European Journal o f Pharmacology, 34 (1975) 397--400
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication EFFECTS OF VARIOUS D E N E R V A T I O N TECHNIQUES ON THE ATP OF THE RAT VAS D E F E R E N S * DAVID P. WESTFALL, KATSUTOSHI GOTO, ROBERT E. STITZEL, J E F F R E Y S. FEDAN and WILLIAM W. FLEMING
Department o f Pharmacology, West Virginia University Medical Center, Morgantown, West Virginia 26506, U.S.A. Received 30 September 1975, accepted 22 October 1975
D.P. WESTFALL, K. GOTO, R.E. STITZEL, J.S. FEDAN and W.W. FLEMING, Effects o f various denervation techniques on the A T P o f the rat vas deferens, European J. Pharmacol. 34 (1975) 397--400. Postganglionic denervation of the vas deferens of the rat by surgical means or pretreatment with 6-hydroxydopamine results in a substantial decrease in the concentration of ATP in the tissue. The loss of ATP cannot entirely be accounted for by the loss of neuronal ATP nor by a compromised blood supply. The results suggest that the postganglionic nerve plays an important r(~le in the regulation of the ATP of the effector cells. 6-Hydroxydopamine Deferential artery Postganglionic denervation
1. Introduction Westfall et al. (1975} have reported that procedures which induce non-specific supersensitivity of the guinea pig and rat vas deferens to drug stimulation produce alterations in the ATP concentration of these tissues. For example, decentralization of the vas deferens or pretreatment of animals with reserpine result in a significant increase in ATP concentration. The effects of postganglionic denervation, however, are more complex in that there are biphasic changes in the ATP concentration. 24 hr after denervation the tissue concentration of ATP was only 20% of control in the rat vas deferens and 50% of control in the guinea pig vas deferens. At subsequent days after denerva* Supported by National Institute of Neurological Diseases and Stroke grant NS 080300 and a grant from the West Virginia Heart Association.
tion there was a gradual rise in the ATP concentration. The purpose of the present investigation was to determine whether this initial, profound loss of ATP is a genuine consequence of adrenergic degeneration or whether the loss is related to other factors such as a compromise of the blood supply to the tissue. This question is raised because the technique that was used to produce denervation also removed the deferential artery. The following procedures were investigated, therefore, for their effect on the concentration of ATP in the rat vas deferens. (1) Denervation by desheathing the tissue in the manner described by Birmingham (1970). This technique removes the deferential artery. (2) Denervation by sectioning the postganglionic nerves emanating from the hypogastric plexus in the manner described by Kasuya et al. (1969). This technique leaves the deferential artery intact. (3) Ligation of the deferential artery without desheathing the tissue. (4) De-
398 nervation by pretreatment with 6-hydroxydopamine (6-OHDA).
2. Materials and methods 2.1. General Sprague--Dawley rats, ranging in weight between 200--400 g, were used for these studies. The rats were killed by a blow on the head and the vasa deferentia were quickly removed and cleaned of connective tissue. The freshly dissected vasa deferentia were homogenized in 6.0 ml of 0.4 N perchloric acid to which had been added 0.1 ml of 1% ascorbic acid and 0.2 ml of 10% ethylenediamine tetraacetic acid. The homogenate was centrifuged at 9000 X g for 15 min. 20 pl aliquots were taken from the supernatant for ATP determinations and the remainder was used for the determination of endogenous norepinephrine. ATP was measured by the method of Stanley and Williams (1969) which utilizes the firefly luciferin--luciferase system in a liquid scintillation spectrometer. Norepinephrine, after separation from deaminated metabolites by ion-exchange chromatography, was analyzed spectrophotofluorimetrically by the method of Bertler et al. (1958).
D.P. WESTFALL ET AL. intended to interrupt the blood supply to the tissue without producing denervation. For these experiments the deferential artery was ligated at a site close to the prostate gland. The artery was severed after ligation. With each of the three procedures, only one tissue was subjected to the surgical manipulation; the contralateral vas deferens remained intact and served as control. The animals were killed at 1, 2, 4 and 7 days after the surgery and the tissues prepared as described above. 2. 3. Pretreatment with 6-OHDA Rats received two injections of 6-OHDA hydrobromide via the dorsal vein of the penis. The first dose was 100 mg/kg; the second dose, administered 24 hr after the first was 250 mg/kg. This treatment schedule has been shown to produce an effective denervation of the rat vas deferens (Westfall and Fedan, 1975). The control tissues for these studies were obtained from animals which received saline injections. The endogenous norepinephrine and ATP concentrations of these tissues were determined at 1 and 7 days after the second injection of 6-OHDA.
3. Results 2. 2. Surgical procedures Tissues were subjected to two different surgical techniques for producing postganglionic denervation. The vasa deferentia of one group of rats were denervated by the m e t h o d of Birmingham (1970). This method involves stripping away the mesentery of the vas deferens leaving the tissue attached at only the epididymal and prostatic ends. The desheathing procedure removes the deferential artery. A second group of tissues was denervated by the method of Kasuya et al. (1969). This technique involves sectioning the postganglionic nerves emanating from the hypogastric plexus. Because this method does not require the desheathing of the tissue, the deferential artery remains intact. A third surgical procedure was
The effects of the three surgical procedures on the norepinephrine and ATP concentrations of the rat vas deferens are summarized in fig. 1. 24 hr after either denervation procedure, the norepinephrine concentration of the vas deferens was reduced by approximately 90% compared to the contralateral intact tissue. At subsequent days after denervation the norepinephrine concentrations were even less. The two denervation methods produced qualitatively similar changes in the ATP concentration of the vas deferens. There was an initial decrease followed by a gradual rise of ATP. However, quantitative differences existed between the two techniques in that denervation by the method of Kasuya et al. (1969) reduced the ATP concentration by 50% rather than by
ATP A F T E R D E N E R V A T I O N
U a,t i
DAYS AFTER: ARTERY LIGATION
DEN (KASUYA et.al. )
Fig. 1. The norepinephrine (NE) and ATP c o n c e n t r a t i o n (as percent of control) at 1, 2, 4 and 7 days after various surgical procedures. The mean NE and ATP concentrations of all controls were 12.7 _+ 0.2 pg/g wet weight and 2.7 _+ 0.06 pM/g wet weight respectively (n = 92). A, Denervation by the m e t h o d of Birmingham (1970). Each p o i n t represents the mean o f 8--16 experiments. B, Ligation of the deferential artery. Each p o i n t represents the mean of 4 experiments. C, Denervation by the m e t h o d of Kasuya et al. (1969). Each p o i n t represents the m e a n of 5--8 experiments. Vertical bars represent standard errors.
80% 1 day after surgery. Ligation of the deferential artery resulted in a 25% decrease in the ATP concentration of the vas deferens. 2 days after artery ligation the ATP concentration was somewhat higher than after one day, but remained significantly less than control. At 4 and 7 days after ligation the ATP concentrations were not significantly different from control. Ligation of the deferential artery also resulted in a 50% decrease in the norepinephrine concentration of the tissue. This loss of norepinephrine persisted for at least seven days after the surgery. Pretreatment of rats with 6-OHDA resulted in a substantial loss of norepinephrine from the vas deferens. At 1 and 7 days after the second injection of 6-OHDA the norepinephrine concentrations were 3.1 + 0.3% (n = 8) and 12.0 + 0.9% (n = 8) of control respectively. These tissues also exhibited a statistically significant reduction in ATP concentration. At 1 and 7 days after 6-OHDA the concentrations of ATP were 69.0 -+ 6.7 and 85.3 + 4.2% of the values obtained in tissues from saline-treated animals.
4. Discussion Both surgical techniques utilized in this study for producing denervation of the vas deferens resulted in an initial decrease (at one day postoperatively) in tissue ATP c o n t e n t followed by a gradual rise (from 2 to 7 days postoperatively). Our estimate of the concentration of neuronal ATP suggests that loss of this fraction contributes only minimally to the change in total tissue concentration of ATP. For example, the concentration of ATP in association with storage of norepinephrine is 0.019 #M/g wet weight (based on a norepinephrine concentration of 13 pg/g and a molar ratio of 4 norepinephrine: 1 ATP). This value is only 0.7% of the total ATP concentration (2.7 pM/g). Even if there is additional ATP in the nerves which is not associated with storage of transmitter, the concentration would be small compared to the total ATP. This view is taken because an analysis of electron micrographs of the vas deferens indicates that the volume of nerve is only 1/200 the volume of muscle (L.
Millecchia, personal communication). The pronounced alterations in the concentration of ATP in the denervated rat vas deferens, therefore, are probably occurring in the smooth muscle. The magnitude of the initial fall in ATP was dependent on the procedure. There was an 80% decrease in ATP following the denervation technique of Birmingham (1970), while the method of Kasuya et al. (1969) produced only a 50% loss of ATP. Because ligation of the deferential artery was found to produce a 25% decrease in the concentration of ATP, it is tempting to ascribe the quantitative differences between the two techniques to the fact that the method of Birmingham removes the deferential artery while the method of Kasuya et al. does not. The results seen after artery ligation are not entirely clear, however, because ligation alone produced a 50% depletion of norepinephrine, indicating that some denervation occurs as a consequence of this procedure. Nevertheless, there is at least a 50% decrease in the concentration of ATP 24 hr after surgical denervation which cannot be explained by a loss of blood supply to the tissue. It could be argued that trauma to the tissue as a result of the surgical procedures initiated the alteration in ATP levels and that the phen o m e n o n is independent of denervation. This does not appear to be the case however, because 'chemical denervation' of the tissue by pretreatment with 6-OHDA also produced a significant decrease in ATP concentration. It can be concluded from these studies that denervation of the rat vas deferens results in a substantial decrease in the concentration of ATP of the tissue irrespective of technique
D.P. WESTFALL ET AL.
used to produce denervation. The loss of ATP cannot be accounted for solely by the loss of neuronal ATP or by a compromized blood supply. Furthermore, interrupting neuronal transmission without producing degeneration of the postganglionic nerves does not produce this change since decentralization or pretreatment with reserpine increase, not decrease, tissue ATP (Westfall et al., 1975). Although the present studies do not delineate the mechanism by which adrenergic nerves control smooth muscle ATP concentration, there is little d o u b t that nerves influence muscle energy stores to a degree not suspected previously.
References Bertler, A., A. Carlsson and E. Rosengren, 1958, A method for the fluorimetric determination of adrenaline and noradrenaline in tissue, Acta Physiol. Scand. 44, 273. Birmingham, A.T., 1970, Sympathetic denervation of the smooth muscle of the vas deferens, J. Physiol. (London) 206, 645. Kasuya, Y., K. Goto, H. Hashimoto, H. Watanabe, H. Munakata and M. Watanabe, 1969, Nonspecific denervation supersensitivity in the rat vas deferens 'in vitro', European J. Pharmacol. 8, 177. Stanley, P.E. and S.G. Williams, 1969, Use of liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase system, Anal. Biochem. 29, 381. Westfall, D.P. and J.S. Fedan, 1975, The effect of pretreatment with 6-hydroxydopamine on the norepinephrine concentration and sensitivity of the rat vas deferens, European J. Pharmacol. 33, 413. Westfall, D.P., T.J.-F. Lee and R.E. Stitzel, 1975, Morphological and biochemical changes in supersensitive smooth muscle, Federation Proc. 34, 1985.