Field-stimulated Rat Vas Deferens Research Articles

Differential blockade of pre- and postsynaptic alpha-adrenoceptors by the 2-R and 2-S enantiomers of WB4101.

Agonists and antagonists of the alpha-adrenoceptor have been shown to possess different degrees of potency at the pre- and postsynaptic levels. The present investigation was undertaken to see whether the two optical isomers of WB4101 likewise exhibit differential blockade at the two receptor sites. This was achieved by measuring the potencies of 2-R and 2-S WB4101 against noradrenaline-induced contractions of the rat isolated vas deferens and against clonidine-induced inhibition of the twitch response in field stimulated rat vas deferens. Results showed that though the enantiomers exhibited widely differing potency, as indicated by calculations of pA2 values, at the postsynaptic level, they possessed almost identical potency presynaptically. In addition, 2-aminomethyl-1,4-benzodioxan itself both exhibited reasonable presynaptic alpha-adrenoceptor blocking activity, whilst being devoid of activity postsynaptically. The values for slope on the Schild plots for 2-R and 2-S WB4101 indicated that they produced classical competitive antagonism at the postsynaptic receptor but at the presynaptic level the values for slope were indicative of a non-competitive type of blockade. Results are discussed in terms of recent suggestions of that agonists and antagonists of the alpha-adrenoceptor may not share a common mechanism or site of action at the presynaptic level.

The prejunctional inhibitory effect of alpha-methylnoradrenaline in the rat vas deferens is not mediated via alpha 2-adrenoceptors.

In field-stimulated rat vas deferens, clonidine (10(-9)-10(-7) M) and alpha-methylnoradrenaline (10(-7)-3 x 10(-5) M) concentration-dependently inhibited twitch response to electrical field stimulation. The inhibitory effects of clonidine and alpha-methylnoradrenaline were similarly reduced by phenoxybenzamine (10(-6) M). However, idazoxan (3 x 10(-7) M) antagonized clonidine but not alpha-methylnoradrenaline-induced responses. The inhibitory effect of alpha-methylnoradrenaline was also not antagonized by phentolamine (3 x 10(-7) M), SK&F 104856 (2-vinyl-7-chloro-3,4,5,6-tetrahydro-4-methylthienol[4,3,2 ef][3]benzazepine) 3 x 10(-6) M) or yohimbine 3 x 10(-7) M). These antagonists however, attenuated the twitch-inhibiting effect of clonidine. The -logKB values were 8.02 +/- 0.05, 6.91 +/- 0.10, and 7.58 +/- 0.07 for phentolamine, SK&F 104856 and yohimbine respectively. In-vivo treatment of rats with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (6 mg kg-1), to inactivate prejunctional alpha 2-adrenoceptors, attenuated clonidine, but not alpha-methylnoradrenaline-induced responses. It was concluded that adrenoceptors are not involved in the prejunctional inhibitory effects of alpha-methylnoradrenaline in the rat vas deferens.

Pre-junctional alpha 2-adrenoceptor activity of B-HT920.

An in-vitro study has been carried out on the pre-junctional alpha 2-adrenoceptor activity of the thiazoloazepine derivative B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]-azepine) using field-stimulated rat vas deferens and guinea-pig ileum. The alpha 2-selective agonists clonidine (an imidazoline derivative) and alpha-methyl noradrenaline (a beta-phenethylamine derivative) were compared. Results show that B-HT 920 is a potent agonist on pre-junctional alpha 2-adrenoceptors and is competitively antagonized by the selective alpha 2-adrenoceptor antagonist yohimbine. The characteristics of the pharmacological responses obtained with B-HT920 indicate that it interacts with the receptor in an imidazoline-like, rather than a beta-phenethylamine-like, manner.

Internal calcium stores and norepinephrine overflow from isolated, field stimulated rat vas deferens

Ryanodine has been shown to selectively inhibit the initial phase of contraction of rat vas deferens smooth muscle stimulated by endogenous release of norepinephrine (NE) (1), and part of this effect could be pre-junctional. To assess this, its effect on NE overflow was measured in the same preparation. NE overflow from electrical field-stimulated isolated rat vas deferens was quantified by electrochemical detection using HPLC. In order to limit pre-junctional autoregulatory mechanisms, α 2 -adrenergic receptors were blocked and P 2x purinergic receptors were desensitized. In these experimental conditions, NE overflow was directly proportional to extracellular Ca 2+ concentration. Ryanodine only induced a modest decrease in NE overflow. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum Ca 2+ -ATPase, slightly increased NE overflow but decreased smooth muscle contraction induced by electrical field stimulation. It is concluded that part of the effect of ryanodine on field stimulation-induced contraction may be due to an inhibition of NE release, although the major inhibitory effect of this alkaloid is post- junctional. For CPA, its inhibitory effect on field stimulation-induced contraction is entirely post-junctional. Its effect on NE overflow suggests that, in this preparation, internal Ca 2+ stores could function to accelerate termination of neurotransmitter release by sequestering cytosolic Ca 2+ .

Internal calcium stores and norepinephrine overflow from isolated, field stimulated rat VAS deferens

Ryanodine has been shown to selectively inhibit the initial phase of contraction of rat vas deferens smooth muscle stimulated by endogenous release of norepinephrine (NE) (1), and part of this effect could be pre-junctional. To assess this, its effect on NE overflow was measured in the same preparation. NE overflow from electrical field-stimulated isolated rat vas deferens was quantified by electrochemical detection using HPLC. In order to limit pre-junctional autoregulatory mechanisms, α 2-adrenergic receptors were blocked and P 2x purinergic receptors were desensitized. In these experimental conditions, NE overflow was directly proportional to extracellular Ca 2+ concentration. Ryanodine only induced a modest decrease in NE overflow. Cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum Ca 2+-ATPase, slightly increased NE overflow but decreased smooth muscle contraction induced by electrical field stimulation. It is concluded that part of the effect of ryanodine on field stimulation-induced contraction may be due to an inhibition of NE release, although the major inhibitory effect of this alkaloid is post- junctional. For CPA, its inhibitory effect on field stimulation-induced contraction is entirely post-junctional. Its effect on NE overflow suggests that, in this preparation, internal Ca 2+ stores could function to accelerate termination of neurotransmitter release by sequestering cytosolic Ca 2+.

Calcium modulatory properties of 2,6-dibutylbenzylamine (B25) in rat isolated vas deferens, cardiac and smooth muscle preparations.

1. In rat isolated vas deferens the new compound 2,6-dibutylbenzylamine (B25) evoked a series of repeating rhythmic contractions. Concentration-response curves constructed for this effect were bell-shaped, indicating a biphasic effect for this compound. By contrast, B25 depressed heart contractility without any visible positive inotropic or chronotropic activity. 2. Experiments with tetrodotoxin, reserpine, capsaicin, alpha-adrenoceptor blocking compounds and other agents permit us to exclude a release of neuromediators or a direct stimulation of post-synaptic receptors to account for the rhythmic effect of B25 in the rat vas deferens. 3. In the same tissue, the increase in 45Ca2+ uptake, the voltage-dependency as well as the dependence of the B25-induced rhythmic activity upon the external calcium concentration indicate a direct activation of voltage-sensitive calcium channels (VSCC). 4. Verapamil paradoxically stimulated the rhythmic effect of B25 in the rat vas deferens. La3+ was inactive while nifedipine was a weak inhibitor. By contrast Ni2+ and Mn2+ ions were good inhibitors (IC50 < 10(-4) M), suggesting that a possible opening of T-type VSCC underlies rhythmic effect of B25. 5. In radioligand binding studies competition experiments with [3H]-nitrendipine indicated that only at high concentrations was B25 able to interact with dihydropyridine-sensitive binding sites of heart and vas deferens smooth muscle. 6. B25 (3-30 microM) counteracted the inhibitory effects of omega-conotoxin GVIA in field-stimulated rat vas deferens.

Norepinephrine release in rat vas deferens: Effect of rauwolscine and BHT 920

Norepinephrine (NE) overflow from field-stimulated rat vas deferens preparations was quantified directly by electrochemical detection using high performance liquid chromatography. The effect of agonist (BHT 920) and antagonist (rauwolscine) of prejunctional α2- adrenoceptors on NE overflow was assessed and compared with their effect on the smooth muscle mechanical response to field stimulation. Increasing the stimulation frequency from 2 to 30 Hz resulted in an increase in muscle tension together with an increase in NE overflow. Addition of 1 μM rauwolscine to the medium resulted in a significant increase in muscle contraction to field stimulation which reached a maximum at 5 Hz. On the other hand, NE overflow increased linearly with the frequency of stimulation within the range studied. Addition of 0.1 μM BHT 920 to the medium significantly decreased the amplitude of contractions at lower stimulation frequencies (2 to 10 Hz) but elicited no significant changes at high frequencies. BHT 920 did not significantly affect NE overflow for all range of stimulation frequency. The simultaneous recording of field-stimulation induced contractions and NE overflow indicates that in the rat vas deferens, rauwolscine acts like a pure α2 adrenoceptor antagonist at a prejunctional level. BHT 920 did not appear to affect selectively prejunctional α2 adrenoceptors but also may activate postjunctional α1 adrenoceptors.

Comparison of the effects of endothelin-1 and Bay k 8644 on twitch contractions of the field-stimulated rat vas deferens

Endothelin-1 (ET-1) potentiated both the fast and the slow components of the twitches of the isolated field-stimulated rat vas deferens, whereas Bay k 8644 potentiated only the fast component. Nicardipine tended to decrease the potentiation. ET-1, but not Bay k 8644, caused membrane depolarization and resting tension elevation, which were suppressed by nicardipine. The excitatory junction potentials (EJPs) were augmented by Bay k 8644 and the effect was not changed by nicardipine. ET-1, however, apparently did not augment the EJPs, because of the accompanying depolarization. These results indicate a difference in mechanism of the potentiating effects of ET-1 and Bay k 8644.

Involvement of calcium ions in the modulation of electrically-induced rat vas deferens contractions by histaminergic drugs

1. 1. The modulatory action of histaminergic drugs: histamine; 2-(2-aminoethyl)thiazole (H 1-agonist); dimaprit (H 2-agonist); diphenhydramine (H 1-receptor antagonist); and cimetidine (H 2-receptor antagonist) on rat vas deferens contractions induced by electrical field stimulation (0.1 Hz, 1 pulse, 1 msec duration, supramaximal voltage) was studied either at different calcium concentrations in the nutrient fluid or after verapamil. 2. 2. Histamine and 2-(2-aminoethyl)thiazole inhibited the electrically-evoked contractions (EEC). This effect was unchanged after verapamil. 3. 3. Diphenhydramine potentiated the EEC. Verpamil added before the H 1-receptor antagonist inhibited this action. 4. 4. The effects of agonists and antagonists of H-receptors decreased with the increasing of calcium concentration. 5. 5. The results obtained suggested the existence of heterogeneity of prejunctional H-receptors. The function of histaminergic drugs on adrenergic neurotransmission in field stimulated rat vas deferens is Ca 2+-dependent.

Structure-function studies on neuropeptide Y and pancreatic polypeptide — evidence for two PP-fold receptors in vas deferens

The biological effects of neuropeptide Y (NPY), rat pancreatic polypeptide (rPP), hybrid analogs of NPY and PP, and C-terminal fragments of NPY were studied in the field-stimulated rat vas deferens model. The results were correlated with paptide binding experiments in Y 1 and PP receptor assays on rat PC-12 cells and Y 2 receptors on porcine hippocampal membranes. NPY and rPP inhibited the electrically induced contractions in the vas deferens with an IC 50 of 25 and 22 nM respectively. However, in contrast to NPY, rPP could not totally block muscle activity. The inhibitory action of the long C-terminal fragments of NPY, NPY-(19–36) and NPY-(11–36), indicated that NPY acts through a Y 2 receptor in the vas deferens. The structural basis for the differential recognition of NPY and PP by Y 2 receptors and partly also by PP receptors, could be defined with hybrid analogs of PP and NPY. The analogs, [Ile 31,Gln 34]PP and [Leu 31,Pro 33]NPY reacted in the vas deferens preparation in accordance with their relative potency in the Y 2 and PP receptor assays. [Ile 31,Gln 34]PP, which bound to the Y 2 receptor like NPY, was also able to block the part of the contractile response which was resistant to rPP. It is concluded that in the vas deferens, PP-fold peptides act through two types of receptors: Y 2 and PP, and that residues in the C-terminal part of the molecules determine the differential recognition of the peptides by these receptor types.

P-583 - Potentiating effects of endothelin and Bay k 8644 on twitch contractions of the field-stimulated rat vas deferens

P-583 - Potentiating effects of endothelin and Bay k 8644 on twitch contractions of the field-stimulated rat vas deferens

Agonists at presynaptic receptors on sympathetic nerves differentially affect two phases of the contractile response in the rat vas deferens

A series of adrenoceptor agonists were investigated for their prejunctional effects on field stimulated rat vas deferens. Tissues were stimulated in 10 s trains of impulses, frequency 10 Hz, every 100 s. This produced a biphasic response comprising an initial twitch followed by a prolonged, plateau phase of contraction. The order of potency for a series of alpha 2-agonists against the twitch phase of contraction was UK14304 greater than clonidine greater than noradrenaline = alpha-methyl noradrenaline greater than B-HT920. The same order of potency was observed against the plateau phase, but approximately 10 fold higher concentrations of agonist were needed. Surprisingly, B-HT920 was inactive against the plateau phase of contraction. Characteristic differences in the slopes and maximum responses of the dose-response curves to the imidazolines (UK14304 and clonidine) and the beta-phenethylamines (noradrenaline and alpha-methyl noradrenaline) were seen against both phases of contraction. It is concluded that the two phases of contraction are influenced by activation of two distinct heterogeneous populations of prejunctional alpha 2-adrenoceptors.

Lack of evidence for ε-opioid receptors in the rat vas deferens

Experiments were performed to test the hypothesis that the field-stimulated rat vas deferens preparation contains opioid receptors, other than of μ-type, which mediate part of the inhibitory effect of β-endorphin. The Piebald Viral Glaxo strain of rats was used. The reported finding that δ-opioid receptors are present in Sprague-Dawley rat was deferens, the effects of which are greatly enhanced in reduced calcium concentrations, could not be replicated in the rat strain used. Reducing the calcium concentration from 2.5 to 1.25 mM improved the response to opioid drugs: all full agonists were about 10 times more potent, the partial agonist normorphine became able to inhibit the twitch completely, and morphine (which behaves as a competitive antagonist in 2.5 mM Ca 2+) appeared to behave as a partial agonist. The pA 2 values for antagonism by naloxone in low calcium of the μ-selective peptide [D-Ala 2, M MePhe 4, Gly(ol) 5] enkephalin and other μ- or δ-selective agonist were consistent with an action at μ-receptors only. The value for β-endorphin was slightly but significantly lower. A similar small discrepancy was found with two other competitive antagonists. The discrepancy remained in the presence of peptidase inhibitors thiorphan, bestatin and bacitracin. Responses to both [D-Ala 2, MePhe 4, Gly(ol) 5]enkephalin and β-endorphin were attenuated by the irreversible antagonists β-funaltrexamine and β-chlornaltrexamine. Co-incubation with [D-Ala 2,MePhe 4, Gly(ol) 5) enkephalin or Mr2266 protected the responses to [D-Ala 2, MePhe 4, Gly(ol) 5]enkephalin and β-endorphin from blockade by β-chlornaltrexamine, and analysis of the curves by the double-reciprocal null method showed that the irreversible antagonists had blocked the same proportion of the receptors for β-endorphin as for [D-Ala 2, MePhe 4, Gly(ol) 5]enkephalin, both in protected and unprotected tissues. The simplest explanation of these results is that β-endorphin and [D-Ala 2, MePhe 4, Gly(ol) 5]enkephalin act at the same receptor, which has the characteristics of a μ-opioid receptor.

Specific [ 3H]propionyl-neuropeptide Y (NPY) binding in rabbit aortic membranes: Comparisons with binding in rat brain and biological responses in rat vas deferens

The binding of biologically active [3H]propionyl-NPY to rabbit aortic membranes was specific and saturable. Scatchard analysis indicated a single class of binding sites with a Kd of 1.1 nM. The rank order of potencies for displacement of [3H]propionyl-NPY binding by NPY analogs in the aorta correlated with their potencies in displacing binding in brain and their activity in inhibiting contractions of the field-stimulated rat vas deferens. However, differences were noted in the absolute or relative potencies of other related polypeptides both in regards to aorta compared to brain NPY binding and NPY binding compared to activity in the vas deferens. Collectively, the results support proposals for heterogeneity of NPY receptors.

2-Amino- and 2-guanidino-1,2,3,4-tetrahydro-1,4-epoxynaphthalenes as conformationally defined analogues of alpha-adrenergic agents.

The exo- and endo-2-amino-5,8-dimethoxy-1,2,3,4-tetrahydro-1,4-epoxynaphthalenes++ + (3b and 4b, respectively) were prepared and evaluated as conformationally defined analogues of the alpha 1-agonist methoxamine. Only compound 3b exhibited significant alpha 1-agonist activity in the field stimulated rat vas deferens assay. Since 3b closely approximates the antiperiplanar form of (1R,2S)-(-)-erythro-methoxamine, the results suggest that methoxamine interacts with the alpha 1-adrenoceptor in the trans extended form. The exo-guanidino derivative 5 was found to be a partial alpha 1-agonist. Among the exo- and endo-2-amino-1,2,3,4-tetrahydro-1,4-epoxynaphthalenes (3a and 4a, respectively) prepared as rigid analogues of norephedrine, compound 3a possessed agonist activity at both alpha 1- and alpha 2-adrenoceptors, whereas 4a was inactive at either receptor.

An endogenous brain substance, CDS (clonidine-displacing-substance), inhibits the twitch response of rat vas deferens

The effect of CDS, an endogenous brain substance that specifically displaces bound [ 3H]clonidine and [ 3H]rauwolscine in rat brain membranes and human platelets, has been tested in isolated, field-stimulated rat vas deferens. CDS, obtained after an extensive purification procedure as a single peak from an HPLC sizing column, inhibited the electrically stimulated rat vas deferens similarly to the inhibitory action of clonidine, an α 2-agonist. The effective dose of CDS as an inhibitor of the vas deferens is equivalent to its effective dose in displacing specifically bound [ 3H]clonidine in rat brain membranes. Furthermore, the CDS inhibition of the twitch response is reversed by two α 2-adrenergic antagonists, yohimbine and phentolamine. From these results, it is suggested that CDS extracted from brain, with affinity for clonidine sites, may be involved in the non-adrenergic fast response of the sympathetic transmission of the vas deferens.

Neuropeptide Y (NPY) binding sites in rat brain labeled with 125I-Bolton-Hunter NPY: Comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens

The binding of biologically active 125I-Bolton-Hunter (BH)-NPY to rat brain membranes was saturable and reversible and regulated by inorganic cations and guanyl nucleotides consistent with other neurotransmitter receptor systems. The concentration of specific 125I-NPY binding differed in various brain regions, being highest in the hippocampus and lowest in the cerebellum. Scatchard analysis of 125I-NPY binding showed a single class of receptor sites with a K d = 0.1 nM and B max of 3 pmole/g tissue in hippocampus. Peptide YY, porcine and human NPY inhibited the specific 125I-BH-NPY binding with IC 50 values of 50–120 pM. In contrast, human NPY free acid and pancreatic polypeptides from human (HPP), rat (RPP) and avian (APP) sources were much weaker (IC 50 ≥ 300 nM). The rank order of potencies for NPY analogs and the inactivity of APP and HPP fragment (31–36) on brain binding appeared to correlate with their relative activities in inhibiting contractions of the field-stimulated rat vas deferens. However, PYY, HPP and RPP exhibited activity in the field-stimulated rat vas deferens indicative of a possible action upon sites distinct from the brain NPY binding site.

Hypothermia-induced supersensitivity to adenosine for responses mediated via A1-receptors but not A2-receptors.

Four isolated tissues were examined in which the responses to adenosine are mediated via either A1- or A2-receptors. The responses examined were the inhibition of cholinergic transmission of field-stimulated guinea-pig ileum (A1), inhibition of noradrenergic transmission of field-stimulated rat vas deferens (A1), inhibition of developed tension of rat paced left atria (A1) and relaxation of carbachol-contracted guinea-pig trachea (A2). Cumulative concentration-response curves for adenosine and 2-chloroadenosine were constructed at 37, 30 or 27 degrees C. When plotted as a percentage of the maximum response, the concentration-response curves were displaced to the left by cooling in the ileum, vas deferens and atria, indicative of supersensitivity. This increase in sensitivity does not arise from inhibition of uptake or deamination by cooling, since it occurs equally for adenosine and 2-chloroadenosine, the latter being immune to these processes. In contrast, the sensitivity of the trachea was not affected (2-chloroadenosine) or reduced (adenosine) by cooling. Thus responses mediated via adenosine receptors of the A1 subtype exhibit hypothermia-induced supersensitivity, whereas those mediated via A2-receptors do not. This suggests a fundamental temperature-dependent difference between the two adenosine receptor subtypes.

Effect of verapamil on the non-adrenergic response of the field stimulated rat vas deferens

A comparison has been made, in the present study, between the effects of verapamil (reported to have smooth muscle depolarizing action) and K+ depolarization on the responses of noradrenaline, ATP and those of field stimulation on the vas deferens obtained from reserpinized rats. Field stimulation of the vas using single pulse (1 ms pulse width; supramaximal voltage) resulted in a fast twitch response reaching a maximum at 300 +/- 20 ms. Verapamil (6 X 10(-6) M) significantly potentiated this response. Verapamil potentiated the twitch component of the biphasic response resulting from field stimulation of the intrinsic nerves with repetitive pulses, while the tonic component was markedly inhibited. Verapamil enhanced the ATP (7 X 10(-5) M) response, while the phasic and tonic components of KCl (5.36 X 10(-2) M)-induced biphasic responses were nearly abolished. While the phasic component of the noradrenaline (7 X 10(-6) M) response remained unaltered in the presence of verapamil, the tonic component was markedly inhibited and rhythmicity following phasic component was markedly enhanced. Partial depolarization, achieved by increasing K+ concentration in the normal Krebs by two-fold i.e., to 11.8 mM, enhanced the responses of ATP, noradrenaline and the twitch resulted from the single pulse stimulation. The finding that verapamil potentiates the contractile response to exogenously applied ATP, which is believed to be the "noradrenergic" neurotransmitter in the vas deferens, suggests that this is the mechanism through which verapamil potentiates the twitch responses to field stimulation of the nerve supply.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-Adrenergic activities of some substituted 2-(aminomethyl)imidazolines.

A series of 2-(aminomethyl)imidazolines related to the alpha-adrenergic antagonist phentolamine was prepared and evaluated for alpha-adrenergic agonist and antagonist activities in the isolated, field-stimulated rat vas deferens. Affinities for alpha-adrenergic receptors were determined by displacement of [3H]clonidine and [3H]prazosin from membrane binding sites of calf cerebral cortex. This series provided a variety of alpha-adrenergic profiles, with some of the (aminomethyl)imidazolines being nonselective alpha 1- and alpha 2-adrenergic antagonists like phentolamine, while others were either nonselective alpha 1- and alpha 2-agonists or mixed alpha 1-agonists/alpha 2-antagonists.