Abstract
The physiological role of neuropeptide Y (NPY) and extracellular adenosine 5′-triphosphate (ATP) in sympathetic neurotransmission is becoming increasingly clear. To assess whether NPY and ATP act as cotransmitters together with noradrenaline (NA) in the sympathetic nerves of the superior mesenteric artery, the changes in perfusion pressure of the arterial mesenteric bed caused by nerve stimulation were recorded. Depolarization of the perivascular superior mesenteric arterial nerves caused frequency- and time-dependent increases in the perfusion pressure that were abolished by guanethidine, which implied the sympathetic origin of these responses. Independent perfusion with either 500 nM BIBP 3226, an NPY Y1 antagonist; 3 μM suramin, a competitive purinoceptor antagonist; or 0.1 nM prazosin, a competitive alpha-1 adrenoceptor antagonist, evoked approximately a 30% reduction in the rise in perfusion pressure caused by the 20- to 30-Hz electrical depolarization of the perimesenteric arterial nerves. Prazosin (0.1 nM) blocked the increases in perfusion pressure caused by electrical stimulation of the perimesenteric nerves but did not significantly reduce the vasomotor effect of exogenous NA. Likewise, 5-methyl urapidil and chloroethylclonidine, alpha-1 adrenoceptor antagonists with selectivity for the alpha-1A and alpha-1B receptor subtypes, respectively, concentration-dependently decreased the increase in perfusion pressure elicited by electrical stimulation of the perimesenteric nerves at concentrations lower than that required to block the vasoconstriction elicited by exogenous NA. The combined perfusion of 3 μM suramin plus 0.1 nM prazosin did not result in a complete inhibition of the physiological response. Only upon the simultaneous application of BIBP plus suramin plus prazosin was the rise in perfusion pressure abolished. These results support the working hypothesis that the sympathetic nerves of the rat mesenteric bed release NPY, ATP and NA that act as postjunctional cotransmitters in this neuroeffector junction.
The classical notion that NA is the only neurotransmitter of the sympathetic nervous system has been convincingly challenged in the past decade. Several lines of evidence support the hypothesis that extracellular ATP, costored in the sympathetic varicosities with NA, triggers fast excitatory postjunctional potentials in a variety of peripheral smooth muscle cells and in the central nervous system (Surprenant et al., 1995). The ATP response is mediated by the activation of ATP-selective P2X membrane receptors (Burnstock and Kennedy, 1985). Suramin, acting as a low-affinity, competitive antagonist of all subtypes of these receptors (Dunn and Blakeley, 1988), has played a pivotal role in the elucidation of the pharmacological properties and physiological role of the growing family of extracellular ATP receptors (Surprenant et al.,1995).
In addition to the colocalization of ATP and NA in synaptic vesicles of sympathetic nerve terminals, numerous immunocytochemical studies also identify the presence of NPY in the large vesicles of these nerve endings. Fried et al. (1986) were the first to demonstrate that NPY is colocalized with ATP and NA in individual peripheral nerve varicosities. NPY is particularly abundant in the perivascular nerve terminals (Ekblad et al., 1984; Edvinsson et al., 1984; Edvinsson, 1985). Since its discovery by Tatemoto et al.(1982), NPY has been linked to sympathetic circulatory control, where it may be of physiological importance in blood pressure homeostasis. As such, NPY Y1 receptors have been implicated in the contraction of vascular smooth muscles (Wahlestedtet al., 1990) leading to elevation of systemic blood pressure (Lundberg and Tatemoto, 1982; Mabe et al., 1985). This notion has been amply supported by studies in isolated smooth muscle preparations, particularly from the cerebral blood vessels (Edvinsson, 1985), where NPY causes well-documented concentration-dependent vasomotor effects. In addition to its direct participation, NPY also potentiates the vasomotor responses of the coliberated NA (Edvinsson et al., 1984; Wahlestedt et al., 1985), offering another interesting mechanism to explain its potent vasomotor effect and its involvement in the control of the peripheral resistance. The recent discovery of BIBP 3226 (Rudolfet al., 1994), a selective and potent nonpeptide NPY Y1 receptor antagonist, has unveiled exciting possibilities for the exploration of the physiology and pathophysiology of this NPY receptor. As a result of the selectivity and competitive pharmacodynamics of this antagonist, BIBP 3226 represents a novel and interesting tool as compared with α-trinositol, a noncompetitive, low-affinity NPY antagonist (Donoso et al., 1993).
Recently, Donoso et al. (in press, 1997) described that the stimulation of the sympathetic nerve terminals from the rat arterial mesenteric bed causes the exocytotic release of NPY to the mesenteric perfusate. Because NPY is known to stimulate vasoconstriction in this vascular territory, we raised the working hypothesis that endogenously released NPY may be involved in the classical vasomotor response elicited by electrical stimulation of the sympathetic nerves surrounding the superior mesenteric artery. The aim of this investigation was to assess whether the endogenously released NPY participates in this vasomotor response, and further, to study the degree to which NPY activity, in this response, is harmonized with the activities of ATP and NA. In support of our hypothesis, a report byZukowska-Grojec and Haass (1987) substantiated that a combined blockade of alpha and beta adrenoceptors resulted in only a 50% inhibition of the severe and prolonged reduction in the mesenteric blood flow upon perimesenteric nerve stimulation. To test the coordinated activity of the neurotrasmitters, NPY, ATP and NA, the following pharmacological tools were used: BIBP 3226, a selective NPY Y1 receptor antagonist; the competitive ATP receptor antagonist suramin; and prazosin, a potent alpha-1 adrenoceptor competitive antagonist. Present results suggest that the sympathetic neurochemical triad is involved in the vasomotor response elicited by stimulation of the perimesenteric sympathetic nerves.
Methods
Animal and drug sources..
All experimental animals were bred at the P. Catholic University Animal Reproduction Laboratories. Adult Sprague-Dawley rats (250–300 g) were housed four to six per cage, maintained on a 12-hr dark/light cycle, at a constant temperature and humidity and fed their standard chow diet ad libitum; rats had free access to food and tap water.
BIBP 3226 was provided by Dr. K. Rudolf, from K. Thomae GmbH, Biberach, Germany. Suramin was donated by Hoechst Pharmaceuticals; prazosin and noradrenaline hydrochlorides were purchased from Sigma Chemical Co. (St. Louis, MO). Chloroethylclonidine dihydrochloride and 5-methyl urapidil were purchased at RBI (Natick, MA). All drugs were dissolved in distilled water and sonicated to assure complete solubility before drug testing.
Perfusion of the rat mesenteric bed and expression of experimental results..
Adult Sprague-Dawley rats (250–300 g) were anesthetized with 40 mg/kg pentobarbitone. The abdomen was opened by a midline incision, and the superior mesenteric artery was cannulated and perfused with warmed (37°C) Krebs-Ringer solution at a rate of 2 ml/min by a Gilson peristaltic pump. The attached mesentery was excised from the intestinal wall and the isolated mesenteric bed was transferred to a warmed chamber, following the procedure detailed by McGregor (1965). The mesenteric fat was not routinely cleaned off the mesenteric bed; only few experiments were performed after the manual removal of the surrounding adipocytes. As there was no substantial difference in the potency of NA or the magnitude of the electrically evoked nerve-induced vasoconstriction, the surgical procedure to remove the mesenteric adipocytes was discontinued. To record variations in the perfusion pressure, a pressure gauge transducer was placed at the entrance of the mesenteric artery. Platinum electrodes were placed around the main channel of the superior mesenteric artery and were later connected to a Grass S44 stimulator. The transducer was connected to a recording polygraph to continually record changes in the perfusion pressure directly from the mesenteric artery.
All experiments were begun with a 4-min perfusion with 70 mM KCl to assess tissue and preparation viability. Next, the preparations were challenged during 10 or 30 sec with electrical pulses of 2, 10, 15, 20 or 30 Hz, or stimulated with trains of 30 Hz for 3, 5, 10, 20 or 30 sec, either alone or in the presence of 500 nM BIBP 3226, 3 μM suramin or 0.1 nM prazosin, or a combination of these antagonists. In either set of experiments, the electrical pulses were delivered regularly every 2 min. All drugs were dissolved in Krebs-Ringer buffer and were continually perfused while the protocols for electrical stimulation of the perivascular nerves progressed. In some of the experiments, a concentration-response curve describing the vasomotor effect of NA was also performed, generating a control with which the efficiency of the alpha-1 adrenoceptor antagonists could be evaluated.
To minimize animal variability and day-to-day variations, the results are expressed, in all cases, as the percent increase in perfusion pressure relative to a standard 70 mM KCl challenge. The vasomotor potency of NA is arbitrarily expressed as the −logarithm of the agonist concentration ± S.E.M. required to increase the perfusion pressure in 50 mm Hg (pD value)
Effect of either BIBP 3226, suramin or alpha-1 adrenoceptor antagonists on the mesenteric artery neuroeffector junction.
To assess the possible participation of NPY, ATP and NA in sympathetic neurotransmission in the arterial mesenteric bed, two separate protocols were developed by use of either BIBP 3226, suramin or prazosin independently.
In the first series of experiments, the perimesenteric arterial nerves were stimulated for 10 sec at regular 2-min intervals with frequencies of 2, 10, 15, 20 or 30 Hz and then, with the same intervals and frequencies, the duration of stimulation was extended to 30 sec. After a 30-min equilibration period with the subsequently added antagonist, the same series of stimuli were repeated in the continual presence of this antagonist: either 500 nM BIBP 3226, 3 μM suramin or 0.1 nM prazosin. A separate preparation was used to study the effect of each drug receptor antagonist. The aim of the second protocol was to investigate the effects of varying the stimulatory duration. The perimesenteric nerves were stimulated at regular 2-min intervals with trains of 30 Hz during 3, 5, 10, 20 and 30 sec before and after a 30-min antagonist equilibration period in the continual presence of the added drug as detailed above. Additional series of experiments were performed to clarify the nature of the alpha-1 adrenoceptor involved in the vasomotor response elicited by electrical stimulation. For this purpose, the isolated arterial mesenteric bed was either incubated with 0.01 to 1 nM 5-methyl urapidil or 1 to 30 μM chloroethylclonidine. As in the other alpha-1 adrenoceptor antagonists, tissues were perfused with these drugs for 30 min and during its continual presence while exogenous NA was applied and during the performance of the electrical stimuli protocols.
Because each preparation was viable through two cycles of a protocol, the first cycle of stimuli served to delineate precise and tissue-specific control conditions (for variations in either frequency or duration of the electrical stimuli), whereas the second was used to analyze the effects of the specific receptor antagonists. This experimental design allowed us to use, in some instances, the paired Students t-test.
In most of the experiments, NA concentration-response curves were performed before and after perfusion with either 500 nM BIBP 3226, 3 μM suramin or 0.1 nM prazosin or the combination of 3 μM suramin plus 0.1 nM prazosin to further specify the individual control conditions. The concentration of NA required to cause an increase in 50 mm Hg was interpolated from each concentration-response curve and was expressed as the −logarithm of the agonist concentration.
In a separate, parallel series of experiments, we assessed the effect of higher antagonist concentrations (30 μM suramin, 10 and 100 nM prazosin) on the vasomotor activity elicited by electrical stimulation of the perimesenteric artery nerve fibers.
Simultaneous perfusion with suramin and prazosin.
To assess the effect of the combined perfusion with 3 μM suramin and 0.1 nM prazosin on the vasomotor responses induced by the electrical depolarizations of the nerve fibers, the two sets of protocols outlined in the previous paragraphs (frequency and duration variations) were performed in preparations simultaneously perfused with 3 μM suramin and 0.1 nM prazosin.
Effect of simultaneous perfusion with BIBP 3226, suramin and prazosin on the vasomotor responses elicited by the perivascular nerve stimulation.
To assess the simultaneous participation of NPY, ATP and NA, arterial mesenteric bed preparations were first challenged with 10 μM NA followed by 10-sec trains of electrical pulses of 2, 10, 15, 20 and 30 Hz, each delivered at regular 2-min intervals. Five minutes later, the same preparations were electrically depolarized with the same trains of electrical stimulation but allowing a 30-sec duration. Immediately after, the preparations were perfused for 30 min with buffer including BIBP 3226 (at either 100 or 500 nM), plus 3 μM suramin and 0.1 nM prazosin. Thereafter, in the continuous presence of this antagonist cocktail, the set of 10-sec and 30-sec trains of electrical stimulation were repeated.
Statistical analysis.
Each experiment was designed to serve as its own control. Several computerized statistical tests were performed; analysis of covariance was used routinely. The paired Student’s t-test was only used when required. In all cases, a P value of less than .05 was assigned significance.
Results
Partial blockage of the vasomotor activity elicited by electrical stimulation of the perimesenteric artery nerve fibers by either BIBP 3226, suramin, alpha-1 adrenoceptor antagonists or a combination of suramin and prazosin.
Electrical stimulation of the perimesenteric nerves caused frequency-dependent increases in the perfusion pressure of the arterial mesenteric bed of the rat. The increases in perfusion pressure elicited by nerve depolarization were abolished in the presence of 1 μM guanethidine (data not shown). The threshold frequency of nerve stimulation that caused a reproducible increase in perfusion pressure was 10 Hz; higher frequencies caused proportional rises in perfusion pressure (figs.1 and 2). In addition, it was observed that as the duration of the 30-Hz train of stimuli was increased from 3 to 20 sec, proportional increases in the perfusion pressure were elicited (fig. 2). This trend was not observed for stimuli surpassing 20 sec of duration.
Perfusion with 500 nM BIBP 3226 significantly blocked by approximately 30% the increase in perfusion pressure elicited by the 20- to 30-Hz train of pulses (see figs. 1A and 2A). Analysis of covariance revealed that the 500 nM BIBP 3226 treatment blocked the frequency-response curve and the duration of the stimuli-curve with a P value less than .005 [F(67,1) = 13.4; and F(67,1) = 26.6], respectively. In the absence of electrical stimuli, this antagonist, at least at the concentrations examined, did not cause per se a change in the perfusion pressure of the preparations. Thus, there is no indication that the peptide has a partial agonist effect, nor that it is integral in the maintenance of perfusion pressure before electrical stimulation of the perimesenteric nerves. Almost identical results were obtained when the perimesenteric nerves were stimulated with the same train of frequencies during 30 sec (data not shown). BIBP 3226 did not modify significantly the concentration of NA required to cause an increase in 50 mm Hg in the arterial mesenteric circulation (table1).
In a parallel fashion, perfusion with 3 μM suramin partially blocked the increase in perfusion pressure elicited by 10 sec electrical stimulation of the perimesenteric arterial nerves (see tracings in figs. 1B and 2B). The frequency-response curve in the presence of 3 μM suramin attained a P value < .005 [F(57,1) = 16.1]; whereas the P value attained with suramin for the duration-response curve was 0.01 [F (73, 1) = 9.1]. Almost identical results were obtained when the frequency-dependency curve was performed with trains of pulses of 30 sec duration (data not shown). Increasing the concentration of suramin 10-fold, did not cause a significantly larger blockade of the pressor response (data not shown). In some experiments suramin even modestly potentiated the vasomotor effect of NA; however, it did not shift significantly the NA concentration-response curve to the right (table 1).
Perfusion of the arterial mesenteric bed with 0.1 nM prazosin significantly dampened the electrically induced increases in perfusion pressure, which caused downward and rightward displacement of the frequency (P < .005) and duration (P < .01) dependency curves. Figure 2C shows the frequency-response curve with pulses delivered during 10 sec. Similar results were obtained when 0.1 nM prazosin was tested in preparations electrically depolarized at different frequencies during 30 sec (data not shown). Augmenting the prazosin concentration 10-, 100- and 1000-fold progressively blocked the vasomotor effect elicited by electrical stimulation of the nerve fibers but never abolishing it. Prazosin (0.1 nM) did not significantly modify the exogenous NA concentration-response curve (table 1). Larger concentrations of prazosin caused concentration-dependent rightward shifts of the NA vasomotor concentration-response curve (data not shown).
To further substantiate the nature of the alpha-1 adrenoceptor involved in the vasomotor effect elicited by the perivascular nerve stimulation, additional experiments were performed with selective antagonists that can discriminate between thealpha-1A and the alpha-1B adrenoceptor subtypes. Tissue incubation with 5-methyl urapidil demonstrated that this antagonist is as potent as prazosin to block the nerve-induced vasoconstriction (fig. 3, left panel), without causing a proportional blockade of the exogenous NA-induced contractions (table 2). In contrast to the graded nature of the 5-methyl urapidil-elicited blockade of the nerve-induced responses, the selective alpha-1B adrenoceptor antagonist, chloroethylclonidine, showed a lesser degree of concentration dependence. Although 1 μM chloroethylclonidine did not essentially modify the nerve-evoked vasoconstriction, treatment with 3 μM elicited a substantial blockade, which caused a flattening of the frequency-response curve (fig. 3, right panel). The magnitude of the blockade was not increased further even by a 10-fold increase in the concentration of chloroethylclonidine. This result is compatible with the alleged alkylation mechanism proposed for the nonequilibrium blockade caused by chloroethylamines reagents. As also observed with the other alpha-1 adrenoceptors antagonists, chloroethylclonidine was less effective antagonizing the NA-induced vasoconstriction than blocking the nerve-evoked responses (table2), except at large concentrations of the alpha-1 adrenoceptor blocker.
Joint perfusion with 3 μM suramin and 0.1 nM prazosin caused a blockade of the increase in perfusion pressure elicited by nerve stimulation which was not additive (fig.4). Analysis of covariance for the curves obtained in the presence of both 3 μM suramin plus 0.1 nM prazosin yielded a P value less than .01 for the frequency-response curve [F(56,1) = 8.9], and a P value < .005 for the analysis of the duration of the electrical stimuli [F(67,1) = 25.2] as compared with the curve obtained before the application of the antagonists.
Blockade of the vasomotor responses evoked by the simultaneous perfusion of BIBP 3226, suramin and prazosin.
The compounded perfusion of the mesenteric bed with 500 nM BIBP 3226, 3 μM suramin and 0.1 nM prazosin obliterated the increases in perfusion pressure caused by either 10- or 30-sec trains of electrical nerve stimulation (figs. 5 and6). The effect of BIBP 3226 was concentration-dependent; although 100 nM BIBP 3226 caused a significant blockade, the effect with 500 nM was more intense (fig. 6), providing circumstantial evidence in favor of the competitive nature of the pharmacodynamics of BIBP 3226. Analysis of covariance established that the effect of either 100 or 500 nM BIBP 3226 plus suramin plus prazosin was significantly different from that observed with only suramin plus prazosin, at either 100 or 500 nM BIBP 3226, in the experiments using a 10-sec train of stimuli or in the experiments using trains of stimulation of 30 sec. The F values for the respective statistical analysis of covariance attained with the 10-sec stimuli are as follows: control vs. suramin plus prazosin [F(86,1) = 111.8, P < .005]; control vs.100 nM BIBP 3226, [F(87,1) = 31.4, P < .005]; control vs. 500 nM BIBP 3226 [F(82,1) =32.8, P < .005]; suramin plus prazosin vs. 100 nM BIBP 3226 [F(56,1) = 12.3, P < .005]; suramin plus prazosinvs. 500 nM BIBP 3226 [F(51,1) = 18.2, P < .005]; and 100 nM BIBP 3226 vs. 500 nM BIBP 3226 [F(52,1) = 13.6, P < .005]. Similar Fvalues were derived for the trains of 30-sec pulses.
Discussion
Several lines of evidence, dating back to the classic pharmacological studies by McGregor (1965), support the notion that the vasomotor response elicited by stimulation of the perimesenteric arterial nerves is specifically caused by the stimulation of sympathetic nerve fibers. Recent data from our group indicate that these vasomotor responses are obliterated after tissue perfusion with 1 μM guanethidine or after the treatment of animals with the sympathetic neurotoxin, 6-hydroxydopamine (Donoso et al., in press, 1997). Furthermore, Donoso et al. (in press, 1997) demonstrated that the release of both NA and NPY to the mesenteric perfusate upon electrical stimulation of the perimesenteric arterial nerves is annulled in rats receiving the pretreatment above. It is unlikely that the adipocytes present in the preparation may release chemicals that could interfere with or facilitate sympathetic neurotransmission. In sum, the present information, in conjunction with recently published data, favors the notion that the stimulation of the perimesenteric arterial nerve fibers cause a sympathetic vasomotor response that involves the coordinated activation of NPY Y1receptors, P2x receptors for extracellular ATP and alpha-1 adrenoceptors. All these receptors are likely localized in the vascular smooth muscle cells (Huidobro-Toro et al., 1990). With regard to the nature of the alpha-1 adrenoceptors involved in the vasomotor responses elicited by electrical nerve stimulation, present results support the notion that both the alpha-1A and thealpha-1B adrenoceptors subtypes are present in the mesenteric circulation, and are likely both involved in the nerve-induced vasoconstriction since either 5-methyl urapidil and chloroethylclonidine (Minneman et al., 1988; Gross et al., 1988) proved potent and efficacious in blocking the said response.
Neither BIBP 3226, suramin, prazosin nor a combination of suramin and prazosin abolished the vasomotor activity elicited by the electrical stimulation of the mesenteric arterial sympathetic nerves. Only in the collective presence of BIBP 3226, suramin and prazosin was the vasoconstriction induced by perimesenteric nerve stimulation obliterated. Because the independent use of a wide range of concentrations of the two antagonists, suramin and prazosin, did not cause complete inhibition of neurotransmission, we reasoned that perhaps their combined application, even at low concentrations, could evoke the desired blockade. Because this was not the case, NPY became the logical and reasonable candidate for the nonadrenergic and nonpurinergic component of this response. As such, 500 nM BIBP 3226 (500 nM) was incorporated, along with 3 μM suramin and 0.1 nM prazosin, into the antagonist cocktail, which resulted in the expected almost full blockade of the neuroeffector junction responses. A parsimonious interpretation of these findings suggests that the combination of all three neurotransmitters (NA, ATP, NPY) is involved in the vasopressor response induced by electrical depolarization of the nerve terminals. However, beyond a simple additive relationship, we hypothesize that some sort of coordinated physiological synergism must be operating, in which in addition to the activation of transmitter-specific receptors, there is an integral dynamic interplay between the transmitters, potentiating a response that each of the three in isolation could not. This interpretation could shed light on the finding that the simple combination of suramin and prazosin did not cause a proportionally larger blockade of the response, as would be expected from the addition of the partial blockades of purinoceptors and adrenoceptors. We are aware that the present study illustrates the antagonist effects related to our hypothesis, provoking us to complement the present findings with positive evidence using the actual cotransmitters to mimick the physiological effect of the sympathetic nerve stimulation.
All three alpha-1 adrenoceptor antagonists studied proved more effective in blocking the nerve-evoked vasoconstrictions, even at high stimulation intensities, than that caused by exogenous NA. Several arguments could be invoked to account for these observations. A pharmacokinetic variant could be related to the access of the agonists and antagonists to the biophase. Pharmacodynamic explanations could be oriented to a differential action of endogenous and exogenous NA at alpha-1 adrenoceptor subtypes. Perhaps the involvement of postjunctional alpha-2 adrenoceptors in the response to exogenous NA could be determinant, because such receptors have been shown to be present in the vasculature of the rat hindlimb (Medgett and Ruffolo, 1987), and in the superior mesenteric artery, but not the rat mesenteric veins (Meynard, C. and Huidobro-Toro, J. P., unpublished observations).
The long-awaited selective, potent and competitive NPY Y1receptor antagonist, BIBP 3226 (Rudolf et al., 1994), has opened new channels for the investigation of the role of the NPY receptors in the maintenance of cardiovascular homeostasis, and particularly for the assessment of NPY’s function in sympathetic neurotransmission. The first report of the in vivocardiovascular effects of this nonpeptide antagonist indicates that in the pithed rat, the antagonist is quite selective, and that i.v. doses in the range of 0.01 to 0.1 mg/kg are required to consistently observe a significant rightward displacement of the NPY pressor dose-response curve (Doods et al., 1995; Mezzano, V. and Huidobro-Toro, J. P., unpublished observations). Lundberg and Modin (1995) demonstrated that BIBP 3226 inhibits sympathetic vasoconstriction in vivo, raising the hypothesis that neuronally released NPY, via its activity at NPY Y1 receptors, is of major importance in the long-lasting component of reserpine-resistant sympathetic vasoconstriction. Parallel results were obtained by Malmstrom and Lundberg (1995) in an in vitro investigation using the isolated guinea pig vena cava and by Racchi et al. (1997)using human mesenteric arteries and veins. These reports substantiate the notion that the vascular actions mediated by NPY released from sympathetic nerve fibers are the result of NPY Y1receptor activation.
In our interpretation of the investigated physiological response, it is imperative to recognize extracellular ATP as a neurotransmitter in the circulatory system. Ramme et al. (1987) reported the first evidence that extracellular ATP is the neurotransmitter in jejunal branches of the rabbit intestinal circulation, demonstrating that nonadrenergic mechanisms indeed operate in sympathetic nerves. Furthermore, Westfall et al. (1995) recently documented that in the rat arterial mesenteric bed, exogenous ATP produces a concentration-dependent increase in perfusion pressure, which supports the notion that ATP receptors present in blood vessels cause contraction in the mesenteric vascular territory. Lending additional support to our hypothesis, Westfall et al. (1995) studied the ability of 1 to 100 nM NPY to facilitate ATP-induced vasoconstriction. The present findings broaden this concept, suggesting that the stimulation of sympathetic fibers implies the coordinated action of ATP, NPY and NA at postjunctional receptors. This concept may have important physiological and clinical implications and is consistent with the views presented by Campbell (1987) and Burnstock (1990).
It is pertinent to ask whether the present observations are limited anatomically to the mesentery and philogenically to rodents. In this context, studies in progress are evaluating whether the principle at hand can be extended to other vascular territories. Recent experimental evidence from our group indicates that in the human saphenous vein, which has a rich NPY immunoreactivity, the peptide potentiates the catecholamine-induced vasoconstriction of in vitro ring preparations (Racchi and Huidobro-Toro, manuscript in preparation). With regard to non vascular tissues, ample evidence, which supports the notion that sympathetic cotransmission is operant in the gastrointestinal and reproductive systems (Ellis and Burnstock, 1990;Torres et al., 1992; Lundberg, 1996), particularly in the vas deferens of various species. In this tissue, ATP and NA are the most significant motor transmitters, ATP being responsible for the fast excitatory postjunctional potential (Donoso et al., 1994). Moreover, the role of ATP in the vas deferens is strongly supported by the recent finding that several members of the growing family of P2X ATP receptors are abundantly expressed in the smooth muscle cells of the vas deferens (Valera, 1994).
Whether the participation of NPY in the regulation of the mesenteric circulation is somehow physiologically related to the central feeding behavior incited by NPY (Clark et al., 1984; Stanley and Leibowitz, 1985), an effect apparently mediated by the newly identified NPY5 brain receptor (Gerald et al., 1996) remains to be elucidated. Of particular interest is the use of the NPY deficiency propagated in mutant mice reported by Erickson et al.(1996). It remains to be clarified whether NPY coparticipates in the minute-to-minute regulation of blood pressure. NPY may be involved in pathophysiological conditions that imply consistent and continual sympathetic discharges. Stress is one such condition. In this context, Zukowska-Grojec et al. (1996) have recently shown that stress-induced mesenteric vasoconstriction can be partially blocked by BIBP 3226, an effect which likely involves the release of NPY.
In summary, the present results indicate that stimulation of perimesenteric sympathetic nerve fibers evokes vasomotor responses of the rat mesenteric circulation that involves the coordinated postjunctional action of NPY in conjunction with extracellular ATP and NA.
Acknowledgments
To Dr. K. Rudolf from K. Thomae GmbH for providing us with a sample of BIBP 3226; R. Miranda for graphical designs; Drs. F. Valenzuela and A. Schliem for editorial assistance
Footnotes
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Send reprint requests to: J.P. Huidobro-Toro, Unidad de Regulación Neurohumoral, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Casilla 114-D, Santiago, Chile.
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↵1 Funded with local intramural grants from VRA, Dirección de Investigación, and CIM, Facultad de Medicina, P. Universidad Católica de Chile, FONDECYT grant 1960502, and Cátedra Presidencial en Ciencias (to J.P.H-T.).
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↵2 Partially supported by a grant from CIM, Escuela Medicina, P. Universidad Catolica de Chile.
- Abbreviations:
- NPY
- neuropeptide Y
- ATP
- adenosine 5′-triphosphate
- NA
- noradrenaline
- BIBP 3226
- (R)-N2-(diphenacetyl)-N-(4-hydroxyphenyl)-methyl-d-arginineamide
- Received October 21, 1996.
- Accepted April 24, 1997.
- The American Society for Pharmacology and Experimental Therapeutics