Introduction

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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 P_2X 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 (Wahlestedt et 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 (Rudolf et 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 -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 by Zukowska-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

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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.

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.

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.

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

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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.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Involvement of NPY, ATP and NA in the vasomotor responses elicited by electrical stimulation of the rat perimesenteric artery nerves. Polygraphic recordings were obtained from three separate protocols; the tracings show changes in the perfusion pressure of the arterial mesenteric circulation. Effect of 70 mM KCl followed by a 2-min pulse with 30 µM NA. The perimesenteric artery nerves were electrically stimulated with trains of 10, 15, 20 and 30 Hz (50 V, 1 msec duration) during 10 sec before and after perfusion with drug receptor antagonists. (A) partial reduction in the vasomotor effect produced by perfusion with 500 nM BIBP 3226; (B) blockade caused by 3 µM suramin; (C) 0.1 nM prazosin.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Partial blockade of the vasomotor response induced by electrical stimulation of the perimesenteric artery nerves by BIBP 3226, suramin and prazosin. Left-side panel shows the changes in the perfusion pressure of the arterial mesenteric bed evoked by a 10-sec train of pulses of varying frequencies recorded before and during a 30-min perfusion with the antagonists. The right-side panel shows the duration of a 30-Hz train of pulses ranged between 3 and 30 sec before and during a 30-min perfusion with each of the antagonists. (A) partial blockade of the vasomotor responses caused by 500 nM BIBP 3226. (B and C) effect of 3 µM suramin and 0.1 nM prazosin, respectively. Symbols in the figures indicate the mean values; bars the S.E.M. The number of separate preparations used in each experimental condition are in parentheses.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Blockade of the nerve-induced vasoconstriction by 5-methyl urapidil and chloroethyclonidine, selective alpha-1A and alpha-1B adrenoceptor antagonists, respectively. Frequency-response curves after 10-sec nerve stimulation periods were performed in the absence and after 30 min of perfusion with varying concentrations of either 5-methyl urapidil (left panel) or chloroethylclonidine (right panel). The antagonist was continually present during the performance of the second frequency response curve. Symbols indicate the mean values; the bars the S.E.M. The number of experiments performed with each concentration of the antagonists in parenthesis.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Effect of the simultaneous perfusion with 3 µM suramin plus 0.1 nM prazosin on the vasomotor responses elicited by the electrical stimulation of the perimesenteric artery nerve fibers. (Upper panel) polygraphic tracing of a typical protocol showing the effect of the simultaneous perfusion with 3 µM suramin and 0.1 nM prazosin on the increase in perfusion pressure elicited by 10-sec trains of pulses of different frequencies. (Lower panel) Effect of the combined perfusion with 3 µM suramin plus 0.1 nM prazosin on the vasomotor responses elicited by 30-sec trains of varying frequencies (left panel) and changes in the perfusion pressure caused by increasing the duration of a 30-Hz train of pulses (right panel) in the presence of the combined perfusion with both antagonists. Symbols depict the mean values, bars the S.E.M.; the number of separate preparations used is in parentheses.

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 and 6). 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 prazosin vs. 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 F values were derived for the trains of 30-sec pulses.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Simultaneous perfusion of the rat arterial mesenteric bed with either 3 µM suramin plus 0.1 nM prazosin, and 500 nM BIBP 3226, 3 µM suramin and 0.1 nM prazosin on the increase in perfusion pressure caused by electrical stimulation of the perimesenteric artery nerve fibers. Representative polygraphic tracings show the increase in perfusion pressure caused by a 4-min pulse of 30 µM NA followed by 10-sec trains of 10, 15, 20 and 30 Hz (left side) or 30-sec trains of electrical pulses (right side). The upper tracing, control, refers to a recording obtained in the absence of the antagonists. The middle tracing was obtained from the same rat when perfused simultaneously with 3 µM suramin plus 0.1 nM prazosin. The lower recording was obtained after the simultaneous perfusion with 500 nM BIBP 3226 plus 3 µM suramin plus 0.1 nM prazosin.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Blockade of the vasomotor response elicited by stimulation of the perimesenteric arterial nerves by the triple combination of BIBP 3226 plus suramin plus prazosin. Increase in perfusion pressure of the rat mesenteric bed elicited by electrical stimulation of the perimesenteric arterial nerves with trains of increasing frequencies during either 10-sec (A) or 30-sec (B) nerve stimulation, before (control) or 30 min after the initiation of the perfusion with either 3 µM suramin plus 0.1 nM prazosin, or these two antagonists plus either 100 nM BIBP 3226 or the combination of 3 µM suramin plus 0.1 nM prazosin plus 500 nM BIBP 3226. Symbols indicate the mean values; bars the S.E.M. The number of separate experiments performed per experimental condition is in parentheses.

	Discussion

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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 Y₁ receptors, P_2x 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 the alpha-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 Y₁ receptor 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 vivo cardiovascular 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 Y₁ receptor 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 P_2X 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.