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Differential effects of neuropeptide Y and opioids on neurogenic responses of the perfused rat mesentery
195 (1991) 365-372 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100261E Europeun Journal of Phar.macologv,
365
c’ 1991
UP 51799
ifferential effects of neuropepti of the perfused rat mesentery Yuanjian Li and Sue P. Duckles Department
of Pharmacology.
College oj Medicine,
Utlicersily
of
California,
Irvine.
CA 9’717.
ti. S.A.
Received 8 January 1991. accepted 15 January 1991
In perfused rat meseniery transmural nerve stimulation activates both adrenergic and capsaicin-sensitive sensory nerves. When adrenergic nerves were blocked with guanethidine and smooth muscle tone was increased. transmural nerve stimulatisn caused a dilator response which was attenuated by tetrodotoxin and abolished by capsaicin. Indomethacin increased the vasodilator response to transmural nerve stimulation, but did not affect the dilation to calcitonin gene-related peptide. Neuropeptide Y (NPY) potentiated vasoconstrictor responses to transmural nerve stimulation. but suppressed capsaicin-sensitive vasodilation. an effect which was unaltered by indomethacin. Opioid agonists selective for ~_r,S or K receptors. DAMGO ([D-Ala’.N-Me-Phe4.Gly’-ol]enkephalin), DPDPE ([D-Pen*.D-Pen’lenkephalin) and ethylketocyclazocine. had no effect on the vasoconstrictor response to transmural nerve stimulation. DAMGO and DPDPE significantly inhibited vasodilator responses to transmural nerve stimulation. but ethylketocyclazocine was without effect. After treatment with indomethacin. DAMGO still inhibited the vasodilator response. but DPDPE was no longer effective. Prejunctional control of transmitter release by NPY’ or opioids is dependent on the specific nerve type as well as, in some cases, the participation of endogenous prostaglandins. Mesentery (rat); Capsaicin; [D-Ala’.N-Me-Phe4,GIy5-ollenkephalin):
CGRP (calcitonin gene-related peptide); Neuropeptide Y; DAMGO DPDPE ([D-Pen’.D-Per?]enkephalin): Ethylketocyclazocine: lndomethacin
1. Introduction
It is clear that peptides mitters and are coreleased vascular nerves, including
coexist with classical transfrom several types of perineuropeptide Y (NPY) with
norepinephrine in sympathetic nerves (Lundberg et al., 1984). vasoactive intestinal polypeptide with acetylcholine in parasympathetic nerves (Lundberg, 1981). and substance P with calcitonin gene-related peptide (CGRP) in primary afferent sensory nerves (Lundberg et al., 1985a). Furthermore, interactions of peptide and non-peptide neurotransmitters and with various autacoids. including prostaglandins. have been shown to occur both pre- and postjunctionally (Mantelli et al., 1990; Burnstock. 1987). It is interesting that NPY and opioids are capable of inhibiting not only norepinephrine released from sympathetic nerves. but also neurotransmission in primary sensory afferent nerves via a prejunctional mechanism (Pohl et al. 1989; Grundemar et al.. 1990; Crain and Shen, 1990). Recently, the presence of a non-adrenergic. noncholinergic vasodilator innervation was demonstrated in
Correspondence to: S.P. Duckles. Department of Pharmacology. College of Medicine. University of California. Irvinr. CA 92717. U.S.A.
the rat perfused mesentery (Manzini and Perretti. 1988: Kawasaki et al., 1988. 1990: Fujimori et al.. 1989). The capsaicin sensitivity of these nerves. the finding thrtt nerve stimulation evokes release of CGRP. as well as the low levels of substance P in this tissue have led to the proposal that CGRP is the principle neurotransmitter of these vasodilator sensory nerves. The purpose of the present study was 3-fold. First. we tested vasoconstrictor and vasodilator responses to transmural nerve stimulation of the perfused rat mesentery and the effect of desensitization to sensory nerve transmitters with capsaicin. Secondly. we investigated the possible contribution of endogenous prostaglandins to vasodilator responses. Thirdly. the modulatory actions of NPY and opioids “r-r responses of sympathetic vasoconstrictor and capsaicin-sensitive vasodilator nerves were compared.
2. Materials
and methods
Mesenteric vasculature of male Sprague-Dawley rats (14-20 weeks old weighing 400 & 70 g) was isolated and prepared for perfusion as described by Kawasaki and Takasaki (1984) with some modifications. Rats were anesthetized with pentobarbital sodium at a dose of 75
it~t~ti~~e and sssocisted vasculsr bed. the mesenteric vs?;&ar bed was flushed with warm Krehs solution and plmced iIt a \vater jacket (volume X0 ml) nlaintained at 37”~. the system was perfused with warm Krebs solutioa~. saturated with 95B 0, and 5C; CO, with the help of ;t peristaltic pump at a rate of 5 + 0.2 ml/min. and superfused by gravity fwd at a rate of 1 t 0.2 ml/min. The Krehs solution had the following composition (in mM): NaCI llS: KC1 4.8: CiiCI, 2.5: KH:PO, 1.2: NaHCO, IS: MgSQ 1.2: EDTA . Na 0.107: and dextrose 11.5. The perfusion pressure was monitored and recorded by a pressure transducer and a Soltec 1242 chart recorder. The tissue was equilihratcd for 60 min before beginning each study. Two platinum eie&rodes. one placed around the superior mesenteric artery and the other resting on the vasculature in a lower part of the intestine. \vere used to create transmural field stimulation. For constrictor responses to trsnsmural nerve stimulation. 3-5 tnin were al!owed between each stimulation train. fn the case of vasodilator responses to transmural nerve stimulation. sufficient time was allowed between each stimulation train for the perfusion pressure to return to a stable level. usually IO-20 min. Drugs were administered by switching the perfusion solution to solution containing drug in the concentrations indicated. Means of four peaks of responses to transmural nerve stimulation that came before administering a drug were used as control responses. 2. I. E_~p~ri~~leittai protoco!s Responses to transmurai nerve stimulation were stable in the absence of drug application. Where indicated, tissues were pretreated with guanethidine (5 x lo-” M) fcr 20 min. or aith indomethacin (lOeh M) for 30 mm hefore testing responses to transmurdl nerve stimulation or CGRP. In these cases. guanethidine and indomethatin remained in the perfusate throughout the remainder of the experiment. Exposure to capsaicin (3 x lo-’ M) was for 20 min. and capsaicin was then washed out. For all studies a paired design was used. that is the same tissue was studied both before and after treatme,lt with capsaicin. indometha~in, guanethidine. NPY or opioid agonists.
The following drugs were used: neuropeptide Y, [DAla~.N-Mu-Phe~.Gly~-ol]enkephalin {DAMGO). [D-
Pen~.D-Fell~]e:nkephalin (DPDPE) and calcitonin generelated peptide (CCRP) (Peninsula); ethylketocyclazotine (EKC) {Sterli~lg-Wil~throp Res. tns.): tetrodoloxin. indomethacin. naloxone and methoxamine HCI (Sigma): ~uanethidine (Ciba Pharmaceutical Co.); and capsaicin (iCN Biomedicals. Inc.). All drugs were dissolved in Krebs solution. except capsaicin and indometh~~~in. which were initially dissolved in ethanol and further diluted in Krebs solution to the proper final concentration. 23. Sturi.h3 Results are expressed as means at S.E.M. Student’s paired t-test was used to determine statistical differences between two means. The level of significance was chosen as P < 0.05.
3. Resul;s 3. f. Vasaco~tst~~~tarand ~?usadiIatarrespmses to tra~~s~lMrul nerve stirmdution In the perfused mesenteric vascular bed, transmural nerve stimulation produced a frequency-dependent increase in perfusion pressure (fig. 1A). This response was completely blocked after treatment with guanethidine (-5 X 10eh M), confirming that it is due to stimulation of adrenergic nerves. Since it has been demonstrated that transmural nerve stimulation can release both dilator and constrictor transmitters, the effect of prior treatment with capsaicin to desensitize the tissue to sensory nerve stimulation was tested. As shown in fig. lB, during treatment with capsaicin (3 x 10m7 M) the presSOT response to transmural nerve stimulation was markedly inhibited. However. after 20 min capsaicin treatment. and when capsaicin was washed out of the bath. there was a marked increase in the pressor response to transmural nerve stimulation. The efficacy c:f our protocol for capsaicin treatment in desensitizing sensory nerves was verified by demonstration that inhibition of transmural nerve stimulation vasoconstrictor responses by capsaicin was abolished by prior exposure to capsaicin, 3 x lo-’ M for 20 min. First exposure to capsaicin caused an inhibition of contractile responses to transmural nerve stimulation of 56 +_78, whereas the second exposure to the same concentration of capsaicin produced no relaxation (n = 3). After blockade of sympathetic responses with guanethidine. methoxamine, 3 x 10. ’ M. was added to increase smooth muscle tone. In this situation, transmural nerve stimulation caused a vasodilator response which was frequency-dependent, especiatly at low frequencies (fig. 2). Vasodilator responses to transmural nerve stimulation were markedly attenuated by tetro-
367
3.2. EJiect oj NPY
2
16
As has been previously reported in a variety of tissues (Vu et al.. 1989). NPY (lo-’ M) significantly potentiated vasoconstrictor responses to transmural nerve stimulation (fig. 4A). This response was not affected by pretreatment with capsaicin (percentage increases in pressure, control and capsaicin. were 110 5 20 and 104 + 14. respectively. P > 0.8. n = 8). in contrast. vasodilator responses (after treatment with guanethidine and in the presence of methoxamine) were significantly depressed in the presence of NPY (fig. 4B). The inhibitory effect of NPY was not affected by pretreatment with indomethacin (fig. 4B) (P > 0.7 for comparison of the effect of NPY in untreated and indomethacin treated tissues). We also tested the effect of NPY on the vasodilater response to exogenous CGRP. Exogenous CGRPinduced vasodilator responses were not affected by NPY treatment (percentage relaxations. control and NPY. were 69 + 6 and 68 f 6. respectively: P > 0.7. n = 4). 3.3. Effects of opioid prptide3.
BEFOFIE
CAP
AFTER
Fig. 1. Frequency dependence of vasoconstrictor responses to transmural nerve stimulation and effect of capsaicin exposure in the perfused mesenteric vascular bed. (A) Contractile responses tu transmural nerve stimulation for trains of 100 pulses (30 V. 2 mb pulse duration) are plotted as increase in perfusion pressure as a function of stimulus frequency. (3) Responses to transmural nerve stimulation (8 Hz. 40 pulses. 70 V. 5 ms) before, during and after exposure to capsaicin (3 x lo-’ M. 20 min) are indicated. Values are means? S.E.M (n = 4). *** P
dotoxin (lo-(’ M) (percentage inhibition was 70 IL 3. n = 5) and completely abolished after treatment with capsaicin (3 x lo-’ M) for 20 min (n = 5). Treatment with indomethacin (lO-h M) to inhibit cyclooxygenase resulted in a significant increase in the vasodilator restonse to transmural nerve stimulation at all frequencies of stimulation (fig. 3A). When relaxation responses to exogenous calcitonin gene-related peptide were studied in the presence of indomethacin. there were no significant differences in vasodilator responses compared to control, regardless of the concentration of CGRP (P > 0.05) (fig. 3B).
Three opioid receptor agonists were selected to represent actions on three types of opioid receptor: DAMGO (p). DPDPE (6) and EKC (K). Concentrations of these agonists which have previously been shown to be selective for their respective receptors were used (Berzetci et al.. 1987: Budai and Duckles. 1988). As shown in fig. 5A. none of these three had any effect on pressor responses to transmural nerve stimulation. In order to rule out any compensatory effect of opioid peptides on release of vasodilator transmitters. the preparation was
50
g
40
g ._ z
30
2 z ti
20
10
0 0.5
1
1.5
2
4
8
16
Frequency (Hz ) o’ 2. Vasodilator resp0nsr.s to transmural IWITT stimulation after Fio treatment with guanethidine (5 x lo- h M) and in the prewnce of methoxamine (3 x 10-h M). Trains of 100 pulsrs (30 V. 2 nib puk duration) were delivered at various frequencies. and data are plotted as percentage relaxation of contraction produced b! metba\amine. Values are meansfS.E.M. (n = 7-12).
Control
Untreated 1
-9.5
8
-8.0 -9.0 -7.5 -8.5 CGRP Concentration (log M)
Fim r ?. Fffect 0f indomethacm (IO ’ M) on vasodilator responses to tran%muml ncrvc stimulation (A) and CCRP (B). (A) transmural ncrrc stimulatu~n wvasapplied at frequencies indicated for 10 s trains $50 V. 2 ms pulse duration) (n =- 4). (3) Vurious ~~~n~entrati~~nsof CGRP =cre te%ted in the ab\encc und presence of indomethacin (n = 41. Rrrlaxatron 1%expressed air ptmxmagr of contraction induced hy meth0xarn1nr (3 x IO ’ M). Tissues were treated with guanethidine t5 x IO ’ M) to hioch adrenergic nrrvcs. * * P < 0.01 for indomrthatin-treated cnmpared to cnntrc~l.
c lndo
Fig. 4. Effect of NPY (IO ’ M) on vasoeonstrictor (A) and vasodilatar (B) responses to transmural nerve stimulation (12 Hz. 60 pulses. 30 V. 2 ms pulse duration). (A) Increase in perfusion pressure for constrictor response to transmurai nerve stimub&on is shown for control and in the presence of NPY (n = 7). (B) Effect of NPY on dilator response to transmural nerve stimulation is shown for untreated tissues and those treated with indomethacin (IOmh M). Dilator responses were measured in tissues treated with guanethidine (5 x fOm6 M) and in the presence of methoxamine (3 x 10-’ M). and relaxation is calcuiated as percentage of contraction to methoxamine (n = 6). Values are given as means+ S.E.M. * * * P ( 0.001 comp~r~~I to control, * P < 0.05 compared to untreated-
ence of indomethacin. DAMGO still inhibited the vasodilator response (fig. 6). However, in the presence of indomethacin, DPDPE was no Ionger effective in inhibiting vasodilator responses to transmural nerve stimulation.
The present study confirms the presence in the rat mesenteric vasculature of two distinct systems of innervation: adrenergic and primary sensory afferent
q
Control Opioids
DPDPE
40 1
3oiTT
TT
20
10
0
Fig. 5. Effect vas~onstrictor with capsaicin was applied at DAMGO (5 X
DAMGO DPDPE MC of opioids on transmural nerve stimulation-induced responses (A) before (n = 6-7) and (B) after treatment (3 x lo-' M) (n = 5). Transmural nerve stimulation 8 Hz for trains of 40 pulses, 70 V, 3 ms pulse duration. lOen M). DPDPE (lo-’ M) and EKC (5 X lo-’ M) were applied for 10 min.
Effect of Opioids on Vasodilation
DA
DP
EKC
DA/I
DPil
Fig. 6. Effect of opioid peptides on vasodifator response to transmural nerve stimulation (8 Hz for trains of 40 pulses. 70 V. 3 ms pulse duration) in the absence and presence of indomethacin (lo-’ M). Tissues were treated with guanethidine (5 x lo-.’ M). and methoxamine (3 x IO-’ M) was added. Responses to transmural nerve stimulation are expressed as a percentage of contractile response to ti~e~h~)xaii~ine (n = 46). DA. DAMGO: DP. DPDPE: EKC. ethylketot)-clazocine: 1. indomethacin. ** P -z 0.01. *** P < 0.001 compared to corresponding response without opioid peptide.
(Kawasaki et al., 1988; 1990). The major transmitter for the vasodilator sensory nerves appears to be CGRP. as transmural stimulation increases the release of CGRPlike immunoreactivity from the perfused rat mesentery Wjimori et al.. 1989) and the CGRP antagonist. CGRP-(8-37). abolishes the vasodilator response to transmural nerve stimulation (Han et al.. 1990). Our findings underscore the simultaneous participation of both systems when nerves are activated by transmural electrical stimulation (Kawasaki et al.. 1990). Thus acute addition of capsaicin, which evokes release of sensory nerve transmitters, causes a decrease in the vasoCOnStriCtOr response to nerve stimulation. After exposure to capsaicin. when responses to sensory nerve stimulation are blocked as confirmed by loss of response to subsequent capsaicin exposure. contractile responses to transmural nerve stimulation are increased. Previous investigators have shown that exposure to capsaicin or exogenous CGRP does not influence norepinephrine release in the mouse vas deferens (AlKazwini et al., 1986) or perfused rat mesentery (Kawasaki et al.. 1990). It is also unlikely that capsaicin directly affects the vasoconstrictor response to nerve stimulation by altering sensitivity to norepinephrine. The pressor response to exogenous norcpinephrine after treatment with capsaicin has beeE shown not to differ significantly from preparations not treated with capsaitin (Kawasaki et al.. 1990). Our results suggest that facilitation of contractiie responses to adrenergic nerve stimulation by capsaicin may be due to desensitization of sensory nerves which results in cessation of release of dilator transmitter, most probably CGRP, and an increased effect of the constrictor transmitter. norepinephrine. Thus in order to study either the adrenergic or sensory nerve system in isolation, it is necessary to block the other type of nerve. As shown by others as well as in the present study, this can be effectively accomplished using guanethidine to block adrenergic nerves or after a brief exposure to capsaici). to block sensory nerve effects. One interesting difference between the adrenegic and sensory nerve systems concerns the time course and frequency dependence of the response to nerve activation. Even with relatively brief times of stimulation. vasodilator responses to sensory nerve stimulation are quite prolonged. with a half-life in excess of 5 min. This is in contrast to responses to adrenergic nerve stimulation, which relax very quickly. The long time course Of vasodilator responses to sensory nerve stimulation in the perfused rat mesentery has been shown to correspond to a prolonged overflow of CGRP from the preparation (Fujimori et al.. 1989). Capsaicin-sensitive vasodilator responses were appreciable at relatively low frequencies of stimulation and reached a maximum at a frequency of 4 Hz. In contrast, for adrenegic nerves higher frequencies of stimulation with the same number
al.. 1990). These findings su,,**est that NPY also inhibits release from sensory nerves via a prejunctionai site of action. although further investigation will he necessary to confirm this hypothesis. Our studies also demonstrated that the effect of NPY to inhibit vasodilator respomes to sensory nerve stimulation is not dependent on production of prostaglandins as addition of indomethacin did not result in any alteration in the effectiveness of N PY. We found that opioid agonists selective for p. 6 or K receptors were quite ineffective against contractile responses to adrenergie nerve stimulation in the perfused mesentery. In order to eliminate a possible influence of vosodiiator responses to sensory nerve activation on pressor responses to adrenergic nerve stimulation, tissues were then pretreated with capsaicin. However, after blockade of sensory nerve effects with capsaicin. aithough contractile responses to adrenergic nerve stimulation were increased. the three opioid agonists still produced no effect. As discussed in more detail below, opioid agonists inhibit sensory nerve vasodilator responses. so it is surprising that this effect is not revealed when adrenergic and sensory nerves are simultaneously activated. However. it is probable that the relatively small inhibitory effect that opioid agonists produce against sensory nerve responses (fig. 6) is quantitatively too small to translate into any alteration in the net vasoconstrictor response. Our findings are similar to previous observations made in the rat tail artery, where the only opioid effects seen were inhibition of contractile responses to adrenergic nerve stimulation with certain opioid agonists. an effect which was ascribed to 4 receptors (Iiies et al., 1987; Bucher et al., 1988). In contrast, p or 6, but not K, receptor stimulation resulted in an inhibition of vasodilator responses to sensory nerve stimulation. These rest&s are in agreement with recent studies showing that both p and S opioid receptor stimulation reduced K’-evoked release of UGRP from the dorsal half of the rat cervical enlargement as well as the guinea-pig lung. but a tc opioid receptor agonist was inactive (Saria et al.. 1988: Pohl et al., 1989). Furthermore, in the guinea-pig atria, evidence was found for the presence of inhibitory 11 and 6 receptors on sensory nerve endings (Mantelli et al., 1989). In contrast. however. in the rabbit iris sphincter muscle. K-selective agonists did attenuate responses to stimulation of sensory nerves (Ueda et al.. 1985). It has been suggested that both the central and peripheral terminals of capsaicin-sensitive sensory nerves possess presynaptic p and 6, but not K, opioid receptors; however. further studies using additional selective agonists and antagonists need to be done to more firmly establish this conclusion for the rat mesenteric vasculature. One of the most intriguing observations of our study is the finding that. in the presence of indomethacin. the inhibitory effect of S rc:*ei;tor stimu!ation by DPDPE
transmitter our >t~di~?; ;&o dmnmttate ti,rn ,)f ~\&k~\!gen;w
prducth
313 importrtllt
participa-
in rrqonses
to sensor!
tlerve &~~d;&w. Tr=zltment \vith indomethncin to iiihibit c;\;~It~3\ygenase results in enhanced vasodilator wsp3nseh to sensory nefie stimuIation_ but does not ;lffKt \-:l?;tdilator responses to exogenous CGRP. This St1Lc:eht3that electrical stimulation of sensory nerves ey,ll;cs the &ase of vasoconstrictor prostaglandins that cclmltcrrlCt the vasodihtor effects of CGRP released from %enx\ry fterves. Aiternotivci~ it is possible that lo&lly producti prost~~gt~~l~dit~s attenuate the release of CGRP. Recent studies hhow that prt~staglimdin E, tF6E1 ). but not PGI,. evokes CGRP retease in the 3ouinea pig heart in vitro ( Franc+Cereeeda. 1989). However. in this case indomethacin would he predicted to produce an attenuation of the vasodiiator response to trammural nerve htimulation. rather than the cnhancement that we c&served. Others have reported previously that responses to sensory nerve stimulation of the perfused rat mrsentery are not altered in the presence of indo~i~tha~in (Kawasaki et al., 1988). Reasons for the dixrepancy with our results are not clear. However. responses to capsaicin of the rabbit ear artery have been shown to he altered after treatment with indomethacin t Xloritoki et al.. 1990). WC also explored the effects of NPY and opioid peptides on vasoconstrictor and vasodilator responses to trammurai nerve stimulation. As others have previously shown (Westfail et al.. 1988: Vu et al.. 1989). NPY potentiated vasoconstrictor responses to nerve stimulation. This response \vas not affected by pretreatment with capsaicin suggesting that the potentiation produced h> NPY is not merely a consequence of inhibition of sensory nerve activation. We have shown in the rat tail artery that the potentiating effect of NPY occurs at a postjunctional site (Vu et al.. 19X9). The potentiation produced b_v NPY has been shown to be non-specific. such that contractile responses to a variety of different substances are increased tWahIestedt et al.. 19S5). It has been variously suggested that this may involve an increase in phosphoinositide turnover and facilitation of intracellular calcium mobilization or activation of membrane calcium channels (Haggbiad and Fredholm. 1987: Wahlestedt et al.. 1985). In contra>t. NPY markedly suppressed the vasodiiator response to transmural neme stimulation. Similar results have recently been obtained in guinea pig isolated left atria and bronchi (Giuliani et al., 1989; Grundemar et al.. 1990). NPY has been shown to decrease release of n~~repinephrine from adrenergic nerves in several tissues (Lundberg et al., 1985b; Pernow et al.. 1987: WestfaIi et al.. 1988). Our studies show that NPY does not affect the vasodilator response to exogenous CARP. and this has also been shown by &hers (Nuki c:
371
was abolished. In contrast the inhibitory effect produced by stimulation of p receptors by DAMGO was unaffected in the presence of indomethacin. Opioid peptides have been reported to increase the production of prostaglandins (Ledda et al., 1989). and prostaglandins are well known to have prejunctional actions (Mantelli et al., 1990). Our findings indicate, however, that prostaglandins may be intimately involved in the effects of only one sub-type of opioid receptor. This suggests that the prejunctional inhibitory mechanisms of 1-1and 6 receptors may not be similar. In summary, the present results suggest that: (1) the contractile response to transmural nerve stimulation in the perfused rat mesentery is the net result of both constrictor and dilator nerve activation, (2) activation of capsaicin-sensitive sensory nerves promotes the local production of vasoconstrictor prostaglandins and (3) prejunctional control of transmitter release by NPY and opioids varies depending on the specific nerve type as well as, in some cases, the participation of endogenous prostaglandins.
Acknowledgements We thank Victor Barrios and Silvia Loyola for their excellent technical assistance. This work was supported by Grant No. PO1 DK 36289 from the National Institutes of Health and by a postdoctoral fellowship from the California Affiliate of the American Heart Association.
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153. Moritoki. H.. H. Takase and A. Tanioka. 1990. Dual effects of capsaicin on responses of the rabbit ear artery to field stimulation.
Br. J. Pharmacol. 99. 152. Nuki, C.. H. Kawasaki and K. Takasaki. 1990. Effect of neuropepttde y on vaso&khon mediated by calcitonin gene-related peptide (CGRP)-containing nerves in the mesenteric rCSistanCC vessel Of the rat. Jap. J. Pharmacol. 53. 125. Pernow, J.. T. Svenberg and J.M. Lundberg. 1987. Action of calcium antagonists on pre- and postjunctional effects of neuropeptide y on human peripheral blood vessels in vitro. European J. Pharmacol. 136. 207.
Vu. H.Q.. D. Budai and S.P. Duckles. 19F9. Neuropeptide Y preferentially potentiates responses to adrenergic nerve stimulation by increasing rate of contraction. J. Pharmacol. Exp. Ther. 251. 852. Wahleatedt. C.. L. Edvinsson. E. Ekhlad and R. Hlkanson. 1985. Neuropeptide Y potentiates noradrenaline-evoked vasoconstriction: Mode of action. J. Pharmacol. Exp. Ther. 234. 735. Westfall. T.C.. J. Martin. X. Chen. A. Ciarleglio. S. Carpentier. K. Henderson, M. Knuepfkr. M. Beinfeld and L. Naes, 1988. Cardiovascular effects and moduhttion of noradrenergic rteurotransmission following central and peripheral administration of neuropeptide Y. Synapse 2. 299.
365
c’ 1991
UP 51799
ifferential effects of neuropepti of the perfused rat mesentery Yuanjian Li and Sue P. Duckles Department
of Pharmacology.
College oj Medicine,
Utlicersily
of
California,
Irvine.
CA 9’717.
ti. S.A.
Received 8 January 1991. accepted 15 January 1991
In perfused rat meseniery transmural nerve stimulation activates both adrenergic and capsaicin-sensitive sensory nerves. When adrenergic nerves were blocked with guanethidine and smooth muscle tone was increased. transmural nerve stimulatisn caused a dilator response which was attenuated by tetrodotoxin and abolished by capsaicin. Indomethacin increased the vasodilator response to transmural nerve stimulation, but did not affect the dilation to calcitonin gene-related peptide. Neuropeptide Y (NPY) potentiated vasoconstrictor responses to transmural nerve stimulation. but suppressed capsaicin-sensitive vasodilation. an effect which was unaltered by indomethacin. Opioid agonists selective for ~_r,S or K receptors. DAMGO ([D-Ala’.N-Me-Phe4.Gly’-ol]enkephalin), DPDPE ([D-Pen*.D-Pen’lenkephalin) and ethylketocyclazocine. had no effect on the vasoconstrictor response to transmural nerve stimulation. DAMGO and DPDPE significantly inhibited vasodilator responses to transmural nerve stimulation. but ethylketocyclazocine was without effect. After treatment with indomethacin. DAMGO still inhibited the vasodilator response. but DPDPE was no longer effective. Prejunctional control of transmitter release by NPY’ or opioids is dependent on the specific nerve type as well as, in some cases, the participation of endogenous prostaglandins. Mesentery (rat); Capsaicin; [D-Ala’.N-Me-Phe4,GIy5-ollenkephalin):
CGRP (calcitonin gene-related peptide); Neuropeptide Y; DAMGO DPDPE ([D-Pen’.D-Per?]enkephalin): Ethylketocyclazocine: lndomethacin
1. Introduction
It is clear that peptides mitters and are coreleased vascular nerves, including
coexist with classical transfrom several types of perineuropeptide Y (NPY) with
norepinephrine in sympathetic nerves (Lundberg et al., 1984). vasoactive intestinal polypeptide with acetylcholine in parasympathetic nerves (Lundberg, 1981). and substance P with calcitonin gene-related peptide (CGRP) in primary afferent sensory nerves (Lundberg et al., 1985a). Furthermore, interactions of peptide and non-peptide neurotransmitters and with various autacoids. including prostaglandins. have been shown to occur both pre- and postjunctionally (Mantelli et al., 1990; Burnstock. 1987). It is interesting that NPY and opioids are capable of inhibiting not only norepinephrine released from sympathetic nerves. but also neurotransmission in primary sensory afferent nerves via a prejunctional mechanism (Pohl et al. 1989; Grundemar et al.. 1990; Crain and Shen, 1990). Recently, the presence of a non-adrenergic. noncholinergic vasodilator innervation was demonstrated in
Correspondence to: S.P. Duckles. Department of Pharmacology. College of Medicine. University of California. Irvinr. CA 92717. U.S.A.
the rat perfused mesentery (Manzini and Perretti. 1988: Kawasaki et al., 1988. 1990: Fujimori et al.. 1989). The capsaicin sensitivity of these nerves. the finding thrtt nerve stimulation evokes release of CGRP. as well as the low levels of substance P in this tissue have led to the proposal that CGRP is the principle neurotransmitter of these vasodilator sensory nerves. The purpose of the present study was 3-fold. First. we tested vasoconstrictor and vasodilator responses to transmural nerve stimulation of the perfused rat mesentery and the effect of desensitization to sensory nerve transmitters with capsaicin. Secondly. we investigated the possible contribution of endogenous prostaglandins to vasodilator responses. Thirdly. the modulatory actions of NPY and opioids “r-r responses of sympathetic vasoconstrictor and capsaicin-sensitive vasodilator nerves were compared.
2. Materials
and methods
Mesenteric vasculature of male Sprague-Dawley rats (14-20 weeks old weighing 400 & 70 g) was isolated and prepared for perfusion as described by Kawasaki and Takasaki (1984) with some modifications. Rats were anesthetized with pentobarbital sodium at a dose of 75
it~t~ti~~e and sssocisted vasculsr bed. the mesenteric vs?;&ar bed was flushed with warm Krehs solution and plmced iIt a \vater jacket (volume X0 ml) nlaintained at 37”~. the system was perfused with warm Krebs solutioa~. saturated with 95B 0, and 5C; CO, with the help of ;t peristaltic pump at a rate of 5 + 0.2 ml/min. and superfused by gravity fwd at a rate of 1 t 0.2 ml/min. The Krehs solution had the following composition (in mM): NaCI llS: KC1 4.8: CiiCI, 2.5: KH:PO, 1.2: NaHCO, IS: MgSQ 1.2: EDTA . Na 0.107: and dextrose 11.5. The perfusion pressure was monitored and recorded by a pressure transducer and a Soltec 1242 chart recorder. The tissue was equilihratcd for 60 min before beginning each study. Two platinum eie&rodes. one placed around the superior mesenteric artery and the other resting on the vasculature in a lower part of the intestine. \vere used to create transmural field stimulation. For constrictor responses to trsnsmural nerve stimulation. 3-5 tnin were al!owed between each stimulation train. fn the case of vasodilator responses to transmural nerve stimulation. sufficient time was allowed between each stimulation train for the perfusion pressure to return to a stable level. usually IO-20 min. Drugs were administered by switching the perfusion solution to solution containing drug in the concentrations indicated. Means of four peaks of responses to transmural nerve stimulation that came before administering a drug were used as control responses. 2. I. E_~p~ri~~leittai protoco!s Responses to transmurai nerve stimulation were stable in the absence of drug application. Where indicated, tissues were pretreated with guanethidine (5 x lo-” M) fcr 20 min. or aith indomethacin (lOeh M) for 30 mm hefore testing responses to transmurdl nerve stimulation or CGRP. In these cases. guanethidine and indomethatin remained in the perfusate throughout the remainder of the experiment. Exposure to capsaicin (3 x lo-’ M) was for 20 min. and capsaicin was then washed out. For all studies a paired design was used. that is the same tissue was studied both before and after treatme,lt with capsaicin. indometha~in, guanethidine. NPY or opioid agonists.
The following drugs were used: neuropeptide Y, [DAla~.N-Mu-Phe~.Gly~-ol]enkephalin {DAMGO). [D-
Pen~.D-Fell~]e:nkephalin (DPDPE) and calcitonin generelated peptide (CCRP) (Peninsula); ethylketocyclazotine (EKC) {Sterli~lg-Wil~throp Res. tns.): tetrodoloxin. indomethacin. naloxone and methoxamine HCI (Sigma): ~uanethidine (Ciba Pharmaceutical Co.); and capsaicin (iCN Biomedicals. Inc.). All drugs were dissolved in Krebs solution. except capsaicin and indometh~~~in. which were initially dissolved in ethanol and further diluted in Krebs solution to the proper final concentration. 23. Sturi.h3 Results are expressed as means at S.E.M. Student’s paired t-test was used to determine statistical differences between two means. The level of significance was chosen as P < 0.05.
3. Resul;s 3. f. Vasaco~tst~~~tarand ~?usadiIatarrespmses to tra~~s~lMrul nerve stirmdution In the perfused mesenteric vascular bed, transmural nerve stimulation produced a frequency-dependent increase in perfusion pressure (fig. 1A). This response was completely blocked after treatment with guanethidine (-5 X 10eh M), confirming that it is due to stimulation of adrenergic nerves. Since it has been demonstrated that transmural nerve stimulation can release both dilator and constrictor transmitters, the effect of prior treatment with capsaicin to desensitize the tissue to sensory nerve stimulation was tested. As shown in fig. lB, during treatment with capsaicin (3 x 10m7 M) the presSOT response to transmural nerve stimulation was markedly inhibited. However. after 20 min capsaicin treatment. and when capsaicin was washed out of the bath. there was a marked increase in the pressor response to transmural nerve stimulation. The efficacy c:f our protocol for capsaicin treatment in desensitizing sensory nerves was verified by demonstration that inhibition of transmural nerve stimulation vasoconstrictor responses by capsaicin was abolished by prior exposure to capsaicin, 3 x lo-’ M for 20 min. First exposure to capsaicin caused an inhibition of contractile responses to transmural nerve stimulation of 56 +_78, whereas the second exposure to the same concentration of capsaicin produced no relaxation (n = 3). After blockade of sympathetic responses with guanethidine. methoxamine, 3 x 10. ’ M. was added to increase smooth muscle tone. In this situation, transmural nerve stimulation caused a vasodilator response which was frequency-dependent, especiatly at low frequencies (fig. 2). Vasodilator responses to transmural nerve stimulation were markedly attenuated by tetro-
367
3.2. EJiect oj NPY
2
16
As has been previously reported in a variety of tissues (Vu et al.. 1989). NPY (lo-’ M) significantly potentiated vasoconstrictor responses to transmural nerve stimulation (fig. 4A). This response was not affected by pretreatment with capsaicin (percentage increases in pressure, control and capsaicin. were 110 5 20 and 104 + 14. respectively. P > 0.8. n = 8). in contrast. vasodilator responses (after treatment with guanethidine and in the presence of methoxamine) were significantly depressed in the presence of NPY (fig. 4B). The inhibitory effect of NPY was not affected by pretreatment with indomethacin (fig. 4B) (P > 0.7 for comparison of the effect of NPY in untreated and indomethacin treated tissues). We also tested the effect of NPY on the vasodilater response to exogenous CGRP. Exogenous CGRPinduced vasodilator responses were not affected by NPY treatment (percentage relaxations. control and NPY. were 69 + 6 and 68 f 6. respectively: P > 0.7. n = 4). 3.3. Effects of opioid prptide3.
BEFOFIE
CAP
AFTER
Fig. 1. Frequency dependence of vasoconstrictor responses to transmural nerve stimulation and effect of capsaicin exposure in the perfused mesenteric vascular bed. (A) Contractile responses tu transmural nerve stimulation for trains of 100 pulses (30 V. 2 mb pulse duration) are plotted as increase in perfusion pressure as a function of stimulus frequency. (3) Responses to transmural nerve stimulation (8 Hz. 40 pulses. 70 V. 5 ms) before, during and after exposure to capsaicin (3 x lo-’ M. 20 min) are indicated. Values are means? S.E.M (n = 4). *** P
dotoxin (lo-(’ M) (percentage inhibition was 70 IL 3. n = 5) and completely abolished after treatment with capsaicin (3 x lo-’ M) for 20 min (n = 5). Treatment with indomethacin (lO-h M) to inhibit cyclooxygenase resulted in a significant increase in the vasodilator restonse to transmural nerve stimulation at all frequencies of stimulation (fig. 3A). When relaxation responses to exogenous calcitonin gene-related peptide were studied in the presence of indomethacin. there were no significant differences in vasodilator responses compared to control, regardless of the concentration of CGRP (P > 0.05) (fig. 3B).
Three opioid receptor agonists were selected to represent actions on three types of opioid receptor: DAMGO (p). DPDPE (6) and EKC (K). Concentrations of these agonists which have previously been shown to be selective for their respective receptors were used (Berzetci et al.. 1987: Budai and Duckles. 1988). As shown in fig. 5A. none of these three had any effect on pressor responses to transmural nerve stimulation. In order to rule out any compensatory effect of opioid peptides on release of vasodilator transmitters. the preparation was
50
g
40
g ._ z
30
2 z ti
20
10
0 0.5
1
1.5
2
4
8
16
Frequency (Hz ) o’ 2. Vasodilator resp0nsr.s to transmural IWITT stimulation after Fio treatment with guanethidine (5 x lo- h M) and in the prewnce of methoxamine (3 x 10-h M). Trains of 100 pulsrs (30 V. 2 nib puk duration) were delivered at various frequencies. and data are plotted as percentage relaxation of contraction produced b! metba\amine. Values are meansfS.E.M. (n = 7-12).
Control
Untreated 1
-9.5
8
-8.0 -9.0 -7.5 -8.5 CGRP Concentration (log M)
Fim r ?. Fffect 0f indomethacm (IO ’ M) on vasodilator responses to tran%muml ncrvc stimulation (A) and CCRP (B). (A) transmural ncrrc stimulatu~n wvasapplied at frequencies indicated for 10 s trains $50 V. 2 ms pulse duration) (n =- 4). (3) Vurious ~~~n~entrati~~nsof CGRP =cre te%ted in the ab\encc und presence of indomethacin (n = 41. Rrrlaxatron 1%expressed air ptmxmagr of contraction induced hy meth0xarn1nr (3 x IO ’ M). Tissues were treated with guanethidine t5 x IO ’ M) to hioch adrenergic nrrvcs. * * P < 0.01 for indomrthatin-treated cnmpared to cnntrc~l.
c lndo
Fig. 4. Effect of NPY (IO ’ M) on vasoeonstrictor (A) and vasodilatar (B) responses to transmural nerve stimulation (12 Hz. 60 pulses. 30 V. 2 ms pulse duration). (A) Increase in perfusion pressure for constrictor response to transmurai nerve stimub&on is shown for control and in the presence of NPY (n = 7). (B) Effect of NPY on dilator response to transmural nerve stimulation is shown for untreated tissues and those treated with indomethacin (IOmh M). Dilator responses were measured in tissues treated with guanethidine (5 x fOm6 M) and in the presence of methoxamine (3 x 10-’ M). and relaxation is calcuiated as percentage of contraction to methoxamine (n = 6). Values are given as means+ S.E.M. * * * P ( 0.001 comp~r~~I to control, * P < 0.05 compared to untreated-
ence of indomethacin. DAMGO still inhibited the vasodilator response (fig. 6). However, in the presence of indomethacin, DPDPE was no Ionger effective in inhibiting vasodilator responses to transmural nerve stimulation.
The present study confirms the presence in the rat mesenteric vasculature of two distinct systems of innervation: adrenergic and primary sensory afferent
q
Control Opioids
DPDPE
40 1
3oiTT
TT
20
10
0
Fig. 5. Effect vas~onstrictor with capsaicin was applied at DAMGO (5 X
DAMGO DPDPE MC of opioids on transmural nerve stimulation-induced responses (A) before (n = 6-7) and (B) after treatment (3 x lo-' M) (n = 5). Transmural nerve stimulation 8 Hz for trains of 40 pulses, 70 V, 3 ms pulse duration. lOen M). DPDPE (lo-’ M) and EKC (5 X lo-’ M) were applied for 10 min.
Effect of Opioids on Vasodilation
DA
DP
EKC
DA/I
DPil
Fig. 6. Effect of opioid peptides on vasodifator response to transmural nerve stimulation (8 Hz for trains of 40 pulses. 70 V. 3 ms pulse duration) in the absence and presence of indomethacin (lo-’ M). Tissues were treated with guanethidine (5 x lo-.’ M). and methoxamine (3 x IO-’ M) was added. Responses to transmural nerve stimulation are expressed as a percentage of contractile response to ti~e~h~)xaii~ine (n = 46). DA. DAMGO: DP. DPDPE: EKC. ethylketot)-clazocine: 1. indomethacin. ** P -z 0.01. *** P < 0.001 compared to corresponding response without opioid peptide.
(Kawasaki et al., 1988; 1990). The major transmitter for the vasodilator sensory nerves appears to be CGRP. as transmural stimulation increases the release of CGRPlike immunoreactivity from the perfused rat mesentery Wjimori et al.. 1989) and the CGRP antagonist. CGRP-(8-37). abolishes the vasodilator response to transmural nerve stimulation (Han et al.. 1990). Our findings underscore the simultaneous participation of both systems when nerves are activated by transmural electrical stimulation (Kawasaki et al.. 1990). Thus acute addition of capsaicin, which evokes release of sensory nerve transmitters, causes a decrease in the vasoCOnStriCtOr response to nerve stimulation. After exposure to capsaicin. when responses to sensory nerve stimulation are blocked as confirmed by loss of response to subsequent capsaicin exposure. contractile responses to transmural nerve stimulation are increased. Previous investigators have shown that exposure to capsaicin or exogenous CGRP does not influence norepinephrine release in the mouse vas deferens (AlKazwini et al., 1986) or perfused rat mesentery (Kawasaki et al.. 1990). It is also unlikely that capsaicin directly affects the vasoconstrictor response to nerve stimulation by altering sensitivity to norepinephrine. The pressor response to exogenous norcpinephrine after treatment with capsaicin has beeE shown not to differ significantly from preparations not treated with capsaitin (Kawasaki et al.. 1990). Our results suggest that facilitation of contractiie responses to adrenergic nerve stimulation by capsaicin may be due to desensitization of sensory nerves which results in cessation of release of dilator transmitter, most probably CGRP, and an increased effect of the constrictor transmitter. norepinephrine. Thus in order to study either the adrenergic or sensory nerve system in isolation, it is necessary to block the other type of nerve. As shown by others as well as in the present study, this can be effectively accomplished using guanethidine to block adrenergic nerves or after a brief exposure to capsaici). to block sensory nerve effects. One interesting difference between the adrenegic and sensory nerve systems concerns the time course and frequency dependence of the response to nerve activation. Even with relatively brief times of stimulation. vasodilator responses to sensory nerve stimulation are quite prolonged. with a half-life in excess of 5 min. This is in contrast to responses to adrenergic nerve stimulation, which relax very quickly. The long time course Of vasodilator responses to sensory nerve stimulation in the perfused rat mesentery has been shown to correspond to a prolonged overflow of CGRP from the preparation (Fujimori et al.. 1989). Capsaicin-sensitive vasodilator responses were appreciable at relatively low frequencies of stimulation and reached a maximum at a frequency of 4 Hz. In contrast, for adrenegic nerves higher frequencies of stimulation with the same number
al.. 1990). These findings su,,**est that NPY also inhibits release from sensory nerves via a prejunctionai site of action. although further investigation will he necessary to confirm this hypothesis. Our studies also demonstrated that the effect of NPY to inhibit vasodilator respomes to sensory nerve stimulation is not dependent on production of prostaglandins as addition of indomethacin did not result in any alteration in the effectiveness of N PY. We found that opioid agonists selective for p. 6 or K receptors were quite ineffective against contractile responses to adrenergie nerve stimulation in the perfused mesentery. In order to eliminate a possible influence of vosodiiator responses to sensory nerve activation on pressor responses to adrenergic nerve stimulation, tissues were then pretreated with capsaicin. However, after blockade of sensory nerve effects with capsaicin. aithough contractile responses to adrenergic nerve stimulation were increased. the three opioid agonists still produced no effect. As discussed in more detail below, opioid agonists inhibit sensory nerve vasodilator responses. so it is surprising that this effect is not revealed when adrenergic and sensory nerves are simultaneously activated. However. it is probable that the relatively small inhibitory effect that opioid agonists produce against sensory nerve responses (fig. 6) is quantitatively too small to translate into any alteration in the net vasoconstrictor response. Our findings are similar to previous observations made in the rat tail artery, where the only opioid effects seen were inhibition of contractile responses to adrenergic nerve stimulation with certain opioid agonists. an effect which was ascribed to 4 receptors (Iiies et al., 1987; Bucher et al., 1988). In contrast, p or 6, but not K, receptor stimulation resulted in an inhibition of vasodilator responses to sensory nerve stimulation. These rest&s are in agreement with recent studies showing that both p and S opioid receptor stimulation reduced K’-evoked release of UGRP from the dorsal half of the rat cervical enlargement as well as the guinea-pig lung. but a tc opioid receptor agonist was inactive (Saria et al.. 1988: Pohl et al., 1989). Furthermore, in the guinea-pig atria, evidence was found for the presence of inhibitory 11 and 6 receptors on sensory nerve endings (Mantelli et al., 1989). In contrast. however. in the rabbit iris sphincter muscle. K-selective agonists did attenuate responses to stimulation of sensory nerves (Ueda et al.. 1985). It has been suggested that both the central and peripheral terminals of capsaicin-sensitive sensory nerves possess presynaptic p and 6, but not K, opioid receptors; however. further studies using additional selective agonists and antagonists need to be done to more firmly establish this conclusion for the rat mesenteric vasculature. One of the most intriguing observations of our study is the finding that. in the presence of indomethacin. the inhibitory effect of S rc:*ei;tor stimu!ation by DPDPE
transmitter our >t~di~?; ;&o dmnmttate ti,rn ,)f ~\&k~\!gen;w
prducth
313 importrtllt
participa-
in rrqonses
to sensor!
tlerve &~~d;&w. Tr=zltment \vith indomethncin to iiihibit c;\;~It~3\ygenase results in enhanced vasodilator wsp3nseh to sensory nefie stimuIation_ but does not ;lffKt \-:l?;tdilator responses to exogenous CGRP. This St1Lc:eht3that electrical stimulation of sensory nerves ey,ll;cs the &ase of vasoconstrictor prostaglandins that cclmltcrrlCt the vasodihtor effects of CGRP released from %enx\ry fterves. Aiternotivci~ it is possible that lo&lly producti prost~~gt~~l~dit~s attenuate the release of CGRP. Recent studies hhow that prt~staglimdin E, tF6E1 ). but not PGI,. evokes CGRP retease in the 3ouinea pig heart in vitro ( Franc+Cereeeda. 1989). However. in this case indomethacin would he predicted to produce an attenuation of the vasodiiator response to trammural nerve htimulation. rather than the cnhancement that we c&served. Others have reported previously that responses to sensory nerve stimulation of the perfused rat mrsentery are not altered in the presence of indo~i~tha~in (Kawasaki et al., 1988). Reasons for the dixrepancy with our results are not clear. However. responses to capsaicin of the rabbit ear artery have been shown to he altered after treatment with indomethacin t Xloritoki et al.. 1990). WC also explored the effects of NPY and opioid peptides on vasoconstrictor and vasodilator responses to trammurai nerve stimulation. As others have previously shown (Westfail et al.. 1988: Vu et al.. 1989). NPY potentiated vasoconstrictor responses to nerve stimulation. This response \vas not affected by pretreatment with capsaicin suggesting that the potentiation produced h> NPY is not merely a consequence of inhibition of sensory nerve activation. We have shown in the rat tail artery that the potentiating effect of NPY occurs at a postjunctional site (Vu et al.. 19X9). The potentiation produced b_v NPY has been shown to be non-specific. such that contractile responses to a variety of different substances are increased tWahIestedt et al.. 19S5). It has been variously suggested that this may involve an increase in phosphoinositide turnover and facilitation of intracellular calcium mobilization or activation of membrane calcium channels (Haggbiad and Fredholm. 1987: Wahlestedt et al.. 1985). In contra>t. NPY markedly suppressed the vasodiiator response to transmural neme stimulation. Similar results have recently been obtained in guinea pig isolated left atria and bronchi (Giuliani et al., 1989; Grundemar et al.. 1990). NPY has been shown to decrease release of n~~repinephrine from adrenergic nerves in several tissues (Lundberg et al., 1985b; Pernow et al.. 1987: WestfaIi et al.. 1988). Our studies show that NPY does not affect the vasodilator response to exogenous CARP. and this has also been shown by &hers (Nuki c:
371
was abolished. In contrast the inhibitory effect produced by stimulation of p receptors by DAMGO was unaffected in the presence of indomethacin. Opioid peptides have been reported to increase the production of prostaglandins (Ledda et al., 1989). and prostaglandins are well known to have prejunctional actions (Mantelli et al., 1990). Our findings indicate, however, that prostaglandins may be intimately involved in the effects of only one sub-type of opioid receptor. This suggests that the prejunctional inhibitory mechanisms of 1-1and 6 receptors may not be similar. In summary, the present results suggest that: (1) the contractile response to transmural nerve stimulation in the perfused rat mesentery is the net result of both constrictor and dilator nerve activation, (2) activation of capsaicin-sensitive sensory nerves promotes the local production of vasoconstrictor prostaglandins and (3) prejunctional control of transmitter release by NPY and opioids varies depending on the specific nerve type as well as, in some cases, the participation of endogenous prostaglandins.
Acknowledgements We thank Victor Barrios and Silvia Loyola for their excellent technical assistance. This work was supported by Grant No. PO1 DK 36289 from the National Institutes of Health and by a postdoctoral fellowship from the California Affiliate of the American Heart Association.
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