In vivo bupivacaine reduces the in vitro sensitivity of ...

In vivo bupivacaine reduces the in vitro sensitivity of the human vas deferens to phenylephrine

'

J. H . KAWAMIBTO, J. A. OWEN, lr2 A. MORAEES,'

AND K. N A K A T S U ~ ' ~

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by China University of Petroleum on 06/04/13 For personal use only.

Departments of Phai-macokogyand Toxicology and Urology, Queen's University, Kingston, Onr., Canrada K7L 3N6 Received September 6, 1985

KAWAMOTO, J. H., J. A. OWEN,A. MORALES, and K. NAKAT~U. 1986, In vivo bupivacaine reduces the in vitro sensitivity sf the human vas deferens to phenylephrine. Can. J. Physiol. Phannacol. 64.46'7-47 1. The isolated human vas deferens (HVD) has been the subject of a limited number of phmacological studies. Furthermore, it has not been established whether the local or general anesthetics used in obtaining the HVD affect the responses of this isolated tissue. Therefore, the aim of this study was to determine if the local anesthetic, bupivacaine (BPV), alters the sensitivity of the HVD to agonists. It was shown that BPV, injected into subjects undergoing vasectomy, was present in the excised HVD and in sufficient concentration to attenuate the phenylephrine (PE)-induced contraction. Bupivacaine assays were done by gas chromatography - mass spectrometry. Following 3 h of equilibration with repetitive washing, 95.0 k 2.1% (SD) of the BPV could be eliminated from the tissue, which correlated well with an observed increase in sensitivity of the HVD to PE. The sensitivity to PE of HVD isolated from subjects under general anesthesia was less than the sensitivity of tissues obtained with bupivacaine and in this case the depressant effect was not reversed by washing. These results indicate that it is important to consider the anesthetic procedure used to acquire the HVD and also the equilibration procedure prior to pharmacologicalstudies.

KAWAMOTO, J. H . , J. A. OWEN, A. MORALES et K. NAKATSU. 1986. In vivo bupivacaine reduces the in vitro sensitivity of the human vas deferens to phenylephrine. Can. J. Physiol. Phmnacol. 64: 467-47 1. Le canal dkferent humain is016 (CDH) a Ct6 le sujet d'un nombre limit6 d'6tudes pharmacologiques. En outre, il n'a pas kt6 ktabli si les anesahCsiques locaux ou gCnkraux utilids en excisant le CDH affectent les rCponses de ce tissu isolt . Par conskquent, le but de cette ktude fut de dktenninersi l'anesthksique local, bupivacaine (BPV), altCrait la sensibilitkdu CDH aux agonistes. On a montrt q w la BPV, injectke B des patients subissant une vasectomie, Ctait prksente dans le CDH excisC et en concentration suffisante pour attknuer la contraction iaduite par la phknylCphrine (PE). On a effectuC des dosages de bupivacdine par chromatographieen phase gazeuse - spectromktriede masse. Aprks 3 h d'un rin~agerkpktitif, on a pu Climiner du tissu 95.0 -+ 2,1% (ET) de BPV, ce qui csmla bien avec une augmentation de la sensibilitk du CDH ? la i PE. La sensibilitk h la PE du CDH isole de su~etssous anesthCsie g6nCrde fut plus faible que la sensibilitt des tissus obtenus avec la bupivacaine et dans ce cas-ci, un rinqage n'inversa pas l'effet dkpresseur. Les rksultats indiquent qu'il est important de considbrer la procaure anesthksique utilisCe pour obtenir le CDH ainsi que la procCdure de rin~ageavant d'entreprendre toute Ctude pharmacologique. [Traduit par la revue]

htroduetion There is a notable lack of basic phmacological studies using isolated human tissue preparations. With an increase in ppularity sf vasectomy as a contraceptive method, the excised and isolated portion of human vas deferens (HVD) is an attractive preparation for experimental pharmacology. It is one of the few healthy human tissues available and its inherent innervation increases its value as a phmacologicd model. Some pharmnacological properties of the isolated HVD have been described in a limited number of papers (Martins et al. 1940; Birmingham 1968; McLeod et d. 1973; Ventura et al. 1973; Hepperlen et al. 1976; Anton and McGrath 1977; Ratnasooriya et al. 1979; Belis et al. 1982), but it is clear that the preparation is far from being fully characterized. In most cases these authors reported observations of dose-dependent contractile responses of the HVD to norepinephrine (NE) and other a-adrenoceptor agonists which could be blocked by phentolamine. In addition, McLeod et al. (1973) showed that the HVD was unresponsive to acetylcholine, 5-hydroxytryptamine, isoproterenol, and histamine, suggesting the absence of receptors for these substances. In a separate study, Birmingham f 1968) demonstrated that the HVD was contracted by electrical stimulation of the intramural nerve, and Anton and McGrath (1977) reported that the isolated HVD responded in a similar fashion to '~ffiliation:Department of Pharmacology and Toxicology, Queen's University, Kingston, Ont., K7L 3N6. 2Affi1iation:&Pmment of Urology, University,Kingston, Qnt., K7L 3N6. 3~uthor to whom all correspondence should be addressed.

either field stimulation or exogenously applied norepinephrine. Taken together with the significant norepinephrine content of the tissue (Birmingham 1968). these reports support the concept of noradrenergic motor innervation in at least the epididymal portion of the HVD. Qn the other hand, histochemical characterization of the isolated HVD fAlm 1982) revealed distinct adrenergic, cholinergic, purinergic, and peptidergic components of innervation; the exact function of each of these has not yet been determined. It was thought that before further pharrnacological characterization of the HVD can be accomplished, it must be determined whether the administration of either local or general anesthetics to the individual for the acquisition of the HVD affects in vitro responses of the isolated tissue. This aspect has not been previously studied, although it was recognized by Ventura f 1973) as a potential problem. Relevant to this question are the reports of Astrom (1964), Vohra (1978), and Grana et al. (1988) who demonstrated the phaumacological activity of local anesthetics on in vitro responses of smooth muscle. Furthermore, general anesthetics have been shown to affect the in v i m responses of smooth muscle to NE and other stimuli (Sprague et aI. 1974; Yang et al, 1973) and general anesthetic procedures nearly always involve the use of other drugs. Clearly, investigation into the potential effects of residual anesthetics is required before the HVD can be used routinely as a pharmacological - model. The majority of the HVD obtained in this center were from subjects who had been injected with the local anesthetic, bupivacaine (BPV), with the remainder acquired from subjects under general anesthesia. Thus, the objectives of this study

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were: (a) $0measure the content of BPV in the excised HVD upon receipt from the operating room and to determine if this BPV can be eliminated from the tissue by extensive washing in the isolated tissue bath; (b) to detemine if this local anesthetic affects isolated HVD responsiveness; (c) to determine if the general anesthesic procedure affects the in vitro responses sf the isslated HVD. Source ofvas deferens Human vas deferens specimens were obtained, with informed consent, from healthy subjects undergoing elective vasectomy at Kingston General Hospital. For the individuals whose surgery was done under local anesthesia, 2 Hnzk of a 0.5% bupivacaine hydrochloride solution were injected into each side of the scrotum, with additional BPV injected as required. Generd anesthesia was conducted in a standard fashion; typically the h g s were sodium thiopentd (3-5 mg/kg i.v.1, fentanyl citrate (5 p,g/kg i.v.), and N20 md 0 2 at Wow rates of 1.5 and 1.OUPgBiln, respectively, with 1.5%halothane throughout. Segments of HVD approximately 1.5 cm in length md approximately 2 cm from the epididymus were excised from both left and right sides. The isolated HVD were placed in cold Dulbecco's phosphate-buffered saline and transported to the Iabsratory. The time from surgical removal to equilibration of the M V D in the tissue bath did not exceed 60 min.

GC-MS analysis ojbacrpivackain~seconcentration BPV was extracted under alkaline conditions with ethyl acetate from the isolated tissue essentially as described by Naito et d.(1977), except that the whole homogenate was extracted rather than just the supernatant, and KOH was not used to precipitate the Cl0;i. The extracted sample was quantitated against a standard curve obtained with a Hewlett-Packwd 5840 gas chromatographic (GC) system interfaced with a 5985B mass spectrometer (MS). The instumentd operating conditions were as follows: carrier gas (prepurified He) flow mte, 25 d/ranin; injection port tempratwe, 250°C; column temperature, 235°C; GC-MS interface temperature, 250°C; ion source temperature, 200°C; ionization was by electron impact ionization (70 eV), and selected ion monitoring, 140 atomic mass units (amu). The column was a 8.8 m X 2 m inner diameter silanized and coiled glass tube, packed with 3% OV-fa on 88/1Wmesh Chromosorb W@. The mean extraction efficiency of BPV from the tissue was 80%with a within-day coefficient of variation of less than 8%. The chomatographic retention time for BPV was reproducible at 3.7 min. No interference was detected at m/z of f 40 amu at this retention time from control rat vas deferens (RVD) or HVD from subjects under general anesthesia. Bso!asated tissue bath experiments The isolated tissues were carefully cleaned of fascia and suspended in 5-mL baths containing Gebs-Henseleit buffer of the following , K + , 5.8; ~ a " , 2.6; M ~ " , B .2; composition (millimola): ~ a +143; C1-, 128; H2P0i, 1.2; HCO,, 25; SO:-, 1.2; glucose, 8 1.1. This buffer was maintained at 37°C and aerated with 95%02-5% CO2throughout the experiment. Isometric tension was recorded with Grass FT.03C force-displacement transducers coupled to a B e e h a n R5 11A physiograph. f i e viability of the tissues was initially assessed with B5Qmkf KC1 prior to obtaining the cumulative dose-response relationships to the relatively selective a*-agonist,phenylephrine (PE). For the cumulative dose-response relationships, the starting concentration sf PE was lw8M ;subsequent doses were given after a steady response had k e n obtained or after 2 min had elapsed since the previous dose, whichever occurred first. For the BPV elimination studies, the HVD were longitudinally bisected and cumulative dose-response relationships to PE of one-hdf of the tissue were detemined following the required equilibration period.

Since PE caused a phasic, rhythmic type of contraction in the tissue, the response measured for each drug dose was the mean sf the third and fourth uniphasic contractions that occurred after addition sf the dmg.

The tissue responses were expressed as a percent s f the absolute maximum response of that particular tissue to PE at any time during the experiment. The ECS0, maximum response, md threshold concentration expressed as an EC5 value were detemined from the doseresponse relationships for each tissue. Dam analysis Statistical significance between two groups was detemined by Student" s-test. Multiple comparisons m o n g groups sf data were conducted by using the appropriate analysis of variance with a subsequent Newman-Keuls test. Mean values were considered to be significantly different if p < 0.05. Sostsce sj c o m p o u ~ s Bupivacaine hy&ocMoride was a gift from Winthop Laboratories of Sterling Drug Ltd. (Aurora, Ont.) and prazosin hydrochioride was a gift from Hzer Canada Ins. (Kirkland, QuC.). Other materials were obtained as follows: e o l u m packing 3% OV-1@on CBPPomomrb W@ $Q/lW,Chromatographic Specialties Ltd. (Brockville, Ont.); phenylephrine hydrochloride, Sigma Chemical Co. Ltd. (St. Louis, MO); and HPLC grade organic solvents, Fisher Scientific Ltd. (Whitby, Ont.). All other chemicals were reagent grade from a variety of sources.

Results Bupivacaine concentration in the isolated human ws deferens The concentration of BPV in the isolated HVD upon receipt from the operating room as detemined by the GC-MS assay ranged from 21.0 to 530.1 nmoVg tissue wet weight, based on analysis of 40tissues (243.7 2 206.2, mean 2 SD). Bupivacaine elimination With this concentration of BPV in the isolated HVD, it was important to determine if the BPV could be eliminated by incubation a d repetitive washing in the isolated tissue bath. Hn this experiment the HVD was cut in two along the longitudinal axis. For one portion the BPV concentration was determined directly, while the other portion was washed extensively for 1-5 h and then the BPV concentration was detemined. Thus, each HVD acted as its own control. The results are shown in Fig B. Clearly, extensive washing resulted in elimination sf BPV from the tissue such that by 3 h about 95% of the original drug concentration was cleared. Responses ofthe isolated human vas deferens The responses of the isolated MVD to both K+ (1 50 mkf) and PE in cumulative doses were qualitatively the same as those described previously (McLeod et al. 1973; Ratnasoof-iya et a%. 1379). Accordingly, prazosin ( 1g7 M)was able to eliminate the responses of the HVD to PE (1 M). As responses of longitudinally bisected vas deferens were monitored as part of the BPV elimination study, it was important to compare the responsiveness of whole and bisected vas deferens. There was no statistically significant difference in the ECgovalue between the whole and bisected vas deferens. Thus, the longitudinal bbisection did not alter the in vitrs sensitivity to PE (data not shown). Cumulative dose-sesponse relationships to PE were obtained using HVD from subjects under BBP Vocal anesthesia after I , % , 3,4, a d 5 h of equilibration to determine if the responsiveness of the MVD changed with equilibration and repetitive washing. Shown in Fig. 2 are the 1-5 h concentration-effect relationships of isolated HVD obtained from subjects treated with BPV, with the data expressed as a percent of the absolute maximum tissue respnse to PE. The EC5 and ECS0 values of the 1-h dose-respnse relationships were significantly different @ < 8.01) from these

TABLE1 . EC5 md ECSovalues s f the isolated human vas deferens to phenylephrine (a) BPV anesthesia =: 8-14)


T i m (h)

(b)General anesthesia (n = 4) EC5 (Fw

Ec5 (PW

NOTE:Values are means f SD. * Significant difference within group at p

ECSO

0.85.

BPV ELIMINATION

4 00

5

6

FIG. 1. Effect s f incubation time with repetitive washing on bupivacaine concentration in the isolated humm vas deferens. The percent BE" remaining refers to the concentration of extractable BPV detected after equilibration expressed as a percent of the extractable eoncentration iwitiaHy in the tissue. Numbers in parentheses represent number of tissues.

values at 2, 3, 4, or 5 h, which were not significantly different from each other (Table la). The apparent difference in the maximum respsnse to PE at 6 x 10-' M between 1 h and 2-5 h was not statistically significant. A comglementq experiment was conducted to determine the acute effects of adding BPV in the tissue bath. The HVD preparations were equilibrated for 3 h with repetitive washing as above to wash out BPV and to allow the HVD to attain its imum sensitivity. At this point, the HVD was considered to be suitable for testing the effects of BPV added to the bath. BPV (6.9 x M ) was added to the bath a d maintained for 45 min; this concentration was calculated to mimic the in vivo exposure to BPV. Following BBV treatment, there was a threefold shift ('p < 0.05) of the PE dose-response relationship to the right and a depression of the maximum respsnse (Fig. 3). The HVD exposed to BPV in this manner contained BPV in the same concentration range as that found in the HVD upon receipt from the operating room (200-300 nmol6g tissue). Therefore, it was

- log

PHENYLEPHRINE

4

(M)

FIG. 2. Effect of equilibration with repetitive washing on the responses of the isolated HVD to phenylephrine. The cumulative Iog dose-response curves presented are after 1 h (O),2 h (A),3 h (a), 4h (E), and 5 h (A>of equilibration. Each point represents the mean of a minimum of eight tissues; vertical lines show SB.To avoid cluttering the graph, representive SD b a s are shown instead of a11 standard deviation ban.

shown that BPV at this concentration attenuated the responses of HVD to PE. Eflects. of genera! anesthesia The sensitivity to PE of HVD obtained from subjects under generd anesthesia were compared with that of HVD isolated from subjects administered BBV for surgery. There were no significant differences among either the ECm or EC5 d u e s or maximum response to PE from the 1, 2, or 3-h dose-response relationships of HVD obtained under general anesthesia (Table Ib). However, the- ECSo value of either the I-, 2-, or 3-41 concentration-effect relationships of the HVD isolated under general anesthesia was significantly greater @ 0.01) than the 2- or 3-kECS0value of the HVD prepared with BPV as the local anesthetic. Thus, HVD from general anesthesia was Iess sensitive to PE than HVD from BPV anesthesia after 3 h of equilibration. It should also be noted that the sensitivity of HVD from


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CAN. J. BHYSIOL. PHARMACBL. VOL. 64, 11986

FIG.3. Depression of phenylephrine-induced contmctisns of HVD by in viigro exposure to bupivacaine. These cumulative log doseresponse curves were obtained following 2 h (0)and 3 h (0) of washing, and following 3 h of washing plus exposure to bupivacaine, 6 . 9 X 1@ Ad, in the tissue bath (A). Each point represents the mean of eight tissues; vertical lines show SD.

general anesthesia did not increase after the 3-h equilibration/ wash period as was the case for HVD from BPV anesthesia.

Discussion The concentration of local anesthetic in the HVD isolated from subjects administered local anesthetic for vasectomy has not been previously reported. In this study, BPV was detected and quantitated in the HVD. The considerable range of concentration of BPV measured in the isolated HVD may reflect differences in the amount of BPV injected into each subject for local anesthetic effect, variability of the injection technique, or variability of phannacokinetics amongst subjects. Nevertheless, the mounts of BPV found were substantial and were considered to be sufficient to affect the responsiveness of the HVD to various stimuli. Therefore, it seemed appropriate to continue the work by attempting to eliminate BPV by a prolonged washing period. In the present work the BPV concentration in the isolated HVD was reduced by incubation with repetitive washing in the tissue bath. Furthermore, the resulting decreased BPV concentration and increased sensitivity of HVD to PE correlated well. This is interpreted to mean that the initial concentration of BPV in the HVD was responsible for the lower sensitivity. This interpretation is further supported by the observations from experiments in which BPV was added to the baths containing HVD. In these experiments the responses of vasa deferentia were depressed after addition of BPV; moreover, this protocol of BPV addition appeared to be appropriate because the tissue concentrations of BPV in HVD following in vitro exposure to BPV were within the range found on surgical excision. We conclude that HVD removed under local anesthesia can contain concentrations of BPV sufficient to cause artifacts. In analogous experiments with rat vas deferens, we also observed that BPV added to the tissue bath depressed the responsiveness of this tissue in a fashion similar to HVD (J. H. Kawamoto, J. A. Owen, A. Morales, and K. Nakatsu, unpublished observations). In addition, responses to PE from RVD of animals subjected to a similar general anesthetic procedure as for the human subjects were attenuated (%.H. Kawamoto, J. A.

Qwen, A. Morales, and K. Nakatsu, unpublished sbservations) . The results of this study indicate that 1 h of equilibration with repetitive washing was insufficient for HVD obtained with BPV as the local anesthetic, and a minimum of 2-3 h is required for full sensitivity. In addition, it was clear that the isolated HVD remains viable for at least 5 h with little change in responsiveness. The action of local anesthetics on smooth muscle is not a novel observation, but information regarding the nature and extent of the action provides part of the foundation of the validity of HVD as a useful phakmacological model. In RVD, low concentrations of procaine, lidocaine, cocaine, and tetracaine (< 1 p M did not induce any contractions in HVD. In contrast, higher concentration of these local anesthesics (> 1 r 2M) have been shown to noncompetitively inhibit the response to NE of the RVD and to competitively inhibit the response to ea2+(Grana et al. 1980). It is hypothesized that the inhibitory action is due to a lower availability of Ca2+ for contractile processes, since increasing the external ~ a " conce8tration has been shown to overcome the effect of local anesthktics (de Jong 1977). Also, it is thought that the inhibitory action is attributed to the wellknown membrane-stabilizing action of the local anesthesia, which render muscle cells less excitable, leading to a general depressant effect (Grana et al. 1980). This action is consistent with the depressant effect of BBV in the isolated HVD. The effect appears to be nonspecific because both the contractile responses to K+ and PE are depressed by BPV, with responses to Kf completely abolished at the concentration of BPV used (J. H. Kawamoto, J. A. Qwen, A. Morales, and K. Nakatsu, unpublished observation). This difference in degree of depression would largely be accounted for by differences in mechanism of deplarization. The isolated HVD obtained from individuals under general anesthesia were less sensitive to BE than HVD isolated under local anesthesia and subjected to prolonged washing. Since the general anesthetic procedure involves many drugs used in combination, any one of them could be responsible for the apparent depressant effect. The inhalational anesthetics, halothane and iwflurane, have been shown to depress contractions of isolated rat uterus and aorta when these gases were bubbled though the isolated tissue bath (Sprague et al. 1974; Yang et al. 1973). However, in this study the volatile anesthetics were administered to the subject and not directly to the isolated tissue; consequently they might not be exbected to have a long-lasting effect on the in vitro responses of the HVD. However, the use of the opioid fentanyl citrate as an adjunct to the fnhalational anesthetics may account for the decreased sensitivity. This compound is reported to have selective a-adrenoceptor antagonistic properties in the isolated rabbit aorta (Toda and Hatona 19'74). This property of fentanyl would be consistent with the decreased sensitivity of the isolated HVD to the al-agonist PE. A review of the medical charts of the vasectomy subjects showed that, with the exception of two individuals, all were clinically normal and were not on any medication. Of the two exceptions, one was being treated with salbutamol for asthma,


KAWAMOTB ET AL.

and the other had been treated for hypertension with hydrochlorothiazide. The responses to PE sf the HVD isolated from these individuals were not different from those of the others. The age of the subjects ranged from 24 to 53 years, and in this range there was no obvious correlation of age and HVD responsiveness to BE. This result confirms the reports of Hepperlen et al. (1976) who also found that the age of the subject from which the HVD was obtained had no effect on the responses of the isolated tissue to norephinephfine . The present data show that the increased responsiveness of the HVD to phenylephrine temporally correlates with a loss of BPV and that the general anesthetic procedure produces a longlasting depression of smooth muscle function. These results do not render the use of isolated HVD invalid, rather they point out some of the difficulties that must be considered;,these have not been specifically addressed in previous studies on HVD. In the long term, the advantages to be gained by developing this healthy and available human tissue preparation may far outweigh the problems encountered.

Acknowledgements This work was supported by the Botterell Foundation. JK is a recipient of an Ontario Graduate Scholarship. ALM,P. 1982. On the autonomic innervation of the human vas deferens. Brain Res. Bull. 9: 673-677.

ANTON,P. G,, and J. C . MCGRATH. 1977. Further evidence for adrenergic transmission in the human vas deferens. J. Physiol. (Landon), 273: 45-55. ASTROM, A. 1964. Influence of some Iocal anesthetics upon the adrenaline contraction of isolated strips of rabbit aorta. Acta Physiol. Scad. 60: 30-38. BELIS,J. A,, J. E. COLBY, and D. P. WESTFALL. 1982. Effects of a-adrenoceptor agents on norepinephrine release from vas deferens of several species including man. Eur. J. Phmacol. 7%:487-490. BIRMINGHAM, A. T. 1968. The human isolated vas deferens: its response to electrical stimulation and to drugs. Br. J. Phmacol. 34: 692P-693P.

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R. H. 1977. Local anesthetics. 2nd ed. Charles C . Thomas Publishing, Springfield, tL. GRANA, E., F. ZONTA, G. D'AGDSTINB, and G. DONDI. 1980. Action of lmal anesthetics on basal tone of rat vas deferens and on resDonse to norepinephrine and calcium. Arch. Int. Pharmacodyn. 247-306319. NEPPERLEN,T. W., W. F. DALSKE, and S. S. LACY.1976. Effect of prostaglandins, nitrofurantoin and Eseherichia eoli on response of human vas deferens to norepinephrine. Fertil. Steril.27: 275-28 1. MARTINS, T., J. R. VAEEE, and A. PORTO. 1940. Pharmacology in vitro of the human vasa deferentia and epididymis: the question of the endocrine control of the motility of the male accessory genitals. J. Urol. 44: 682-698. MCLEOB, D., D. G. REYNOLDS, and G. E. BEMAREE.1973. Some pharmacologic characteristics of the human vas deferens. Invest. Urol. 10: 338-341. NAITO, E., M. MATSUKI, and K. SHIMOJI. 1977. A simple method for gas chromatographicdetermination of lidocaine in tissues. Anesthesiology, 47: 466-467. RATNASOORIYA, W. D., R. M. WABSWORTH, and D. P. GILMORE. 1979. The effect of sympathomirnetic drugs on contractility of the vas deferens in vitro and in vivo. J. Reprod. FertiB. 56: 633-641. SPRAGUE, D. W., J. C. YANG,and S. M. NGAI.1974. Effects of isoflurane and halothane on contractility and the cyclic 3'5'adenosine monophosphate system in rat aorta. Anesthesiology, 40: 162-166. TODA, N., and Y. HATANO. 1977. Alpha-adrenergicblocking action of fentanyl on the isolated aorta of the rabbit. Anesthesiology 46: 41 1-416. VENTURA, W. P., M. FREUND,J. DAVIS,and C. PAN NU^. 1973. Influence of norepinephrine on the motility of the human vas deferens: a new hypothesis of sperm transport by the vas deferens. Fertil. Sted. 24: 68-77. VOHRA, M. M. 1970. An analysis of the contractile responses of the rat vas deferens to xylocaine (lidocaine) and procaine. Ew. J. PharmaC O 9: ~ . 14-20. YANG,J. C.,L. TRINER, Y. VULLIEMOZ, M. VEROSKY, and S. H. NGAI.1973. Effects of halothane on the cyclic 3',5'-adenosine monophosphate (cyclic AMP) system in rat uterine muscle. Anesthesiology, 38: 244-248. DE JONG,