 M. In the guinea-pig taenia coli, the compounds shifted the concentration-response curve of ADPS to the right in a parallel manner, but in the one case where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent K _d values were between 10 and 786 M. The removal of ATP from the medium by vas deferens tissue was decreased only by suramin, NF023 and BSt101, with IC_25% values between 170 and 590 M.The results indicate that P_2X-purinoceptor affinity, P_2Y-purinoceptor affinity and the ecto-nucleotidase effect all increase with the size of the molecule. BSt101 resembled NF023 in potency at all three sites, indicating that the possession of a second naphthalene-trisulphonate group is not a prerequisite for relatively high affinity. NF023 is interesting because it is P_2X- versus P_2Y-selective and, in addition, the compound with the highest P_2X- versus ecto-nucleotidase-selectivity presently available.     

P2-purinoceptor antagonists: II. Blockade of P2-purinoceptor subtypes and ecto-nucleotidases by compounds related to Evans blue and trypan blue

Effects of Evans blue and four derivatives as well as of trypan blue and four derivatives, mostly smaller fragments but two compounds with an additional ethylene bridge in the center of the molecule, were studied on contractions of the rat vas deferens elicited by ,-methylene ATP (,-McATP; mediated by P_2X-purinoceptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5-O-(2-thiodiphosphate) (ADPS; mediated by P_2Y-purinoceptors), and the degradation of ATP by rat vas deferens tissue.All compounds shifted the concentration-response curve of ,-MeATP in the rat vas deferens to the right, and most compounds increased the maximum of the curve. Each member of the Evans blue series was similar in potency to the corresponding member of the trypan blue series. Where three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from 1. The apparent K _d values were between 0.8 and 385 M. In the guinea-pig taenia coli, only the members of the trypan blue group were relatively potent, shifting the concentration-response curve of ADPS to the right in a surmountable manner. In 2 of 3 cases where three concentrations were tested, the slope of the Arunlakshana-Schild regression was lower than 1. Apparent K _d values in the trypan blue group were between 5.2 and 324 M. The removal of ATP from the medium by vas deferens tissue was decreased mainly by the members of the Evans blue group, with IC_25% values between 13 and 158 (in 1 case >1000) M.The results indicate that the position of the sulphonate residues at the terminal naphthalene rings of these compounds hardly influences P_2X purinoceptor affinity but greatly influences P_2Y affinity and ecto-nucleotidase blockade. Among active compounds, apparent purinoceptor affinity and ecto-nucleotidase blockade increase with the size of the molecules up to Evans blue and trypan blue themselves; introduction of a central ethylene bridge does not result in a further gain in potency. NH01, the desmethyl derivative of Evans blue, seems to be interesting because it is the compound with the highest P_2X- versus P_2Y-selectivity presently available.     

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery

1. The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Suramin (1 μM–1 mM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi-effective concentrations of α,β-methyleneATP (α,β-meATP) (5 μM), 2-methylthioATP (2-meSATP) (100 μM) and adenosine 5′-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to α,β-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.
3. Contractions evoked by uridine 5′-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).
4. Desensitization of the P2X₁-receptor by α,β-meATP abolished contractions evoked by 2-meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.
5. Responses to α,β-meATP (5 μM) and 2-meSATP (100 μM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.
6. ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β-meATP (5 μM), 2-meSATP (100 μM) and ATP (1 mM) in a concentration-dependent manner.
7. These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X₁-receptors and G-protein-coupled P2Y-receptors.