Rabbit Vas Deferens Research Articles

Distribution of spermatozoa in the rabbit vas deferens.

To ascertain whether sperm transport through the rabbit vas deferens (vas) takes place during sexual rest, the number of spermatozoa in the ampullary. abdominal and scrotal segments of the vas and in the cauda epididymis was determined after varying periods of sexual rest and following sexual stimulation. Immediately after ejaculation, the abdominal and scrotal portions contained equivalent amounts of spermatozoa. With increasing sexual rest, the total amount of sperm in the vas increased, with the greatest number found in the scrotal section and the least in the ampulla. This gradient in numbers was maintained during sexual rest. After sexual stimulation without ejaculation, the sperm number in the vas greatly increased and was large enough to account for the number of spermatozoa in an ejaculate. A significant shift in the gradient was observed due to increased sperm in the abdominal vas. The results indicate that during sexual rest the spermatozoa which are not stored in the epididymis are transported into and through the vas which maintains a decreasing sperm gradient towards the urethra. During sexual stimulation, spermatozoa are moved from the epididymis into the vas from where they are removed rapidly at ejaculation. The vas deferens subsequently reestablishes the sperm gradient maintained during sexual rest. This implicates the vas as an active organ during sexual inactivity.

Further evidence for adrenergic transmission in the human vas deferens.

1. Isolated portions of human vas deferens responded to field stimulation of the intramural nerve fibres or to exogenously applied noradrenaline with rhythmical contractions of both longitudinal and circular muscle layers. 2. In guinea-pig and rabbit vas deferens field stimulation produced an initial rapid 'twitch' response which was not found with human vasa. 3. Responses of the human vas to field stimulation were depressed by the alpha-adrenoceptor blocking agents phentolamine and yohimbine. 4. It is concluded that the motor innervation of the human vas deferens is adrenergic and the relevance of this to the physiological operation of the tissue is discussed.

Effect of neuronal uptake inhibitors on the adrenergic-neuron blockade produced by guanethidine in rabbit vas deferens.

The effect of neuronal-uptake inhibitors on the guanethidine-induced inhibition of responses of rabbit vas deferens to transmural stimulation was investigated. The twitch and sustained responses were inhibited by about 70% by 1.2 X 10(-5) M guanethidine. Prior or subsequent exposure to the noradrenaline-uptake inhibitors, 3.3 X 10(-5) M cocaine, 10(-5)M nisoxetine, and 10(-7)M desipramine prevented or reversed these effects of guanethidine. The depressant effects of guanethidine were not modified by the serotonin-uptake inhibitor, 10(-6)M fluoxetine. The uptake of 5 X 10(-8)M (-)-[3H]noradrenaline by rabbit vas deferens was significantly reduced by guanethidine, cocaine, nisoxetine, and desipramine, but not by fluoxetine. It was concluded that guanethidine inhibited both the twitch and sustained responses of the vas by producing adrenergic-neuron blockade and that these effects were dependent on continued transport into adrenergic neurons.

Intravas device studies in rabbits: I. Effect of surgical procedures on adrenergic innervation of the vas deferens

The effect of intravas device placement on the adrenergic innervation of the vas deferens was studied in the rabbit. Utilizing varying methods for intravas device insertion (non-transected vs. transected vas), segments of vasa were removed at autopsy and examined histologically for the fluorescent histochemical demonstration of norepinephrine. In each surgical procedure, fluorescence was maintained above the device site or area of transection. Intraluminal placement of an intravas device without vas transection reduced, but did not abolish, innervation below the device site; fluorescence below the device was abolished, however, if the vasa were transected. Comparative studies with vasectomized rabbits resulted in a similar loss of fluorescence below the area of transection.It is concluded that transection of the rabbit vas deferens for intravas device insertion results in a loss of adrenergic innervation below the point of transection; this effect can be mimicked by vasectomy. Intravas device insertion without transection retained some of the neuromuscular integrity of the vas smooth musculature.

The potentiating effects of 4-aminopyridine on adrenergic transmission in the rabbit vas deferens

The effects of 4-aminopyridine on adrenergic transmission in rabbit vas deferens were investigated. 10 −5–10 −3 M 4-aminopyridine markedly potentiated the sustained, secondary contractile response to transmural stimulation and significantly increased the amount of (±)-[ 3H]-metaraminol released at 5 Hz, but had little effect on responses to exogenous (−)-noradrenaline. 10 −5–10 −4 M 4-aminopyridine did not significantly alter the accumulation or efflux of (−)-[ 3H]-noradrenaline in rabbit heart. It is suggested that 4-aminopyridine potentiated responses of rabbit vas deferens to transmural stimulation mainly by increasing transmitter release, possibly as a result of prolongation of the action potential. Responses to transmural stimulation were also potentiated by 2- and 3-aminopyridine and by 3,4-diaminopyridine but not by pyridine, aniline, 4-dimethylaminopyridine, 4-aminoethylpyridine or 2-aminopyrimidine.

Effects of rubidium on responses of rabbit vas deferens to transmural stimulation and to noradrenaline

The effects of 2 mM RbCl on adrenergic transmission in rabbit vas deferens were investigated. Rubidium, but not potassium, markedly potentiated the sustained, secondary contractile response to transmural stimulation at 2–16 Hz. This effect was not abolished by indomethacin. Significant spontaneous desentization to (−)-noradrenaline occurred, but this was prevented by the addition of 2 mM RbCl or KCl. Addition of 2 mM RbCl, but not of 2 mM KCl, significantly increased the amount of [ 3 H] (±)- metaraminol released on transmural stimulation at 5 Hz, without affecting the uptake of [ 3 H] (±)- metaraminol over a 30 min period. It is suggested that rubidium mainly potentiated responses of rabbit vas deferens to transmural stimulation by increasing transmitter release, possibly as a result of prolongation of the action potential.

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.

Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine

This investigation was carried out to test the hypothesis that the brain contains a substance that functions as an endogenous mediator at central morphine receptor sites. The extraction and purification of a low molecular weight substance are described. This substance inhibits neurally evoked contractions of the mouse vas deferens and guinea pig myenteric plexus. The inhibitory action of this substance was antagonized by the 3 narcotic antagonists naloxone, naltrexone and MR 1302 at nanomolar concentrations. The narcotic antagonists alone had no effect on the assay tissues. The purified substance had no effect on the guinea pig or rabbit vas deferens, tissues which do not possess morphine receptor sites. The pharmacological activity of the substance is destroyed by car☐ypeptidase and leucine aminopeptidase, it is hydrophilic and behaves as an ampholyte in ion exchange systems. These observations and the production of ninhydrin positive products on acid hydrolysis indicate the presence of a small molecular weight ( < 700) peptide. The morphine-like substance was unevenly distributed in the brain, the highest concentrations occurring in the striatum, midbrain, pons and medulla. No activity could be detected in extracts of cerebellum, liver or lung. It is suggested that the compound isolated in this investigation forms part of a central pain suppressive system and may also have a wider neurochemical role in the brain.

Variation in noradrenaline output with changes in stimulus frequency and train length: role of different noradrenaline pools.

1 During adrenergic nerve stimulation the output/pulse of noradrenaline from the rabbit vas deferens and portal vein is not constant but increases as the stimulus frequency or train length is increased. Depending upon the stimulus frequency and train length the fractional release of noradrenaline may vary from less than 10(-7) to greater than 10(-4).2 Endogenous tissue stores of noradrenaline were labelled by incubation with (-)-[(3)H]-noradrenaline and [(14)C]-tyrosine. The output/pulse of newly synthesized [(14)C]-noradrenaline remained constant as the train length was increased whilst the output/pulse of [(3)H]-noradrenaline increased under the same conditions. This phenomenon was independent of the stimulus frequency. Newly synthesized noradrenaline also appeared in the superfusate following nerve stimulation more rapidly than exogenously loaded noradrenaline.3 Both [(3)H]-noradrenaline and [(14)C]-tyrosine were found to label an easily releasable store of noradrenaline. Mobilization from this store was the same at low and high frequencies of nerve stimulation.4 It is concluded that at least two functional stores of noradrenaline exist within the adrenergic nerve ending. Newly synthesized noradrenaline is probably only a minor constituent of transmitter output under normal conditions of adrenergic nerve activity.5 At least two mechanisms control the amount of noradrenaline released per pulse during nerve stimulation. Facilitation of release with increasing train lengths appears to be due to the mobilization of transmitter from a secondary store. Facilitation of release with increasing stimulus frequency is not dependent on mobilization from any particular store and at present there is no satisfactory explanation for this phenomenon.

Differential labelling of intraneuronal noradrenaline stores with different concentrations of (-)-3H-noradrenaline.

Isolated preparations of the rabbit vas deferens were incubated with (--)-(3)H-nor- adrenaline. A preferential release of (3)H-noradrenaline was demonstrated after electrical stimulation of the intramural nerves in those tissues labelled with 10 ng/ml (3)H-noradrenaline. There was no preferential release of (3)H-noradrenaline when the vasa were initially labelled with 100 ng/ml (3)H-noradrenaline. It is concluded that low concentrations of (3)H-noradrenaline do not mix homogeneously with the total tissue store but enter a small easily releasable store.

Stereoselectivity of catecholamine uptake in noradrenergic and dopaminergic peripheral organs

The stereoselectivity of noradrenaline uptake was studied in several adrenergically innervated peripheral organs of the rabbit and rat, as well as in the isolated rabbit retina. (−)- and (+)-noradrenaline bitartrate, and (−)- and (+)-amphetamine surfate, were compared as relative antagonists of the neuronal membrane catecholamine uptake mechanism in the various organs, in order to determine stereoselectivity at both the α and β carbons of noradremaline. It was not possible to demonstrate stereoselectivity unless optimal conditions prevailed. These included the use of finely divided tissues (<0.5 mm on a side), of linear uptake rates, and of non-saturating levels of amines. In the rabbit vas deferens and iris/ciliary, where noradrenaline is the predominant catecholamine and is present in high endogenous concentrations, there was a marked preference for (−)-noradrenaline and for (+)-amphetamine, in contrast with the rabbit retina where dopamine is the chief catecholamine and in which no stereoselectivity was demonstrable. These results confirmed earlier findings in the rat brain in which catecholamine uptake was stereoselective in noradrenergic brain regions but not in the corpus striatum, a dopaminergic region (Coyle and Snyder, 1969). Cardiovascular tissues of the rabbit exhibited a reversal of the usual stereoselectivity, i.e. (+)-noradrenaline had more affinity than (−)-noradrenaline in the rabbit atrium, left ventricle and thoracic aorta, and (−)-amphetamine was more potent an inhibitor of catecholamine uptake than (+)-amphetamine in the thoracic aorta, Amphetamine stereoisomers were equipotent in the tissues of the rabbit heart. Unlike cardiovascular tissues of the rabbit, ventricular slices of the rat displayed a marked preference for (−)-noradrenaline and for (+)-amphetamine.

Evaluation of mechanisms controlling the release and inactivation of the adrenergic transmitter in the rabbit portal vein and vas deferens.

1. A method is described for the detection and assay of picogramme quantities of noradrenaline. This involves transferring Krebs solution containing noradrenaline to a cascade system where the catecholamine may be bioassayed on superfused preparations of the rabbit aorta and iliac artery.2. Electrical field stimulation of the rabbit vas deferens and portal vein caused the release, into the bathing medium, of a material which was identified by pharmacological and chemical tests as noradrenaline.3. Cocaine (0.3-5 mug/ml) caused a marked increase in noradrenaline output after electrical stimulation of the portal vein and vas deferens. This effect appeared to be maximal at a concentration of 2.4 mug/ml; when the cocaine concentration was increased above 10 mug/ml the noradrenaline output was greatly reduced.4. Phenoxybenzamine (5 mug/ml) caused a 4-8 times greater increase in noradrenaline output than cocaine; however, the increase in output due to phenoxybenzamine was much smaller in tissues pretreated with cocaine.5. Corticosterone (20 mug/ml) increased noradrenaline output by 30-40% in untreated vas deferentia, but caused a 300% increase in output in tissues pretreated with cocaine. Cocaine also caused a much greater increase in output in tissues pretreated with corticosterone than in untreated tissues.6. Treatment with pargyline plus tropolone caused a 100-200% increase in noradrenaline output; this effect was not modified by cocaine, but was abolished when the tissues were pretreated with either phenoxybenzamine or corticosterone.7. When tissues were stimulated for 240 pulses at 1-16 Hz, the output per pulse of noradrenaline increased linearly with the logarithm of the frequency of stimulation. This relationship between frequency and output was seen in both untreated tissues, and in tissues treated with cocaine, phenoxybenzamine, corticosterone or pargyline plus tropolone.8. It is concluded that cocaine enhances output by blocking the neuronal reuptake of noradrenaline, and corticosterone by blocking the extraneuronal uptake and subsequent metabolism of noradrenaline. Phenoxybenzamine acts by blocking both neuronal and extraneuronal uptake mechanisms. There appears to be a dynamic balance in the distribution of noradrenaline between the two uptake mechanisms after the release of the transmission from the nerve endings.9. It is calculated that more than 90% of the noradrenaline released by nerve stimulation (240 pulses at 2-16 Hz) is inactivated by neuronal and extra neuronal uptake mechanisms.10. It is calculated that the fraction of the total noradrenaline store that is released by one pulse at 2 Hz is 6.6 x 10(-5) in the portal vein and 5.6 x 10(-5) in the vas deferens; the corresponding values at 16 Hz were 15.9 x 10(-5) and 16.2 x 10(-5).

Occurrence and properties of catechol-0-methyl transferase in adrenergic neurons.

Abstract—A study has been made of the catechol‐O‐methyl transferase activity of some peripheral tissues after sympathetic denervation. A fall in catechol‐O‐methyl transferase activity was found in some organs, e.g. rat and rabbit vas deferens, cat nictitating membrane and rabbit submaxillary gland but not in mouse heart and spleen. It was found that suboptimal concentrations of S‐adenosylmethionine did not reveal a significant difference between normal and denervated organs but at optimal concentrations a fall was seen in some organs. Catechol‐O‐methyl transferase activity was present in bovine splenic nerve and in adrenal medulla. It is suggested that the fall in enzyme activity after denervation indicates a neuronal cellular localization. A kinetic study of catechol‐O‐methyl transferase from normal and denervated rat vas deferens suggested that the neuronal and extraneuronal catechol‐O‐methyl transferase had different kinetic properties and an estimation of the kinetic constants of the neuronal enzyme was made.

Noradrenaline metabolizing enzymes in normal and sympathetically denervated vas deferens.

Abstract—A surgical technique for sympathetically denervating the vas deferens has been evaluated biochemically. A slight fall in soluble muscle protein content and no significant change in DNA content of the operated vas deferens were found. This indicates that the surgical procedure causes only a slight degree of tissue damage and may be useful for investigating the cellular localization and properties of noradrenaline metabolizing enzymes. In three species examined (rat, guinea pig and rabbit), monoamine oxidase activity of the vas deferens fell by approximately 50 per cent after denervation. The time course of the fall in monoamine oxidase activity of rat vas deferens was parallel to that of the disappearance of noradrenaline suggesting that this proportion of the total enzyme activity had a neuronal localization. The remaining enzyme activity is presumably located extraneuronally.Significant falls in catechol‐O‐methyl transferase activity were found in rat and rabbit vas deferens after denervation but not in guinea pig. The rabbit and rat vas deferens had respectively approximately 60 and 30 per cent of the catechol‐O‐methyl transferase activity associated with the sympathetic nerves. A complete loss of DOPA decarboxylase and tyrosine hydroxylase activities occurred in rat vas deferens after denervation, suggesting that these noradrenaline synthesizing enzymes have an entirely neuronal localization.

Effects of ionic alterations on various sympathetic nerve-smooth muscle preparations.

SummaryIsolated preparations of guinea pig, rat and rabbit vas deferens, and Finkleman preparations of rabbit ileum and rat and guinea pig colon have been used to study the effects of ionic alterations on responses to added noradrenaline and to stimulation of sympathetic nerves. With vas deferons preparations it was found that, depending on the species used, these responses were affected differently by certain of the ionic alterations. Intestinal preparations from the three species behaved more consistently.Species difierences in vas deferens preparations were seen in response to the following ionic changes: (i) reduction in sodium chloride (replaced by sucrose); (ii) absence of potassium; (iii) increasing potassium to 15 and 20 mM, and (iv) raising calcium to 5–10 mM. Lowering calcium to 0·25–0·5 mM depressed the responses of vas deferers from all three species.Intestinal preparations showed a uniform depression in 7·5–10 mM calcium. Raised potassium depressed the response to sympathetic nerve stimulation in all three species, though the response to noradrenaline was affected differently with different species.The interactions of drugs and ions also showed differences with the three species. While 15 mM potassium could reverse the blocking action of both guanethidine and procaine on guinea pig vas deferens, it was almost ineffective on rat vas deferens, and on intestinal preparations raised potassium increased the blocking action of these drugs. High calcium, which enhanced the effects of sympathetic nerve stimulation in varions tissues, failed to modify the blocking action of guanethidine.

THE EFFECT OF PROSTAGLANDIN E1 ON RESPONSES OF SMOOTH MUSCLE TO CATECHOL AMINES, ANGIOTENSIN AND VASOPRESSIN.

Reduction of the pressor responses to adrenaline in the rabbit following administration of prostaglandin E(1) has been confirmed. The effect is, however, nonspecific since noradrenaline, angiotensin and vasopressin are also antagonized. Analogous responses were observed in blood flow experiments on the cat hind limb but not on the rabbit isolated auricles or the rabbit isolated duodenum. Contractions of the cat nictitating membrane produced by sympathetic preganglionic stimulation or by adrenaline were not decreased following injection of prostaglandin E(1) but the relaxation period was shorter. Contractions of the rabbit vas deferens induced in vivo by adrenaline were smaller after prostaglandin E(1) and this effect tended to be long-lasting.