A pharmacological investigation of the biphasic nature of the contractile response of rabbit and rat vas deferens to field stimulation

It has been demonstrated previously with the vas deferens of the guinea-pig that the first and second phases of the contractile response to motor nerve stimulation are preferentially antagonized by the P₂-purinoceptor antagonist arylazido aminopropionyl ATP (ANAPP₃), and the α₁-adrenoceptor antagonist prazosin, respectively. We have now investigated the effect of the two antagonists on the biphasic contraction in the vas deferens of two other species; rabbit and rat. ANAPP₃, in a concentration which antagonized responses to exogenously applied ATP but not those to exogenous norepinephrine, preferentially reduced the initial phasic response of the rabbit vas deferens to motor nerve stimulation without significantly reducing the secondary, tonic phase of the response. Prazosin had the opposite effect; antagonizing the response to norepinephrine but not to ATP and reducing the tonic response to motor nerve stimulation without significantly reducing the initial phasic response. Results obtained with the rat vas deferens were similar. The present results combined with previous findings suggest that ATP and norepinephrine act as cotransmitters in the vas deferens of several species.     

Characterization of postjunctional alpha-adrenoceptors in the isolated aorta of the snake Bothrops jararaca

1. To characterize the alpha-adrenoceptors present in Bothrops jararaca aorta, selective alpha-adrenoceptor agonists and antagonists were used.2. The relative order of potency of the tested agonists was noradrenaline > phenylephrine > clonicline.3. Prazosin was more potent than phentolamine and yohimbine in antagonizing noradrenaline response, since the PA₂ values were 9.91, 7.59 and 7.33, respectively.4. Yohimbine was also able to block the contraction produced by phenylephrine, an alpha-1 agonist.5. These results suggest the existence of an alpha-1 subtype of adrenoceptor in this preparation which, however, presents some different characteristics from those described for mammals.     

Differential contractile response of normal vas deferens of rodents in correlation to their calcium and electrolyte levels

The contractile pattern of the vas deferens in three different rodents, rat, guinea pig and mouse was studied in response to adrenaline and noradrenaline. The left vas deferens of rat was more responsive to the graded doses of adrenaline and noradrenaline than the right. The same was also true for guinea pig and mouse vas deferens. This differential response has been correlated with the greater concentrations of calcium and sodium in the right vas deferens in rats and guinea pigs and it might also be related to the levels of membrane-bound and intracellular calmodulin-bound calcium. It is suggested that the left vas deferens might possess more calmodulin-bound calcium than the right, which might have instead, more membrane-bound calcium.     

The effect of cocaine on kinetics of α<Subscript>1</Subscript>-adrenergic contractile response in different portions of the rat vas deferens

The effect of cocaine (10 μM) on the kinetics of contractile response to noradrenaline (NA) was studied in the rat epididymal and prostatic vas deferens. Cocaine caused an acute increase in vas deferens adrenergic sensitivity primarily due to blockage of NA neuronal capture. The presynaptic action prevailed in the epididymal half: the EC 50 value decreased 32-fold with a slight increase of the maximum adrenergic response more evident in the prostatic half. In the presence of cocaine, the prostatic contraction to NA was mediated not by a single pool of α₁-adrenoceptors like in epididymal vas deferens but by two. Its high affinity pool had the same affinity as α₁-adrenoceptors of the epididymal half, the affinity value of the low one was 36-fold less, and the total maximal response of both pools increased 4.5-fold. The differences in cocaine effect on the rat epididymal and prostatic vas deferens contractions to NA appear to be caused by the different sources of Ca²⁺ involved in these responses.     

A radiolabeled-ligand-binding technique for the characterization of opioid receptors in the intact mouse vas deferens

The mouse vas deferens has served as a useful bioassay for examining the properties of opiate receptor subtypes. However, recent data indicate that the response of the vas deferens to opiates may be mediated by one or more of the several opiate receptors found in this preparation. Although a number of techniques can be utilized to assess the relative contribution of these receptors to the response of the mouse vas deferens to opiates (e.g., selective tolerance and naloxone antagonism studies), a radiolabeled-binding technique would provide an independent means of more completely characterizing the opiate receptor profiles in this preparation. Up to the present, however, there has been only limited success in developing a binding assay utilizing crude membrane fractions of the mouse vas deferens. To circumvent these problems, we have developed a binding technique utilizing the intact vas deferens. In contrast to results obtained with membrane fractions, we found highly specific (90–95%) and saturable binding of d-[2-³H]alanine, 5-d-leucine enkephalin, a ligand selective for delta opiate receptors, to the intact vas. Scatchard analyses indicated a single class of binding sites with an apparent K_d of 1.5 nm and a B_max of approximately 12 pmol/2 vas. The selectivity of binding was also examined. Naltrexone was 40 times less potent than unlabeled 2-d-alanine, 5-d-leucine enkephalin in displacing binding, whereas morphine and ethylketocyclazocine were 300 and 500 times less effective, respectively. This technique, coupled with the mouse vas deferens bioassay, should provide a more complete characterization of opioid receptor populations than has heretofore been possible.     

Action of colchicine on cholinergic and adrenergic mechanisms in guinea pig vas deferens

Guinea pig vas deferens responds to externally applied acetylcholine (ACh) or noradrenaline (NA) by a small rapid contraction (phasi phase) and then a large contraction (tonic phase). The phasic phase was not affected by removal of external Ca²⁺, but tonic phase depended on external Ca²⁺. At lower temperatures the two components became larger and detectable separately. The tonic phase induced by ACh at low temperature (at 20°C) was greatly depressed by brief treatment with colchicine (0.5 μM – 5 μM), although the tonic phase at high temperature (at 37°C) was not affected. Na-induced contraction (phasic or tonic phase) was not changed by the colchicine-treatment. High K⁺ (40 mM)-contracture, which in many cases consisted of a single phase and depended on external Ca²⁺, was also not affected by brief treatment with colchicine. Culture of vas deferens for 3 days in the presence of colchicine, increased the phasic phase of ACh- and NA-induced contractions significantly, but reduced the tonic phase of contractions induced by ACh and NA. Colchicine also reduced high K⁺-contracture, the decrease depending on the period of culture with colchicine. Organ culture with colchicine did not affect the amounts of m-ACh and α-Ad receptors or the IC₅₀ value of ACh and NA on ³H-ligand binding. These results suggest that colchicine specifically interacts with some steps in m-ACh and α-Ad receptor-responsor (e.g. ionophore) coupling without affecting the receptor number or affinity of the receptors for agonists. The mechanisms of action of colchicine are discussed in relation to m-ACh and α-Ad receptor functions.     

Neurochemical evidence for two types of presynaptic alpha2-adrenoceptors

Neurochemical and pharmacological evidence has been obtained that noradrenergic varicosities (in mouse and rat vas deferens) and cholinergic varicosities (in the Auerbach's plexus) contain heterogenous alpha₂-adrenoceptors through which the release of [³H]noradrenaline and [³H]acetylcholine can be modulated. The quantitative data also support the hypothesis that different noradrenaline and xylazine sensitive alpha₂-adrenoceptors are present prejunctionally in the vas deferens and Auerbach's plexus preparations. Prazosin, although it has a presynaptic inhibitory effect on alpha₂-adrenoceptors of noradrenergic axon terminals, has no effect on cholinergic axon terminals. These data suggest that there are two different types of alpha₂-adrenoceptors at the presynaptic axon terminals.Special Issue Dedicated to Dr. Abel Lajtha     

Effects of endothelins on nerve-mediated contractions of the mouse vas deferens

The effects of 3 endothelins (ETs) on sympathetic nerve-mediated responses were investigated in the mouse isolated vas deferens. ET-1, ET-2 and, to a lesser extent, ET-3 (0.3–30 nM) caused marked and sustained potentiation of responses to field stimulation at 0.1 Hz, but had little effects, if any, on baseline tension. Incubation with nicardipine (30 nM) strongly inhibited the development of twitch potentiation by the ETs. Twitches potentiated beforehand by ET-1 (10 nM) displayed marked resistance to inhibition by nicardipine, so that 10 μM of nicardipine only reversed part of the effect of ET-1. ET-1 also enhanced both components of the response to high frequency field stimulation (2 to 16 Hz) and contractions induced by submaximal concentrations of noradrenaline, ATP or KCl. All effects of ET-1 were mimicked by Bay K 8644, an activator of L-type Ca++ channels. It is concluded that ETs increase the efficacy of sympathetic neurotransmission in the mouse vas deferens by, at least in part, a postjunctional mechanism which involves activation of L-type Ca⁺⁺ channels.     

Development of clonidine-tolerance in the rat vas deferens: Cross tolerance to other presynaptic inhibitory agents

Possibility of the development of clonidine-tolerance in the peripheral nervous tissue was examined using vas deferens isolated from rats chronically treated with clonidine. Rats were treated with clonidine for 10 days by adding the drug to drinking water (10 μg/ml). For the control rats, drug-free tap water was provided. Electrically evoked twitch response of vas deferens was suppressed by adenosine, β-endorphine and α₂-adrenergic agonists, such as clonidine and B-HT 933, both in control and clonidine-treated groups. Vas deferens isolated from clonidine-treated rats showed significantly lower responsiveness to the inhibitory effects of clonidine and B-HT 933 compared to those from control rats. Vas deferens from clonidine-treated rats also was less responsive to adenosine and β-endorphin, both of which interact with presynaptic inhibitory receptors other than α₂-adrenergic and muscarinic cholinergic stimulation responsiveness of the postsynaptic smooth muscle to both α-adrenergic and muscarinic cholinergic stimulation did not change after 10 days of treatment with clonidine. These results suggest that clonidinetolerance can be induced in the peripheral nervous system by chronic treatment of this drug and that the tolerance is not specific to α₂-adrenergic agonists. Some common pathway in the inhibitory mechanisms of various agents or possible interactions between the different types of presynaptic inhibitory receptors may be involved in this phenomenon.     

Effect of cocaine on the contractile response to noradrenaline in the epididymal and prostatic regions of the vas deferens

In the prostatic half of the rat vas deferens, the response to noradrenaline under the cocaine effect revealed a phasic and a tonic components, whereas in the epididymal portion of the vas deferens there only occurred the tonic component. Cocaine increased the maximal tonic contractile response to noradrenaline in the epididymal portion and the maximal phasic response--in the prostatic one. Mechanisms of direct postsynaptic action of cocaine are discussed.     

Tetrapeptide-amide analogues of enkephalin: the role of C-terminus in determining the character of opioid activity

The opioid activities of tetrapeptide-amide analogues of enkephalin /H-Tyr-D-Met-Gly-Phe-NH₂; H-Tyr-D-Nle-Gly-Phe-NH₂/ were studied in isolated, electrically stimulated longitudinal muscle strip of guinea-pig ileum and mouse vas deferens preparations in vitro and in vivo in the rat tail-flick test. Their effects were compared to those of the parent L-Pro⁵-NH₂ containing analogues, and to other enkephalin derivatives substituted with D-Met in position 2 and L-amino/imino acids of different character in position 5. It was found that whilst the opioid receptor in mouse vas deferens preferred aliphatic residues of acidic character at the C-terminus of pentapeptides and was highly sensitive to the removal of C-terminal amino acid, the other systems were either much less responsive to these changes, or the effects were opposite to those found in mouse vas deferens.     

Intracellular effect of β3-adrenoceptor agonist Carazolol on skeletal muscle,a direct interaction with SERCA

Carazolol (CZL) is a known agonist of β3 and antagonist of β1 and β2 adrenoceptors (AR), used in the animal production industry to improve meat quality by reducing animal stress and skeletal muscle (SM) proteolysis. Here we sought to better understand the direct effect CZL has on SM. We study CZL effect on calcium (Ca²⁺) regulation by enzymatic activity kinetics of the Ca²⁺-ATPase (SERCA), in isolated sarcoplasmic reticulum (SR) from SM and on the mechanical properties of isolated muscle. In isolated SR from SM previously incubated with 0.03 mM CZL, but absent during SR isolation and during SERCA activity determination, the activity was reduced by 45%. Thermal analysis of SERCA activity with CZL shifted the transition temperature of inactivation (T_i) from T_i = 47 to 44 °C. When isolated SR from fast and slow SM was exposed to CZL, inhibition of SERCA occurred in a dose dependent manner. Slow and fast SM T_i of SERCA shifted to a lower temperature in the presence of CZL and a second transition appears at temperatures <40 °C. In isolated extensor digitorum longus (EDL) and soleus muscles, CZL reduces the contraction force and increases susceptibility to fatigue. However, recovery force after fatigue in either muscle was higher. Our results suggest that Carazolol penetrates the plasma membrane and interacts with SERCA, thus having an important effect on skeletal muscle function. The inhibition of SERCA may lead to a decrement in SR Ca²⁺-release promoting further failure in muscle contraction.     

The effect of caesium on adrenergic transmission in rabbit vas deferens.

The effect of caesium on the responses of rabbit vas deferens to transmural stimulation was investigated. The tissue responded to transmural stimulation with a phasic spike contraction followed bya sustained contractile response. The sustained response was inhibited by phentolamine and guanethidine and thus apparently results from noradrenaline release from adrenergic nerves. Addition of 2-5mM Cs+ greatly potentiated this secondary response without altering the sensitivity of the tissue to added (minus)-noradrenaline. This potentiation was not due to Cs+ decreasing the neuronal uptake of noradrenaline, or by Cs+ altering prostaglandin synthesis. Addition of 2mM Cs+ significantly increased the amount of (plus or minus)-[3-H] metaraminol released from tissues in response to transmural stimulation (5 Hz). It is suggested that caesium potentiated responses of rabbit vas deferens to transmural stimulation by increasing the amount of transmitter released per nerve impulse, possibly as a result of prolongation of the action potential.     

Comparative study on the effect of morphine and the opioid-like peptides in the vas deferens of rodents: species and strain differences, evidence for multiple opiate receptors

The effect of an opiate alkaloid and an opioid-like peptide was studied on the electrically evoked twitching of the vas deferens of 3 common laboratory rodents. Normorphine and the synthetic opioid peptide D-Alanine₂ methionine enkephalinamide (D-Ala₂) produced dose dependent inhibitions of the twitching response in the mouse vas deferens. In the rat vas, while β-endorphin (β-EP) caused an inhibitory effect in three strains of rats to a similar degree, morphine produced a dose related enhancement of the twitching. In the guinea pig, both morphine and β-EP caused an increased in the muscular twitch. The results are interpreted in terms of an heterogenous mixture of opiate receptors present in the vas deferens from these rodents. The mouse appears to contain mainly δ receptors while the rat has mostly ε receptors characterized by their specificity and sensitivity to the action of β-EP. The guinea pig vas deferens has apparently lost the sensitivity to the inhibitory influence of the opioids, suggesting the absence of μ or δ opiate receptors in this tissue.     

The effects of the ionophore A-23187 on the rat vas deferens

The calcium ionophore A-23187 induced spontaneous, rhythmic contractions in the rat isolated vas deferens in a concentration-dependent manner. Contractions were blocked by phentolamine and were abolished following pretreatment with reserpine. In tissues preloaded with [3H]noradrenaline, A-23187 (10 microM) caused a time-dependent increase in the release of tritium. The findings suggest that A-23187-induced contractions in the rat vas deferens are secondary to the release of endogenous noradrenaline from the adrenergic nerves, as are contractions induced in this preparation by X-537A (another calcium ionophore) described earlier by other investigators.     

Inhibitory effect of prostaglandin E2 on noradrenaline secretion from sympathetic nerves as a function of external calcium

The calcium dependence of the inhibitory effect of prostaglandin E₂ on sympathetic neurotransmitter secretion was studied in isolated superfused field stimulated guinea-pig vas deferens, by determination of the nerve stimulation induced rise in efflux of total tritium after preincubation of the tissue with tritium-labelled 1-noradrenaline. Reuptake of noradrenaline was blocked by desipramine and normetanephrine, added to the medium. Reduction of the calcium in the superfusion medium strongly depressed the nerve stimulation induced rise in efflux of tritium. Addition of the alpha-adrenoceptor blocking drug phentolamine markedly raised the efflux of tritium on nerve stimulation, even at low calcium. The inhibitory effect of prostaglandin E₂ on nerve stimulation induced rise in efflux of tritium was inversely related to the calcium concentration. The results are consistent with the possibility that prostaglandin E₂ depresses neural secretion by facilitating efflux of intraaxonal calcium.     

Instantaneous reactions of guinea-pig vas deferens preparation to X-irradiation

Summary In the mechanical-inactive guinea-pig vas deferens X-rays (25 kV) at threshold doses of about 100 kR initiated phasic activity and an immediate increase in tone. After addition of acetylcholine, noradrenaline or adrenaline to the rinsing solution a slight contractile activity of vas deferens appeared and the preparation reacted to X-irradiation at a threshold dose of 3 to 5 kR (dose-rate 20 kR/min) with increased phasic contractions and with a dose and dose-rate dependent tonic contraction. After repeated irradiation a sensitization was observed. X-rays produced tonic contractions of the vas deferens preparation up to a total dose of about 200 kR (fractionated irradiation). An irreversible contraction of the vas appeared after continuous exposure to X-ray doses larger than 500 kR (dose-rate 20 kR/min).     

Tetrandrine,a calcium antagonist of Chinese herbal origin,interacts with vascular muscle α1-adrenoceptor

The effects of tetrandrine (TET) on the contractile responses of rat aortic rings and perfused rat mesenteric arteries to phenylephrine (PE) were investigated. TET inhibited the maximal contraction to PE in a concentration-dependent manner. TET significantly inhibited the transient contraction in Ca²⁺-free medium presumably due to release of intracellular Ca²⁺ after activation of α₁-adrenoceptors. However, it caused a stronger inhibition of the sustained contraction in Ca²⁺-containing medium presumably the result of Ca²⁺ influx. TET has no inhibitory effect on caffeine-induced transient contraction. Radioligand receptor binding study using isolated dog aortic muscle membranes indicated that TET inhibited the binding of ³H-prazosin in a competitive manner, hence showing that TET interacted directly with the α₁-adrenoceptors. Thus, TET affected PE-induced aortic contractions by multiple mechanisms, inhibiting interaction of PE with α₁-adrenoceptors and interfering with PE-induced responses involving both Ca²⁺ entry and release.     

Effects of veratrine and paeoniflorin on isolated mouse vas deferens

In this study, we attempted to identify the interactions and mechanisms between veratrine and paeoniflorin on isolated mouse vas deferens. Paeoniflorin had no effect on isolated mouse vas deferens. Veratrine (1 x 10(-5) approximately 1 x 10(-3) g/ml) could directly induce contraction of isolated rat and mouse vas deferens. The concentration induced by veratrine (1 x 10(-5) g/ml) was completely inhibited by Ca2+-free solution and verapamil (1 x 10(-5) M), in both the epididymal and the prostatic portions of isolated mouse vas deferens. Naloxone (1 x 10(-5) M) did not alter the contraction induced by veratrine (1 x 10(-5) g/ml) in either the epididymal or the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) inhibited the contraction induced by veratrine (1 x 10(-5) g/ml) in both the epididymal and the prostatic portions of isolated mouse vas deferens. Paeoniflorin (4.8 x 10(-5) g/ml) potentiated norepinephrine (1 x 10(-5) M)-induced phasic contraction in the epididymal portion, but decreased contractions in the prostatic portion. Paeoniflorin (4.8 x 10(-5) g/ml) increased KCI (56 mM)-induced phasic contraction in the epididymal portion, but decreased the tonic contraction in either the epididymal or the prostatic portion. Veratrine (1 x 10(-5) g/ml)-induced contractions could be decreased by pretreatment with ryanodine (1 x 10(-5) M) in both the epididymal and the prostatic portions. Pretreatment with the combination of paeoniflorin (4.8 x 10(-5) g/ml) and ryanodine (1 x 10(-5) M) did not potentiate the inhibition of paeoniflorin in the veratrine-induced contraction in both the epididymal and the prostatic portions of isolated mouse vas deferens.     

Bradykinin modulates the release of noradrenaline from vas deferens nerve terminals

To asses whether bradykinin influences the release of noradrenaline from the adrenergic varicosities of the vas deferens, tissues were loaded with 3H-noradrenaline. Upon electrical depolarization bradykinin increased in a concentration-dependent fashion, the overflow of tritium from the mouse or rat vas deferens. The 3H-overflow is dependent on the external Ca2+concentration suggesting neuronal release of 3H-noradrenaline. The present results add evidence to the hypothesis that bradykinin modulates the release of noradrenaline from peripheral sympathetic nerve terminals via the activation of a presynaptic mechanism.