Pharmacologic characterization of SK&F 104078, a novel alpha-2 adrenoceptor antagonist which discriminates between pre- and postjunctional alpha-2 adrenoceptors

SK&F 104078 has been reported to be a moderately potent antagonist at postjunctional alpha-2 adrenoceptors in canine saphenous vein, rabbit saphenous vein and canine saphenous artery (KB = 76-150 nM). In contrast, SK&F 104078 has been found to have essentially no affinity for prejunctional alpha-2 adrenoceptors in the guinea pig atrium. To characterize further the pharmacology of SK&F 104078 we have examined its effects in several additional alpha-2 adrenoceptor models and on several non alpha adrenoceptor-mediated vascular responses. SK&F 104078 does not block the neuroinhibitory effect of alpha methylnorepinephrine in the guinea pig ileum. In contrast, in the rat vas deferens, high concentrations of SK&F 104078 (3-30 microM) antagonized the neuroinhibitory effect of UK 14,304; however, the antagonism was not competitive. At concentrations up to 1 microM, SK&F 104078 did not potentiate [3H]overflow from guinea pig vas deferens or guinea pig atrium prelabeled with [3H]norepinephrine, indicating no prejunctional alpha-2 adrenoceptor blocking activity in these tissues. SK&F 104078 is a competitive antagonist at alpha-1 adrenoceptors, and at 5-hydroxytryptamine2 receptors, as demonstrated by blockade of norepinephrine- and serotonin-induced contraction in the rabbit aorta, with KB values of 155 and 20 nM, respectively. At concentrations up to 10 microM, SK&F 104078 does not depress angiotensin II-induced contraction of rabbit aorta. At 1 microM, no depression of the response to Ca++ in depolarized rabbit aorta is observed, although a significant inhibition of this response is seen at 10 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta2-adrenoceptor-mediated prejunctional facilitation and postjunctional inhibition of sympathetic neuroeffector transmission in the guinea pig vas deferens.

This study examines the role of prejunctional and postjunctional beta-adrenoceptors in the modulation of sympathetic cotransmission in the guinea pig vas deferens. The prejunctional involvement of beta-adrenoceptors was evaluated by testing the effects of several agonists and antagonists on the nerve stimulation-evoked overflow of ATP and norepinephrine (NE) from the "in vitro" vas deferens. The nonsubtype-selective beta-adrenoceptor agonist isoproterenol and the beta2-subtype-selective agonist clenbuterol increased, to a similar degree, the overflow of ATP and NE, while the beta1-subtype-selective agonist xamoterol and the beta3-subtype-selective agonist BRL 37 344 had no effect. Pretreatment with ICI 118, 551, a beta2-subtype-selective antagonist, abolished the facilitation of cotransmitter release by isoproterenol and clenbuterol, while the beta1-subtype-selective antagonist atenolol had no effect. Activation of beta-adrenoceptors by either isoproterenol or clenbuterol, but not by xamoterol and BRL 37 344, reduced the amplitude of contractions evoked by exogenously applied ATP. Pretreatment with propranolol or ICI 118, 551, but not atenolol, prevented these inhibitory effects. Isoproterenol in lower concentrations produced dose-dependent reduction of the purinergic but not the adrenergic phase of nerve stimulation-induced contraction of the guinea pig vas deferens. When applied in concentrations greater than 1 microM, isoproterenol, but not clenbuterol, actually produced a concentration-dependent facilitation of contractions evoked by both nerve stimulation and exogenously applied ATP. Antagonists of alpha-adrenoceptors blocked these facilitatory effects. Together, these results demonstrate that beta2-adrenoceptors can influence sympathetic neuroeffector transmission both prejunctionally, where they facilitate equally well the release of sympathetic cotransmitters and postjunctionally, where they inhibit smooth muscle contractions evoked by ATP.     

Reserpine-induced postjunctional supersensitivity in rat vas deferens and caudal artery without changes in alpha adrenergic receptors

Caudal artery and vas deferens from rats treated chronically with reserpine (1 mg/kg/day i.p.) were used to study drug-induced postjunctional supersensitivity in smooth muscle. There was no change in contractile sensitivity of the rat vas deferens after 1 day of reserpine treatment; however, there were similar increases in sensitivity 4 and 7 days after reserpine treatment. The potencies of phenylephrine, methacholine and potassium chloride in causing contraction of the vas deferens were significantly increased by 4.9-, 19.5- and 1.23-fold, respectively, after 7 days of chronic treatment. This treatment also increased the potencies of phenylephrine, serotonin and potassium chloride in causing contraction of rat caudal artery by 1.8-, 1.7- and 1.23-fold, respectively; however, the potency of clonidine was unchanged after 7 days of reserpine treatment. There was no change in sensitivity to phenylephrine 1 day after reserpine treatment but sensitivity was significantly increased after 4 days. There was no significant change in maximum contractile response in either tissue to any of these agents after chronic reserpine treatment. Scatchard analysis of saturation isotherms of specific [125I]BE 2254 binding to membrane fractions from rat vas deferens and caudal artery showed no change in the density or affinity of alpha-1 adrenergic receptors after 1, 4 or 7 days of chronic reserpine treatment. In addition, no change was observed in specific [3H]rauwolscine binding to either tissue after 7 days of chronic reserpine treatment, suggesting no change in alpha-2 adrenergic receptors. These data indicate that changes in alpha adrenergic receptors are not involved in postjunctional supersensitivity of smooth muscle caused by chronic reserpine treatment.     

Effect of capsaicin on smooth muscles of rat vas deferens: involvement of calcitonin gene-related peptide?

The effect of capsaicin on the rat vas deferens was examined in relation to the role of endogenous calcitonin gene-related peptide (CGRP) in modifying the contractility of smooth muscles. Capsaicin attenuated the twitch response of the rat vas deferens induced by the transmural nerve stimulation (TNS) in vitro. The effect of capsaicin was transient and developed tachyphylaxis rapidly. Capsaicin inhibited the contraction induced by the direct electrical stimulation of the innervated tissues in the presence of tetrodotoxin but not of the surgically denervated tissues. CGRP-like immunoreactive nerves were demonstrated to be present in the rat vas deferens. Exogenously applied CGRP inhibited both the TNS-induced twitch response and the contraction induced by direct electrical stimulation. Both capsaicin and CGRP slightly hyperpolarized the resting membrane potential of smooth muscle cells but did not affect the amplitude of the excitatory junction potentials. The intensity of CGRP-like immunoreactivity was reduced markedly after the in vitro incubation of the tissue with capsaicin. No CGRP-like immunoreactive nerves were detected in the denervated tissues. Thus, capsaicin inhibited both the TNS-induced twitch response and the contraction induced by direct stimulation of the smooth muscles only when CGRP-like immunoreactive nerves were normally present. These results suggest that capsaicin releases endogenous CGRP and that the released CGRP inhibits the contraction of the rat vas deferens by acting directly on smooth muscle cells but not on the sympathetic nerves.     

Differential cotransmission in sympathetic nerves: role of frequency of stimulation and prejunctional autoreceptors.

Recent reports have suggested that sympathetic nerves may store separately and release independently the cotransmitters ATP and norepinephrine (NE). It is conceivable therefore that the quantity of each neurotransmitter that is released from the nerves is not fixed but rather may vary, possibly with the frequency of stimulation. To test this hypothesis we studied the concomitant release at various frequencies and cooperative postjunctional actions of ATP and NE during the first 10 s of electrical field stimulation of the guinea pig vas deferens. We found that at lower frequencies (8 Hz), prejunctional inhibition of the release of NE, which occurs via alpha2-adrenoceptors, modulates the ultimate composition of the cocktail of cotransmitters by limiting the amount of NE that is coreleased with ATP. As the frequency of stimulation increases (above 8 Hz), the autoinhibition of the release of NE is overridden and the amount of NE relative to ATP increases. The smooth muscle of the guinea pig vas deferens reacts to changes in composition of the sympathetic neurochemical messages by increasing the amplitude of its contractions due to the enhancement by NE of the contractile responses triggered by ATP. This evidence suggests that the prejunctional alpha2-adrenoceptor may function as a sensor that "reads" the frequency of action potentials produced during a burst of neuronal activity and converts that information into discrete neurochemical messages with varying proportions of cotransmitters. The mechanism for decoding the informational content of these messages is based on the cooperative postjunctional interactions of the participating cotransmitters.     

Opioid receptor selectivity of beta-endorphin in vitro and in vivo: mu, delta and epsilon receptors

The relative contributions of mu and delta opioid receptors in the response to Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe- Lys-Asn - Ala-Ileu-Ileu-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu (B-endorphin) were assessed as reductions in B-endorphin potency in the presence of mu and delta receptor selective antagonists in the guinea pig ileum, mouse vas deferens, rat vas deferens and in analgesic and gastrointestinal transit time tests in mice. We used the nonselective antagonist naloxone, the mu antagonist D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) and the delta antagonist N,N,diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864) in each test system at concentrations that effectively antagonized the respective mu and delta agonists, Tyr-Pro-N-MePhe-D-Pro-NH2 and Tyr-D-Pen-Gly-Phe-D-Pen. In the guinea pig ileum, the inhibitory effects of 1 microM B-endorphin were blocked by 1 microM CTP and 1 microM naloxone, but not by 1 microM ICI 174,864. In the mouse vas deferens, B-endorphin (0.2 microM) was antagonized by 1 microM CTP, 1 microM ICI 174,864 and by 1 microM naloxone. In contrast, in the rat vas deferens, B-endorphin (0.01-1 microM) produced potent inhibitory actions that were blocked by 1 microM naloxone, but not by 1 microM-CTP or by 1 microM ICI 174,864. The mu agonist, Tyr-Pro-N-MePhe-D-Pro-NH2 (0.1-10 microM), like B-endorphin, also had inhibitory actions in the rat vas deferens, but its effects were blocked by 1 microM CTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additional evidence for functional subclassification of alpha-2 adrenoceptors based on a new selective antagonist, SK&F 104856.

SK&F 104856 (2-vinyl-7-chloro-3,4,5,6-tetrahydro-4-methyl-thieno[4,3,2ef][3] benzazepine) shows a similar selectivity profile to the previously reported alpha adrenoceptor antagonist, SK&F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4,5-tetrahydro-3- benzazepine), having the ability to block alpha-1 and postjunctional alpha-2 adrenoceptors, although having little or no activity at most prejunctional alpha-2 adrenoceptors. SK&F 104856 is more potent than SK&F 104078, and lacks the 5-hydroxytryptamine receptor antagonist activity associated with the earlier compound. The postjunctional vs. prejunctional selectivity of SK&F 104856 at alpha-2 adrenoceptors in the same tissue preparation was demonstrated in both canine and human saphenous vein. Concentrations substantially higher than those required to block postjunctional alpha-2 adrenoceptor-agonist induced vasoconstriction had no effect on the ability of norepinephrine, acting on prejunctional alpha-2 adrenoceptors, to inhibit stimulation evoked transmitter overflow in the human tissue, and only a small effect in the canine vein. As observed with SK&F 104078, SK&F 104856 has some prejunctional alpha-2 adrenoceptor antagonist activity in the rat vas deferens, although the receptor dissociation constant is nearly 50-fold higher than that at the postjunctional alpha-2 adrenoceptor in the canine saphenous vein. The results obtained with SK&F 104856 provide additional evidence to support the premise that alpha-2 adrenoceptors can be functionally differentiated. Because SK&F 104856 can selectively antagonize certain alpha-2 adrenoceptor-mediated responses, this agent may be a useful tool to evaluate the functional roles of the multiple alpha-2 adrenoceptor subtypes that have been identified in biochemical and molecular studies.     

Atrial natriuretic factor inhibits adrenergic and purinergic neurotransmission in the rabbit isolated vas deferens

This study tests the hypothesis that atrial natriuretic factor (ANF) acts to inhibit neurotransmission in the rabbit vas deferens. The vas deferens is a unique model of autonomic neurotransmission in that it is composed of primarily nonvascular smooth muscle and has both a purinergic or twitch contraction and an adrenergic or phasic contraction associated with its response to electrical stimulation. In this study ANF was found to inhibit both adrenergic and purinergic neurotransmission in the rabbit vas deferens. ANF inhibited both the electrically induced phasic contraction and electrically induced norepinephrine release in a concentration-dependent manner over the ANF concentration range of 10(-10) to 10(-7) M. ANF at a concentration of 10(-7) M had no effect on norepinephrine-induced or ATP-induced contractions. Therefore, the neuromodulatory effect of ANF in the rabbit vas deferens appears to be prejunctional, on the release of the neurotransmitters norepinephrine and ATP from the nerve terminal and not postjunctional on the smooth muscle. Neither the alpha-2 antagonist rauwolscine nor the cyclooxygenase inhibitor indomethacin had any effect on the inhibitory effect of ANF on electrically induced twitch or phasic contractions. Additionally, ANF did not affect vasa deferentia prostaglandin E production. Therefore, the inhibitory neuromodulatory ANF effect is not mediated via alpha-2 adrenergic receptors or prostaglandin E production. The observed inhibitory neuromodulatory effects in this study may be involved in the hypotensive effects of ANF including natriuresis, diuresis and vasodilation.     

Adenosine affects sympathetic neurotransmission at multiple sites in vivo

We examined the effects of adenosine and its analogs on sympathomimetic responses of pithed rats to electrical stimulation of preganglionic sympathetic nerves (ES) or to injections of nicotine, phenylephrine (PE) or isoproterenol (ISO). Four physiological indices of sympathetic neurotransmission were measured: blood pressure, heart rate and contractions of smooth muscle in vas deferens and eyelid. Elevation of arterial adenosine levels from 1.5 to 2 to 3 microM caused a 2- to 3-fold potentiation of nicotine-induced increases in blood pressure, heart rate and smooth muscle tension. Higher adenosine concentrations (3-4 microM) produced a smaller potentiation of the effects of nicotine. At 2 to 3 microM, adenosine had no effect on sympathomimetic responses to ES or PE. Higher concentrations (3-4 microM) attenuated pressor responses to ES and PE and the contractile responses of the vas deferens to ES; these levels also potentiated positive chronotropic responses to ISO. The adenosine analogs N-cyclopropylcarboxamido adenosine (N-CPCA), 2-chloroadenosine (2-CLA) and R- and S-phenylisopropyl adenosine (R-PIA and S-PIA) also reduced pressor responses to both ES and PE, with the potency order: N-CPCA greater than R-PIA greater than 2-CLA greater than S-PIA. These analogs exhibited this same potency series in attenuating contractile responses to ES in the vas deferens. However, all four analogs potentiated, at the lower doses tested, the contractile response of the vas deferens to PE; at higher concentrations, inhibition predominated. N-CPCA enhanced the chronotropic effects of ISO and ES.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel peptidic mu opioid antagonists: pharmacologic characterization in vitro and in vivo

A series of six synthetic octapeptides, structurally related to somatostatin, demonstrate high affinity and selectivity for mu opioid receptors in radioligand binding assays. The compounds, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), D-tetrahydroisoquinoline carboxylic acid (D-Tic)-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (D-Tic-CTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (D-Tic-CTOP) and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (D-Tic-CTAP), were tested in vitro and in vivo for agonist and antagonist potency and selectivity. In vitro bioassays included the guinea pig ileum, mouse vas deferens and rabbit vas deferens. In vivo tests included hotplate antinociception and gastrointestinal transit inhibition, performed in mice. In vitro, all six derivatives were competitive, highly selective mu antagonists (pA2 values from 6.4-7.9). The compounds demonstrated varying degrees of intrinsic agonist activity especially in the mouse vas deferens, the least active being CTAP and D-Tic-CTAP, which showed no mu or kappa agonist actions, and delta activity only at very high (greater than 3 microM) concentrations. In vivo, none of these compounds showed antinociceptive actions when administered i.c.v. in mice. All were competitive mu antagonists in the hotplate antinociception test.(ABSTRACT TRUNCATED AT 250 WORDS)     

Norepinephrine and potassium induced calcium translocation in rat vas deferens

To understand calcium regulation in smooth muscle, we studied both potassium- and norepinephrine-mediated alterations in the movement of calcium in the smooth muscle of rat vas deferens. We employed 45Ca to measure agonist-mediated calcium influx and efflux, as well as tissue calcium content. In addition we labeled tissues with [3H]myoinositol to measure the effect of norepinephrine on inositol phosphate generation. Stimulation of the vas deferens with 50 mM potassium caused a rapid influx of 45Ca (6-fold). Norepinephrine stimulation, even at a concentration maximal for contraction of the tissue (1 mM), did not result in any alteration in 45Ca influx by itself but inhibited potassium-stimulated 45Ca influx (IC50 = 3 microM). This alpha receptor-mediated effect of norepinephrine was not diminished by either pretreatment with reserpine or adrenergic denervation. Studies of the efflux of 45Ca from vas deferens revealed that efflux was not affected by potassium but was significantly stimulated by norepinephrine. Alpha receptor stimulation of vas deferens smooth muscle caused a marked elevation in the appearance of inositol phosphates, particularly inositol trisphosphate, that was not dependent on extracellular calcium. We conclude that norepinephrine does not stimulate calcium influx in vas deferens smooth muscle but leads to the release of calcium from intracellular stores via formation of inositol trisphosphate and that the resulting increase in intracellular calcium may lead to inactivation of the potential-dependent calcium channel.     

Multiple cellular mechanisms mediate the effect of lobeline on the release of norepinephrine

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished. The effect of lobeline on the basal release was [Ca(2+)]-independent, insensitive to mecamylamine, a nicotinic acetylcholine receptor antagonist, and to desipramine, a noradrenaline uptake inhibitor. However, lobeline-induced release was temperature-dependent: at low temperature (12 degrees C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [(3)H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC(50) values of 1.19 +/- 0.11 and 6.53 +/- 1.37 microM, respectively. Lobeline also inhibited Ca(2+) influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an alpha(1)-adrenoceptor agonist, contracted the smooth muscle of the vas deferens and enhanced stimulation-evoked contraction. Both effects were inhibited by lobeline. Our results can be best explained as a reversal of the monoamine uptake by lobeline that is facilitated by the increased intracellular NE level after lobeline blocks vesicular uptake. At high concentrations, lobeline acts as a nonselective Ca(2+) channel antagonist blocking pre- and postjunctional Ca(2+) channels serving as a counterbalance for the multiple transmitter releasing actions.     

Neuromodulatory effect of the atrial natriuretic factor clearance receptor binding peptide, cANF(4-23)-NH2 in rabbit isolated vasa deferentia.

This study tests the hypothesis that atrial natriuretic factor (ANF) and the ANF clearance receptor binding peptide, cANF(4-23)-NH2 (cANF), inhibit adrenergic and purinergic neurotransmission in the rabbit isolated vas deferens by a pertussis toxin (PTX)-sensitive mechanism. The vas deferens is a unique model used in the study of autonomic neurotransmission inasmuch as it has both a purinergic or twitch contraction and an adrenergic or tonic contraction associated with its response to electrical stimulation. Both ANF and cANF (10(-11) to 10(-6) M) inhibited electrically induced purinergic and adrenergic contractile force generation in a concentration-dependent manner. The ANF effect on both purinergic and adrenergic contractions was blocked by PTX (100 ng/ml). The cANF effect on the adrenergic contraction was also PTX-sensitive. Both peptides also attenuated evoked norepinephrine release in a concentration-dependent manner by a PTX-sensitive mechanism. cANF (10(-7) M) had no effect on norepinephrine- or ATP-induced contractions as was shown previously for ANF (10(-7) M). Therefore, the inhibitory effects of ANF and cANF appear to be prejunctional, on the release of the neurotransmitters, norepinephrine and ATP, from the nerve terminal and not postjunctional on the smooth muscle. An effect of cANF on neurotransmission suggests that the reputed "silent" ANF clearance receptor has biological activity. PTX-sensitivity suggests the involvement of a guanine nucleotide-binding protein in mediating the neuromodulatory effect of atrial peptides.     

Evidence for heterogeneity between pre- and postjunctional alpha-2 adrenoceptors using 9-substituted 3-benzazepines

A series of alpha adrenoceptor antagonists, including both reference compounds and the novel benzazepine antagonists, SK&F 86466 (6-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benzazepine) and two of its 9-substituted derivatives, SK&F 101253 and SK&F 104078, were tested in vitro for affinity at central and peripheral alpha adrenoceptor subtypes. Peripheral alpha-1 adrenoceptor antagonist potency of these agents, as assessed by the receptor dissociation constant (KB) against norepinephrine-induced contraction in the rabbit aorta, correlated with the Ki value for inhibition of [3H]prazosin binding to central alpha-1 adrenoceptors in rat brain homogenates. Central alpha-2 adrenoceptor affinity, measured as the Ki for inhibition of [3H]rauwolscine binding to rat brain homogenates, correlated well with antagonist activity at peripheral postjunctional alpha-2 adrenoceptors as reflected by the KB against B-HT 920-induced contraction in canine saphenous vein. The 9-substituted benzazepines, SK&F 101253 and SK&F 104078, produce preferential blockade of postjunctional vs. prejunctional alpha-2 adrenoceptors in peripheral models. The high affinity of SK&F 104078 for postjunctional alpha-2 adrenoceptors in the canine saphenous vein was confirmed by its ability to inhibit [3H]rauwolscine binding to postjunctional alpha-2 adrenoceptors in this tissue. The observation that the Ki values for these antagonists against [3H] rauwolscine binding correlate with their KB values at the postjunctional alpha-2 adrenoceptors, rather than those at the prejunctional neuroinhibitory alpha-2 adrenoceptor, suggests a pharmacologic similarity between the postjunctional vascular alpha-2 adrenoceptors and the central [3H]rauwolscine binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative assessment of the pre- and postsynaptic alpha adrenoceptor antagonist potency of amitriptyline

The pre- and postsynaptic alpha adrenoceptor blocking affinity of amitriptyline was determined in isolated tissues by Schild regression analysis. In the absence of uptake-1 blockade with cocaine, amitriptyline treatment (3 X 10(-8)-3 X 10(-6) M) affected only marginally norepinephrine concentration-response curves in the rat anococcygeus muscle; a result suggesting opposing pharmacological effects (uptake-1 blockade and alpha blockade). After cocaine (3 X 10(-5) M) treatment, amitriptyline (3 X 10(-8)-3 X 10(-6) M) antagonized competitively concentration-response curves to norepinephrine, yielding a postsynaptic pKb of 7.51. A similar pKb was obtained when methoxamine was the agonist. Presynaptic alpha blocking affinity was determined by using the field-stimulated rat vas deferens. Stimulus conditions were chosen which minimized the inhibition of twitch height by high concentrations of cocaine. Using these conditions (10 Hz, 200 msec duration at 100-sec intervals), amitriptyline antagonized competitively clonidine inhibition of field-stimulated twitch contractions, yielding at pKb of 5.23. The presynaptic pKb was not changed in the presence of theophylline (10(-4) M). Amitriptyline was also observed to increase the release of [3H]norepinephrine from the field-stimulated rat anococcygeus muscle pretreated with cocaine. Although this effect primarily reflects alpha blockade, other biochemical and presynaptic mechanisms may be involved. Comparing the pre- and postsynaptic alpha blocking affinities indicates that amitriptyline has 191 X greater affinity for post- than presynaptic alpha adrenoceptors (i.e., alpha-1 much greater than alpha-2). The relevance of these observations to the mechanism of action and side effects of amitriptyline are discussed.     

CH-38083, a selective, potent antagonist of alpha-2 adrenoceptors

The selectivity and specificity of CH-38083 [7,8-(methylenedioxi)-14-alpha-hydroxyalloberbane HCl], a berbane derivative for alpha adrenoceptors has been studied and compared with yohimbine and idazoxan in peripheral tissues and in the central nervous system. In isolated tissue experiments CH-38083 was a competitive antagonist at presynaptic alpha-2 adrenoceptors on the axon terminals of the rat vas deferens (pA2 against xylazine = 8.17 +/- 0.06) and of the longitudinal muscle strip of guinea pig ileum (pA2 against xylazine = 8.07 +/- 0.20). As far as its postsynaptic alpha-2 adrenoceptor antagonistic activity is concerned its affinity in rat vas deferens (pA2 = 4.95 +/- 0.11) against l-phenylephrine and in rabbit pulmonary artery (pA2 = 5.38 +/- 0.33 against l-norepinephrine) was markedly less than that displayed for presynaptic sites. From pA2 values obtained in rat vas deferens the calculated alpha-1/alpha-2 adrenoceptor selectivity ratios for yohimbine, idazoxan and CH-38083 were 4.7, 117.5 and 1659, respectively. CH-38083 failed to show any affinity for histamine and muscarinic receptors and it even potentiated the effect of serotonin on atropinized longitudinal muscle strip of guinea pig ileum. It enhanced the release of [3H]norepinephrine from electrically stimulated mouse vas deferens loaded previously with labeled [3H]norepinephrine. In binding studies carried out in rat brain membrane preparations using [3H]prazosin and [3H]idazoxan, the selectivity ratios (Ki alpha-1/Ki alpha-2) proved to be 32.5, 289.5 and 1368 for yohimbine, idazoxan and CH-38083, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prejunctional inhibitory effect of a dopamine D-2 agonist, N-0437, on vascular adrenergic responses

N-0437 [2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin], a potent and selective agonist for D-2 dopamine receptors, was used to investigate inhibitory prejunctional dopamine receptors in the rat tail artery and rabbit ear artery. N-0437 inhibited contractile responses to transmural nerve stimulation in a frequency dependent manner. Thus, N-0437 profoundly inhibited responses to nerve stimulation in the rat tail artery at a frequency of 1 Hz (ED50 = 1.6 nM), but had minimal effects when nerves were stimulated at 6 Hz. The D-1/D-2 dopamine agonist, apomorphine, exhibited a similar frequency dependent inhibitory effect but with less potency (ED50 = 30 nM at 1 Hz). In concentrations up to 1 microM, N-0437 had no effect on responses to exogenously applied norepinephrine, but N-0437 inhibited [3H] norepinephrine efflux induced by transmural stimulation. Inhibitory effects of N-0437 were blocked by the D-2 antagonist sulpiride but not by the D-1 antagonist SCH 23390. Furthermore, the selective D-1 agonist SKF 38393 did not inhibit vascular responses to adrenergic nerve stimulation. These data indicate that the inhibitory effects of N-0437 are via activation of D-2 dopamine receptors that inhibit norepinephrine release. Thus, N-0437 shows potency and selectivity of action for prejunctional D-2 dopamine receptors in vascular tissues. The frequency dependence of the actions of N-0437 suggest that the level of sympathetic activity is an important variable in determining effectiveness of prejunctional modulation.     

Nucleotide modulation of norepinephrine release from sympathetic nerves in the rat vas deferens.

The effects of a number of purinoceptor agonists and antagonists on norepinephrine (NE) overflow were examined in the electrically field-stimulated rat vas deferens. The P1 receptor agonists adenosine and 2-chloroadenosine and the P2 receptor agonists ATP and beta, gamma-methylene ATP all reduced the overflow of NE, which was quantified by high-performance liquid chromatography-electrochemical detection techniques. The P1 receptor antagonist 8-(p-sulfophenyl)-theophylline (8-SPT) and the P2 receptor desensitizing agent alpha, beta-methylene ATP blocked the inhibitory effects of both P1 and P2 receptor agonists. The pyrimidine nucleotide UTP also inhibited NE overflow and this effect was antagonized by 8-SPT. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine potentiated and adenosine deaminase blocked the inhibitory effect of adenosine on NE overflow but neither had any effect on the ability of the adenine nucleotides to inhibit NE overflow. These results indicate that adenine nucleotides can act per se, without conversion to adenosine, on a prejunctional receptor to inhibit the release of NE. Because the effects of the adenine nucleotides are antagonized by 8-SPT, it appears that they act at the same receptor as the adenine nucleosides. UTP apparently acts at this receptor as well. These findings suggest that prejunctional purinoceptors on the sympathetic nerves of the rat vas deferens differ from P1 or P2 receptors as usually defined and thus may represent a unique class of receptor (P3) as has been suggested for the prejunctional receptors of the rat caudal artery.     

Inhibitory effect of calcitonin gene-related peptide on excitation and contraction of smooth muscles of the rat vas deferens

Calcitonin gene-related peptide (CGRP) inhibits the twitch response of the rat vas deferens induced by nerve stimulation. Inasmuch as CGRP-like immunoreactive nerves were demonstrated to be present in the rat vas deferens, the effect of CGRP on the contractile activity of the tissue was pharmacologically and electrophysiologically analyzed in vitro. The twitch response of the vas deferens induced by transmural nerve stimulation was inhibited by rat CGRP, porcine CGRP and human CGRP (hCGRP). Both ED50 values and the extent of the maximum inhibition of the twitch response were the same for these CGRP groups. hCGRP (10(-8) to 10(-7) M) hyperpolarized the resting membrane potential of smooth muscle cells but did not affect the amplitude of excitatory junction potentials induced by nerve stimulation. Neither the frequency of occurrence nor the amplitude of spontaneous junction potentials were affected by hCGRP. The threshold potentials for the generation of action potentials were less negative in the presence of hCGRP. hCGRP reduced slightly the contraction of the tissue induced by norepinephrine and by adenosine 5'-triphosphate. hCGRP reduced markedly the contractions induced by direct electrical stimulation of both denervated and normal tissues in the presence of tetrodotoxin. These results indicate that CGRP does not affect the release of a sympathetic neurotransmitter. CGRP appears to attenuate the contraction of the rat vas deferens through suppression of both the excitability and excitation-contraction coupling of the smooth muscle.     

Evaluation of tamsulosin and alfuzosin activity in the rat vas deferens: relevance to ejaculation delays

The effect of two alpha-adrenergic receptor antagonists widely employed in the therapy of benign prostatic hyperplasia, tamsulosin [(-)-(R)-5-[2-[[2-(0-ethoxyphenoxy) ethyl]amino]propyl]-2-methoxybenzenesulfonamide] and alfuzosin [(+/-)-N-[3-[(4-amino-6,7-dimethoxy-2-quinazolinyl) methylamino]propyl] tetrahydro-2-furancarboxamide], was investigated in the rat vas deferens. Because several clinical studies have shown that tamsulosin causes ejaculatory disorders, this study also evaluated the possible mechanisms implicated in these disorders by comparing the effect of tamsulosin with that of alfuzosin. Tamsulosin competitively antagonized the contractions induced by noradrenaline in vitro in the epididymal portion of the vas deferens with a potency pA(2) value of 9.2 +/- 0.8. In the prostatic portion, tamsulosin increased the amplitude of intermittent spikes induced by exogenous noradrenaline (100-1000 microM). In both portions of the vas deferens, alfuzosin behaved as an alpha-adrenergic antagonist blocking the contractions induced by exogenous noradrenaline without altering spikes. The administration of tamsulosin (3 microg/kg i.v.) significantly reduced the contractions evoked by electrical pulses in the epididymal portion, whereas it increased those produced in the prostatic portion. Intravenous tamsulosin antagonized the contraction produced by exogenous noradrenaline, whereas alfuzosin administration (10 microg/kg i.v.) did not change the electrically induced contractions in both portions of the rat vas deferens and did not antagonize the contractions produced by exogenous noradrenaline. The fact that tamsulosin unusually enhances noradrenaline-induced intermittent spike contractions and nerve stimulation-induced twitches in the prostatic portions might be linked to its greater propensity to cause sexual dysfunctions.