Angiotensin effects on vas deferens adrenergic and purinergic neurotransmission

European Journal of Pharmacology, 146 (1988) 261-269

261

Elsevier EJP 50125

Angiotensin effects on vas deferens adrenergic and purinergic neurotransmission G e o r g e J. T r a c h t e Department of Pharmacology, University of Minnesota-Duluth, Medical School, Duluth, MN 55812, U.S.A.

Received 14 May 1987, revised MS received 29 September 1987, accepted 17 November 1987

Angiotensin effects on purinergic and adrenergic neurotransmission in the rabbit vas deferens were examined. Both angiotensins inhibited the non-adrenergic, and potentiated the adrenergic, neurogenic contraction. Angiotensin III inhibited the non-adrenergic neurogenic contraction to a greater extent than angiotensin II at all concentrations tested (maximal inhibition being 42 + 4 vs. 17 + 3% for angiotensin II). Angiotensin II was more potent than angiotensin III at potentiating adrenergic neurotransmission. Neither peptide altered the postjunctional action of either putative neurotransmitter, ATP or norepinephrine. These results are inconsistent with the hypothesis that ATP and norepinephrine are released in constant ratios. Furthermore, the different pattern of angiotensin responses is consistent with the existence of at least two separate angiotensin receptors with markedly different affinities for angiotensin 1I and angiotensin III. Angiotensin II; Angiotensin III; Purinergic neurotransmission; Adrenergic neurotransmission; Norepinephrine; Adenosine triphosphate

1. Introduction The vas deferens has been used as a model preparation for testing angiotensin effects on adrenergic nerves by a number of investigators (Magnan and Regoli, 1979; Sybertz and Peach, 1980). The vas deferens is one of the most densely innervated preparations in the body and the innervation of the longitudinal muscle is almost exclusively adrenergic (Furness and Iwayama, 1972; Anton et al., 1977). Therefore, the use of this preparation for assessing angiotensin effects on adrenergic nerves appeared logical. However, it has been shown that the neural response of the vas deferens is biphasic, consisting of a rapid twitch contraction followed by a sustained phasic contraction (Swedin, 1971). The twitch contraction is

* To whom all correspondence should be addressed.

resistant to adrenoceptor antagonists but is prevented by an A T P receptor antagonist, arylazidoaminopropionyl A T P (Fedan et al., 1981). The twitch contraction resembles an ATP-induced contraction (Kazic and Milosavljevik, 1980) and both are eliminated by the calcium channel blocker, nifedipine (Blakeley et al., 1981). The sustained neurogenic contraction is sensitive to a-adrenoceptor antagonists (Blakeley et al., 1981; Fedan et al., 1981). These results have been interpreted to indicate that the nerves of the vas deferens release two neurotransmitters, A T P and norepinephrine, in response to electrical stimulation. It is believed that the neurotransmitters originate from the same nerve because norepinephrine and ATP are stored together in adrenal granules (Douglas, 1968) and because the sympatholytic, 6-hydroxydopamine, eliminates both neurogenic contractile phases (Fedan et al., 1981; Sneddon et al., 1982).

0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

262 The purpose of this study was to assess the effect of the angiotensins on the adrenergic and non-adrenergic neurogenic responses. We previously have reported that angiotensins II and III had opposite effects on the integrated contractile response to nerve stimulation (Trachte et al., 1984). Therefore this preparation appeared to be appropriate for testing for multiple angiotensin receptor subtypes. Furthermore the effect of anglotensin peptides on purinergic neurotransmission has not been determined. The final goal of the study was to indirectly test the hypothesis of cotransmission of the two neurotransmitters. We reasoned that if the neurotransmitters originated in the same vesicles they would be affected identically by agents such as the angiotensins because their exocytotic release would necessarily be coupied to reflect their intragranular concentrations,

2. Materials and methods 2.1. General Vasa deferentia were removed from rabbits killed with pentobarbital sodium (50 m g / k g ) injected i.v. The vasa deferentia were placed in Krebs bicarbonate buffer of the following cornposition (in mM): NaC1 112, KC1 4.5, N a H C O 3 25, NaH2PO 4 1.2, MgSO4 0.5, glucose 10 and CaCI2 2.5. Each vas deferens was placed in an organ bath containing the Krebs bicarbonate buffer maintained at a temperature of 37 ° C. The vas deferens was attached to a glass anchor, passed through platinum ring electrodes and connected to a Ft 03c transducer for recording isometric force generation. One gram of resting force was added to each preparation.

dotoxin sensitive (Trachte et al., 1987) and are eliminated by agents which destroy adrenergic neurons (unpublished results). The preparations were stimulated for 10 s every 2 min. Angiotensins were added cumulatively at 10 min intervals to determine their effects on neurotransmission. The chart speed was increased during electrical stimulation to discern the two components of the response. 2.3. Postjunctional effects The putative neurotransmitters, ATP and norepinephrine, were administered in the presence and absence of the angiotensins to test for a postjunctional effect. Curves were constructed by adding ATP at 10 5, 1 0 - 4 o r 10 - 3 M at 90 min intervals to control or angiotensin-treated vasa deferentia. The 90 rain interval between doses was necessary because of tachypylaxis. Another P2 purinoceptor agonist, a,j~-methylene-ATP was similarly utilized to test for postjunctional effects of the angiotensins. Norepinephrine concentration-response curves, in the presence and absence of the angiotensins, were obtained by adding increasing concentrations of norepinephrine at 4 min intervals. The vasa deferentia were not electrically stimulated during this experiment. 2.4. Statistics Comparison of curves was performed by the analysis of variance for repeated measures (Dixon, 1983). Individual values and ECs0s were compared by Student's paired t-test. 2.5. Materials

2.2. Electrical stimulation Nerves were activated by electrical pulses of 10 mV (at the electrode), at a frequency of 2 Hz and a stimulus duration of 1 ms. Swedin (1971) found electrical stimuli with durations of less than 10 ms to selectively affect nerves and not smooth muscle. We have confirmed that our stimulus parameters selectively activate nerves because they are tetro-

Angiotensins II and III were purchased from Bachem Chem. Co. (Torrance, CA) and the carboxy-terminal hexapeptide and pentapeptide derivatives of angiotensin II were purchased from Chemical Dynamics Corp. (South Plainfield, N J). Norepinephrine, a,fl-methylene ATP and ATP were purchased from Sigma Chemicals (St. Louis, MO).

263

3. R e s u l t s

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3.1.Effectoftheangiotensinsonneurotransmission A typical response to angiotensins II and III is shown in fig. 1. Neither angiotensin affected basal force, indicating an absence of a postjunctional effect on the smooth muscle of the preparation. Both angiotensins depressed the initial (nonadrenergic) contraction in a concentration-dependent manner. Angiotensin II potently augmented the adrenergic contraction and angiotensin III elevated it at concentrations of 100 n M or greater. Angiotensin III consistently depressed the nonadrenergic contraction to a greater extent than angiotensin II. These results indicate that the angiotensins have qualitatively similar effects on this preparation but quantitatively the effects are quite different. The angiotensins also affect the two neurogenic c o m p o n e n t s oppositely, depressing the non-adrenergic while enhancing the adrenergic, The effect of angiotensins II and III on nonadrenergic neurogenic contractions is presented in fig. 2. The average basal force was 1.77 + 0.16 g. Angiotensin II maximally depressed this contractile c o m p o n e n t 17--+ 3%. Angiotensin III had a

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muchlargereffectdepressingforce42-+4%.The two curves were statistically different (P < 0.001). Angiotensin I I I effects were greater than those of angiotensin II at all concentrations tested (P < 0.05). Nevertheless, angiotensin II was more potent with an EC~0 of 3.0-+0.55 nM, while that to Angiotensin effects on the adrenergic neurogenic contraction are shown in fig. 3. Basal force averaged 1.01 -+ 0.12 g. Angiotensin II potentiated force generation at all concentrations tested. The largest effect observed was a potentiation of 144 -+ 22%. Angiotensin III was less potent, only increasing force at concentrations exceeding 10 nM and the largest observed increase was 102 -+ 20%. The curves were statistically different (P < 0.001). The ECs0 for the two peptides was 6.6 -+ 1.6 n M for angiotensin 1I and 100 -+ 21 n M for angiotensin I I I (P < 0.001), assuming that the largest effect observed was the maximal effect.

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scntially cquipotcnt manner. The greatest ohserved depressions averaged 3~ ± ~% for the hcxapcptidc and ~0 + ~% for the pentapcptidc. Both a~cnts were as effective, or more so, than angiotcnsin II at supprcssinB these contractions, They were less potent requiring concentrations greater than I p M to have a significant effect, The effect of these agents on adrcncrgic ncurotransmission is presented in fig. 5. Both pcpfidcs potentiated adrcncr~c ncurotransmJssion at conccntrations exceeding 1 /~M. The greatest effects observed wcrc 5 9 ± ]6% potentiation for the hc×apcptidc and 56 ± 27% potentiation for the pentapeptide. The potentiation was not as great as that associated with angiotensin II or III.

3.2. Postjunctional actions of the angiotensins The effect of angiotensins II and I I I (100 nM) on the contractile response to exogenously administered ATP, a,/3-methylene-ATP and norepinephrine are shown in figs. 6, 7 and 8. Neither angiotensin significantly influenced the contractile response to ATP, or a,/3-methylene-ATP. Similarly,

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neither angiotcnsin altered the response to norcpincphrinc, as indicated in fig. ~. The anglotcnsin Ill curve appears depressed but did not differ significantly from vehicle treatment when either the curve or the E ~ 0 wcrc compared. These resultsindicate that angJotcnsins do not influence the post~unctiona] effect o~ the putative ncurotransmittersin this preparation.

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Figure 9 is a composite graph of the angiotensin actions on both the adrenergic and the nonadrenergic neurogenic contractions. Angiotensin II primarily enhanced adrenergic responses which is indicated by a vertical rise on the graph. In contrast, angiotensin II only modestly depressed non-adrenergic neurogenic contractions, as indicated by a horizontal distribution in the figure. Angiotensin III had the opposite spectrum of ac-

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tivity, primarily inhibiting non-adrenerNc neurogenic contractions but potentiating adrenergic responses to a smaller degree than angiotensin II. The smaller angiotensins had activities intermediate between those of angiotensins II and III.

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As mentioned in the Introduction, numerous studies have concluded that two neurotrans~tters a r e involved in the response to electrical stimulation in the vas deferens of guinea pigs and rabbits. T h e e,-e~reva;1;n" o~;n;one,, is t h a t y*~et~ a r e released together. T ~ s is supported by the fact that sympatholytic agents such as guanethidine (Swedin, 1971; Furness, 1974; McGrath, 1978)and 6-hy-

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droxydopa~nc (Fcdan ctal., ]98]; Sncddon ct a]., ]982) cfi~natc both electrically induced contracfi]c eventS. R c s c ~ n c selectively cIJm~natcd the a~rcncr~Jc ~curo~c~c co~tractJon w i t h o u t a ] t c r J ~

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(% of control) Fi~. 9. Composite ~raph showin~ an~iotcns~ncffccts on both the non-adrcncr~c and adrcncr~c ncuro~cnJc contractions. Values arc mcans~S.E.M. The number o~ preparations per ~roup ~s presented in o(hcr fi~urcs. ~n~iotcnsJn 11 primarily potentiatedadrcncr~Jc ncurotransmJssion, represented by an upward dc~cction, and sli~htl~ inhibited non-adrcncr~ic ncurotransmission. Both the pcnta- and hcxapcpfidc ~ra~mcnts potentiated adrcncr~ic ncuro~ransmiss~onmore than they inb~bit~d non-adrcncr~Jc responses. ~n~Jotcnsin III primarily inhibited non-adrcncr~ic events. Onl~ (hc responses to the three lowest an~otcnsin II and 111 concentrations arc shown becausethe non-adrcncr~Jcrcsponscto thesea~cnts rcvcrsc~ at hi~hcr concentrations.

Grath, ]978; Sncddon and Wcsffall, 1984). Thus

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a common origin ~or the two n c u r o t r a n s ~ t t c r s ~n the vas de~crcns at present, U n t i l t ~ s stud~ there wcrc two pubfishcd reports w ~ c h arc clca~ly in conflict w i t h the bypothcsis o~ coupled corc1casc o~ ncurotransmittcrs in the vas dc~crcns. I n i t i a l l y , Swcdin (]971) ~ound prosta~Iandin E 2 to almost totally abolish the non-adrcncr~c contraction at a concentration w h i c h slightly potentiated the adrcncr~ic contracfion in electrically stimulated ~uinca pi~ vas de6crcns. The prosta~landin somewhat potentiated the response to exogenous norcp~ncphdnc but not ~nou~h to account ~or the totally opposite effects o~ the autocoid on the two neuronal phases. ~ o r c

ported that immature rat vasa dc6crcnfia r~spondcd to an electrical stimulus w i t h a twitch contraction that was resistant to ~-adrcnoccptor antagonists. ~orcp~ncphrJnc contracted the immaturc vas dc~crens, indicafin~ that a postjunctional alteration Jn sc~sJtJv~t~ was ~ot responsible ~or the absence o~ the adr~ncr~ic contractile phase. The a u t h o r s ' c o n c l u s i o n was that J n s u f f i c i c n [ norcpincphrinc was bcm~ rc]cascd from nerves to cause a response but the non-adr~ncr~c ncurotransmitter was bc~n~ released in adcquatc amounts to mediate the twitch contraction. There~orc, nerves Jn th~ immature rat vas dc~crcns appear to release a non-adrcncr~ic ncurotrans-

267

mitter, presumably ATP, in the absence of norepinephrine. These two studies seriously question the hypothesis of cotransmission of norepinephrine and ATP from the same secretory vesicles which would require them to be released in a constant ratio, Our results are the first report of an agent having opposite effects on the two contractile responses to low frequency (2 Hz) nerve stimulation in the vas deferens. All the angiotensins tested were observed to inhibit the non-adrenergic and to potentiate the adrenergic neurogenic contraction, The potentiative effect predominated with angiotensin II and the inhibitory effect was the most obvious effect of angiotensin III. Since neither of the angiotensins altered the postjunctional effects of the putative neurotransmitters, it is assumed that these effects were of a prejunctional nature. Such a scenario is suggestive of separate origins of the two neurotransmitters in order to explain the simultaneous potentiative and inhibitory effect of the angiotensins on their release.

lease of adrenergic neurotransmitters from nerves (Bennelli et al., 1964; Peach, 1977) and adrenal medulla (Feldberg and Lewis, 1964; Westfall, 1977). The potentiative effect of the angiotensins on the adrenergic neurogenic contraction of the vas deferens was expected from the numerous reports of the potentiative action of angiotensin II on vas deferens neurotransmission (Bennelli et al., 1964; Magnan and Regoli, 1979; Sybertz and Peach, 1980). Our results (Trachte et al., 1984; Saye et al., 1986; current study) and most reports in the literature attribute the potentiative action to an increase in the release of norepinephrine and not to a postjunctional action of the angiotensins. This is consistent with angiotensin actions on other adrenergic nerves. 4.4. Effect of angiotensin fragments on neurotransmission

The hexapeptide and pentapeptide carboxyterminal fragments of the angiotensins had impotent effects on vas deferens neurotransmission.

4.2. A ngiotensin effects on purinergic neurotransmission

They altered neurotransmission in a manner intermediate between that of angiotensin II and III, being slightly more likely to enhance adrenergic

As mentioned above, the angiotensins inhibited the putative purinergic component of neurotransmission. Angiotensin III was more effective at inhibiting this component but angiotensin II was more potent. The smaller fragments also effectively inhibited neurotransmission although they were extremely impotent. These conclusions about angiotensin effects on purinergic neurotransmis-

neurogenic contractions than to inhibit nonadrenergic neurogenic contractions. Both of these agents were capable of inhibiting the non-adrenergic response at least as effectively as angiotensin II. Therefore both compounds have full intrinsic activity in comparison to angiotensin II but were 1000-fold less potent. This is in agreement with

sion are indirect because ATP release was not actually measured. Attempts to measure neuronal ATP release have met with frustration because the majority of labelled adenosine released in response to nerve stimulation originates from the smooth muscle (Fredholm et al., 1982). We conclude that angiotensins inhibit the neuronal release of ATP but recognize that direct studies must be performed to ascertain this conclusion,

4.5. Angiotensin receptors

4.3. Angiotensin effects on adrenergic neurotransmission

Angiotensins are known to potentiate the re-

findings in numerous other systems (Peach, 1977).

The different pattern of angiotensin responses observed in this preparation is unique. The angiotensins have similar effects in most systems tested but the peptides smaller than angiotensin II have lower potencies. Because of the dual response of the vas deferens it is possible to test the similarity in the angiotensin responses quantitatively. Our findings are that the angiotensins exhibit totally different patterns of responses. This fact is inconsistent with the presence of only one homogenous angiotensin receptor in this preparation, as has been concluded from previous work (Magnan and

268

Regoli, 1979) unless the processing or coupling of the receptors to biological responses differs for angiotensins II and III. Furthermore, the action of angiotensin III on the non-adrenergic neurogenic response is greater than that of angiotensin II. This has only been observed at two other sites, the adrenal glomerulosa (Goodfriend and Peach, 1975) and the central nervous system (Fink and Bruner, 1985). We speculate that the vas deferens contains at least two angiotensin receptors with markedly different affinities for angiotensins II and lII. One of these receptors may resemble those in adrenal glomerulosa or brain. 4. 6. Summary In conclusion, angiotensins were observed to inhibit non-adrenergic neurotransmission in the vas deferens, presumably by a prejunctional mechanism of action. Angiotensin III depressed n o n adrenergic neurotransmission t o a greater e x t e n t than angiotensin II, a finding which is relatively unique. The patterns of angiotensin responses o n the non-adrenergic and adrenergic neurogenic r e s p o n s e s w e r e completely distinguishable which is suggestive of the existence of multiple angiotensin receptor subtypes in this preparation. The angiotensins potentiated adrenergic neurotransmission simultaneously while inhibiting non-adrenergic neurotransmission. The opposing effects o n n o n adrenergic and adrenergic neurotransmission a r e in conflict with the hypothesis that the neurotransmitters of the vas deferens are coreleased from the s a m e secretory granules if angiotensin actions a r e indeed prejunctional, as our results suggest. The vas deferens preparation represents a model system for investigating the cotransmission hypothesis. Additionally the unique different pattern of responses to the angiotensins in this preparation identify it as a very promising preparation for the study of angiotensin receptors.

Acknowledgements The expert technical assistance of E. Stein and R. Marzsalek is greatly appreciated, as is the manuscript preparation by S. Kurki. This work was supported by Public Health Services Grant No. R01 HL 34600 from NHLBI.

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musculotropic responses to angiotensin peptides in normal and sodium-restricted rabbits, Circ. Res. 46, 836~ Trachte, O.d., E. Stein and M.d. Peach, 1987, Alpha adrenergic receptors mediate angiotensin-induced prostaglandin production in the rabbit isolated vas deferens~ J. Pharmacol. Exp. Ther. 240, 433. Trachte, G.J., E.J. Sybertz, M. Michener, S.B. Binder and M.J. Peach, 1984, Angiotensin Ill-induced modulation of neurogenic responses in the rabbit vas deferens and portal vein, Naunyn-Schmiedeb. Arch. Pharmacol. 326, 327. Westfall, T.C., 1977, Local regulation of adrenergic neurotransmissiom Physiol. Rev. 57, 659.