Introduction

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Stimulation of the sympathetic nerves of the guinea pig vas deferens evokes a biphasic contractile response, which is mediated by two neurotransmitters, ATP and norepinephrine (NE) (Fedan et al., 1981; Sneddon and Westfall, 1984). The first phase of the neurogenic contraction is a twitch, which is mediated by ATP. NE mediates the second, tonic phase of the neurogenic contraction. Until recently, it has been a general belief that both transmitters are stored in the same synaptic vesicles and therefore upon release are presented at their specific receptors simultaneously and in constant proportions despite the frequency-dependent amplitude modulation of their release (Stjarne, 1989; Stjarne et al., 1994).

However, results from our recent studies on the kinetics of neuronally released cotransmitters from the guinea pig vas deferens demonstrated that ATP is released transiently at the beginning of nerve stimulation (Todorov et al., 1994, 1996). Quickly following its release, ATP is degraded by neuronally released nucleotidases (Todorov et al., 1996, 1997). The release of NE occurs later in the train of stimuli and is maintained throughout the course of nerve stimulation. This demonstrates that the release of ATP and NE from sympathetic nerves of the guinea pig vas deferens does not occur simultaneously, at least at the relatively low frequencies of nerve stimulation (up to 8 Hz). The purinergic, twitch-like contraction reflects the early and transient release of ATP, whereas the adrenergic, tonic contraction follows the kinetics of release of NE (Todorov et al., 1994; 1996). To explain the temporal disparity in the release of cotransmitters we suggested that sympathetic nerves may store ATP and NE in separate synaptic vesicles and release them via independent mechanisms (Todorov et al., 1996; Westfall et al., 1996). If the release of cotransmitters is indeed differential, it is conceivable that during the short-lasting bursts of sympathetic nerve activity ATP may be released either alone or together with NE, thus producing neurochemical messages with variable cotransmitter composition.

In the current study we quantified the amounts of endogenous ATP and endogenous NE that are coreleased within the first 10 s of nerve stimulation at various frequencies. We also used antagonists of the postjunctional P2X-purinergic and ₁-adrenergic receptors to pharmacologically dissect the effects of ATP and NE and to estimate the contribution of each cotransmitter to the initial twitch-like contractile response of the guinea pig vas deferens. Together, the results from transmitter release and contraction experiments indicate that at relatively low frequencies of stimulation, the twitch contraction is mediated exclusively by ATP but at higher frequencies of stimulation (e.g., above 8 Hz) NE contributes to the response by enhancing the postjunctional action of ATP.

We also examined the potential role of prejunctional ₂-adrenoceptors in regulating the composition of the transmitter "cocktail" that is released at various frequencies. The results indicate that at relatively low frequencies of stimulation prejunctional inhibition of the release of NE limits the amount of NE that is coreleased with ATP. At higher frequencies of stimulation (e.g., above 8 Hz) the autoinhibition of the release of NE is overridden and the amount of NE relative to ATP in the transmitter cocktail increases.

	Materials and Methods

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Tissue Preparation. Male albino guinea pigs (350-400 g) were sacrificed by decapitation. The vasa deferentia were removed, cleaned of the connective tissue, and the lumen exposed by section along the longitudinal axis.

Recording of Contractile Responses in a Superfusion System. The prostatic half of one vas deferens was placed in a horizontally oriented chamber (inner volume of 600 µl) equipped with two platinum ring electrodes for producing electrical nerve stimulation. One end of the tissue was fixed in place and the other attached by silk surgical suture to a Grass force-displacement transducer (FTO3). The tissue was superfused (2 ml min¹) with Krebs' solution (37°C) of the following composition: 150 mM NaCl, 4.6 mM KCl, 1.2 mM MgCl_4, 2.5 mM CaCl_2, 24.8 mM NaHCO₃, 1.2 mM KH₂PO₄, and 5.6 mM glucose, bubbled with 95% O₂, 5% CO₂. An initial load of 0.5 g was applied to the tissue by means of a micromanipulator. After an equilibration period of 45 min electrical field stimulation (EFS) at 8, 16, 32, and 64 Hz with pulse duration of 0.1 ms and supramaximal voltage was applied. Each tissue was stimulated at only one of these frequencies for 20 s.

Recording of Contractile Responses in an Organ Bath. In some experiments vasa deferentia were fixed with one end to a holder and incubated in chambers filed with 5 ml Krebs' solution (37°C) bubbled with 95% O₂, 5% CO₂. The other end of the tissue was attached to a force-displacement transducer for registration of the contractile effects produced by bolus injection (50 µl) of exogenously applied ATP and/or NE.

Overflow Experiments. Three tissues were loaded in a Brandel superfusion chamber with a volume of 200 µl. Whatman 541 filters were cut to fit both ends of the chamber, which was then inserted vertically into a thermostatic block (36°C) with platinum "screen" electrodes at each end. The tissues were superfused from bottom to top (2 ml min¹) with Krebs' solution bubbled with 95% O₂, 5% CO₂ and allowed to equilibrate for 45 min. When used, drugs were added to the superfusing solution. Sympathetic nerves were stimulated once for 20 s by EFS at 8, 16, 32, or 64 Hz with pulse duration of 0.1 ms and supramaximal voltage. Samples of the superfusate (~320 µl) were collected at 10-s intervals before and during sympathetic nerve stimulation in ice-cold test tubes.

HPLC Assays. The ATP, ADP, AMP, adenosine, and NE content of the superfused samples were analyzed as described previously (Todorov et al., 1996). Briefly, to measure the overflow of endogenous purines, chloroacetaldehyde (10 µl) was added to a 200-µl aliquot of the superfused samples to form 1, N⁶-etheno derivatives (e-purines) of ATP, ADP, AMP, and adenosine present in the sample. The e-purines were then separated on a gradient HPLC system equipped with a Waters Resolve radial pack cartridge (8NV Ph 4 µ; 8 × 10 mm). The amount of each compound was quantified by the means of a Shimadzu (RF 535) fluorescent monitor at an excitation wavelength of 230 nm and an emission wavelength of 420 nm. Buffer solutions consisted of 0.1 M phosphate (KH₂PO₄₎, pH 6.0 (buffer A) and 75% 0.1 M phosphate and 25% methanol (buffer B). The nucleotides and adenosine were separated using a gradient in which the concentration of buffer B was increased from 0 to 100% in 8 min according to Waters gradient profile 7. To measure the overflow of NE, 90-µl aliquots from the same samples were acidified with 10 µl of 1 M perchloric acid and filtered through a 0.22-µm Cameo 3N syringe filter into Waters limited volume inserts by centrifugation at 1000g for 1 min. The catecholamines were then separated on a isocratic HPLC system equipped with an ESA catecholamine HR-80 column, and the amount of NE quantified using an ESA model 5100 Coulochem electrochemical detector. The mobile phase for separation consisted of the following: 50 mM Na₂PO₄, 0.2 mM EDTA, 3 mM 1-heptanesulfonic acid, and methanol 3%, v/v, in deionized water. The pH was adjusted to 2.6 with phosphoric acid. Identification of individual peaks was by comparison with the retention times of known e-purines or catecholamines standards and the concentration was determined by peak area per pmol compared with standards. Standards were run with each set of samples. The HPLC equipment was controlled by and data collected by a HP Vectra XU computer equipped with a LAC/E card and Millenium 2010 Chromatography Manager software from Waters Corp. Results were normalized for volume and tissue weight and the data calculated as pmol mg¹.

Chemicals. The following chemicals, adenosine 5'triphosphate (disodium salt), ,-methyleneadenosine 5'-triphosphate (lithium salt), chloroacetal, desipramine, idazoxan (hydrocholride), ()norepinephrine (bitartrate salt), prazosin (hydrochloride), and yohimbine (hydrochloride) were purchased from Sigma Chemical Co. (St. Louis, MO), and suramin (hexasodium) was purchased from Research Biochemicals International (Natick, MA).

	Results

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The release of cotransmitters and the corresponding smooth muscle contractions of the guinea pig vas deferens are presented in Fig. 1. Increasing the frequency of nerve stimulation results in a proportional increase of the amplitude of the twitch contractions of the guinea pig vas deferens (Fig. 1A). The size of the contractions, as determined by measuring the areas under the curve of the twitch contractile responses, evoked by EFS at 16, 32, and 64 Hz, increased 3-, 6-, and 8-fold, respectively (Fig. 1B), over the contraction evoked by 8 Hz. However, we found that at these frequencies relatively constant amounts of purines are released during the first 10 s of nerve stimulation (Fig. 1C). The overflow of purines evoked by EFS at 64 Hz is only 0.7-fold higher than that at 8 Hz (Fig. 1D). At the same time, there is a dramatic 8-fold increase in the concomitant overflow of NE (Fig. 1, C and D). Thus, the molecular ratio of ATP/NE changes in a frequency-dependent manner and reflects the addition of NE to relatively constant amounts of ATP (Fig. 4D). This increasing amount of NE in the neurochemical message correlates with the appearance of a prazosin-sensitive component of the twitch response (Fig. 1, A and B). Twitch contractions with lower and relatively constant amplitudes remain once the postjunctional effects of NE are eliminated by prazosin (Fig. 1A). ATP is apparently the mediator causing these contractions, because they are abolished in the presence of suramin (Sneddon, 1992) or after desensitization of the P2X-purinergic receptors with , -methylene ATP (-m ATP) (Kasakov and Burnstock, 1982; Allcorn et al., 1986). The twitch contractions evoked at higher frequencies, even in the absence of prazosin, are also abolished after pretreatment with -m ATP indicating that they are evoked by ATP (Fig. 2, A and B). One possible explanation of these results is that activation of the postjunctional P2X-purinoceptors by ATP alone evokes the neurogenic twitch contraction and the role of the coreleased NE may be that of a positive modulator of the contractile response.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Effect of EFS at 8, 16, 32, and 64 Hz on the mechanical responses of the guinea pig vas deferens and the overflow of purines and NE. A, original recordings of contractile responses to EFS under superfusion conditions. The amplitude of the control biphasic contractile responses increases in a frequency of stimulation-dependent manner. The initial twitch contraction is attributed to the postjunctional action of the neurotransmitter ATP via P2X-purinoceptors. The second or tonic phase reflects the postjunctional action of NE via 1-adrenoceptors. Accordingly, the second phase of the contractile responses of the same tissue preparations is abolished in the presence of the 1-adrenoceptor antagonist prazosin (1 µM). The twitch contraction evoked by EFS at 8 Hz remains unaffected by prazosin. The twitch contractions evoked by 16, 32, and 64 Hz are progressively reduced by prazosin. Addition of idazoxan (1 µM), an antagonist of the prejunctional 2-adrenoceptors, produces a slight increase in the amplitude of the purinergic twitch contractions. B, mean results of the neurogenic twitch contractions of vas deferens evoked by EFS at 8, 16, 32, and 64 Hz (n = 12). The difference between the curves demonstrates the appearance and increase of a prazosin-sensitive, therefore, adrenergic component of the twitch contraction evoked by increasing frequencies of EFS. C, concomitant overflow of purines and NE during the first 10 s of EFS at 8, 16, 32, and 64 Hz (n = 8). The curves demonstrate that changeable proportions of purines and NE appear in the overflow evoked by EFS at different frequencies. D, results from the overflow experiments presented as percentage of the overflow of cotransmitters evoked by EFS at 8 Hz. The overflow of NE increases significantly more than the overflow of purines as frequency of stimulation increases.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effect of P2X-purinoceptor desensitization on the neurogenic contraction of the guinea pig vas deferens evoked by EFS at 8, 16, 32, and 64 Hz (n = 4). Pretreatment with the P2X-purinoceptor-desensitizing agent -m ATP (100 µM) abolishes the twitch contractions evoked by EFS at lower frequencies (8 and 16 Hz) and greatly reduces the twitch contractions at higher frequencies (32 and 64 Hz; A and B). Note that the tonic, adrenergic component of the neurogenic contraction remains after desensitization of the P2X-purinoceptors (A).

To test this hypothesis we studied the contractile effects of cocktails of chemicals that contained different proportions of ATP and NE. Application of ATP alone at a concentration of 100 µM evokes a transient contraction of the guinea pig vas deferens (Fig. 3). When the concentration of ATP was held constant at 100 µM but NE was applied with ATP, the contractions increased in parallel with the increase of the concentration of NE in the cocktail (Fig. 3, upper traces). Significant enhancement of the contractile response evoked by ATP is observed at concentrations of NE, which do not induce contractile effects on their own (Fig. 3, lower traces). Pretreatment of the tissue preparations with prazosin abolishes the potentiating effect of NE (data not shown), which suggests that the enhancement of the contractile responses to ATP is most likely mediated via postjunctional ₁-adrenergic receptors.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Effect of NE on the contractile response of the guinea pig vas deferens evoked by exogenous ATP (n = 6). Injection of a 50-µl solution of ATP (100 µM) evokes a transient contraction of vas deferens. A cocktail (50 µl) that contains both ATP (100 µM) and increasing concentrations of NE (0.01-10 µM) evokes contractions with increasing amplitudes (upper traces). Injections of NE in concentrations that produce significant facilitation of the contractile responses (0.01-1 µM) do not produce contractile response on their own (lower traces). Higher concentrations of NE (10 µM) produce small initial and a larger but delayed contractile responses. The increased amplitude of contractions produced by cocktails of ATP and NE indicates facilitation of the ATP evoked response in the presence of NE.

Pretreatment with antagonists of prejunctional ₂-adrenoceptors, such as idazoxan or yohimbine (the data for idazoxan are shown in Fig. 4), enhances both the overflow of NE and the prazosin-sensitive component of the twitch contraction of the guinea pig vas deferens but not, to any appreciable extent, the overflow of purines. Maximal enhancement by ₂-adrenoceptor antagonists of the contractile response and the overflow of NE is observed when the tissues are stimulated at 8 Hz (Fig. 4, A-C). The enhancing effects of idazoxan decline gradually with increasing frequency of nerve stimulation (Fig. 4, B and C). Thus, after elimination of ₂-adrenoceptor-mediated inhibition of NE release, the guinea pig vas deferens responds to nerve stimulation at 8, 16, 32, and 64 Hz with twitch contractions of similar amplitudes (Fig. 4, A and B). The overflow of NE after ₂-receptor blockade is also similar at 8, 16, 32, and 64 Hz (Fig. 4C). The concomitant overflow of purines (Fig. 4C) and the amplitude of the purinergic contraction that remains after addition of prazosin (Fig. 4A) are not significantly altered by idazoxan. Thus, removing prejunctional ₂-adrenoceptor-mediated inhibition of NE release eliminates the frequency-dependent variation in the ratio of ATP and NE and the frequency-dependent variation of the amplitudes of the corresponding contractile responses (Fig. 4D).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effect of inhibition of the prejunctional 2-adrenoceptors on the nerve-stimulated mechanical responses of the guinea pig vas deferens and the overflow of purines and NE. A, control contractile responses to EFS at 8, 16, 32, and 64 Hz are compared with those in the presence of idazoxan (1 µM). The application of this 2-adrenoceptor antagonist produces enhancement of both phases of the neurogenic contraction. The enhancement of the twitch contraction is more pronounced at lower frequencies of stimulation (8 and 16 Hz) and gradually declines with increasing frequency (A and B). Prazosin (1 µM), an antagonist of postjunctional 1-adrenoceptors, eliminates the effects of idazoxan and further reduces the amplitudes of the twitch contractions evoked by EFS at 16, 32, and 64 Hz (A) below the control levels (n = 12). After the combined idazoxan/prazosin treatment, the guinea pig vas deferens responds to EFS at different frequencies with twitch-like contractions only slightly higher in amplitude than these obtained after treatment with prazosin alone (Fig. 1, A and B). C, concomitant overflow of purines and NE from idazoxan-treated (1 µM) preparations during the first 10 s of EFS at 8, 16, 32, and 64 Hz (n = 8). Note the relatively constant levels of overflow of NE during stimulation at 8, 16, and 32 Hz. At all frequencies of nerve stimulation the overflow of NE is almost equal to that evoked at 64 Hz. This relatively constant overflow of NE is accompanied by a relatively constant (similar to the control) overflow of purines (C). Therefore, the ratio of purines/NE in the overflow after elimination of 2-adrenoceptor autoinhibition becomes relatively independent of frequency and similar to the ratio at 64 Hz (D).

	Discussion

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Neurons in both the central (Hokfelt, 1989) and peripheral (Stjarne, 1989; Lundberg, 1996) nervous system release a complex mixture of chemicals, including multiple neurotransmitters. The reason why nerves should release more than one transmitter is not immediately obvious to us nor is the mechanism by which it occurs. In this context Mayer and Baldi (1991) suggested, in analogy with strategies for encoding information in technical systems, that individual neurons may be able to control the "informational" content of their neurochemical messages by varying the proportions of the participating cotransmitters (see also Mayer, 1994). Such a model for cotransmission implies that the neurons should have a mechanism for selective, nerve activity-dependent regulation of the release of each of the participating cotransmitters (i.e., an encoding mechanism). It follows that to respond properly to the changeable informational content of the plurichemical message the target cells should be able to adjust the amplitude of their response according to the cotransmitter composition of each message (i.e., decoding mechanism).

In this study we examined this model for cotransmission in experiments with the guinea pig vas deferens. Previously reported results from studies on the kinetics of release of cotransmitters from this tissue (Todorov et al., 1994, 1996) have shown that at lower frequencies of stimulation (up to 8 Hz) NE is released in very small amounts compared with ATP. This finding suggests that if action potentials were fired at low frequency during the short-lasting bursts of sympathetic nerve activity, ATP would be the only mediator engaged in the process of neurotransmission. To approximate the expected duration of nerve activity under physiological conditions, we quantified the release of cotransmitters and the resulting smooth muscle contraction that occur within the first 10 s of EFS. The results show that when applied for a period of 10 s, EFS at 8 Hz produces release of ATP, accompanied with trace amounts of NE. The twitch contraction recorded under these conditions is not changed in the presence of ₁-adrenoceptor antagonist prazosin, however, it is abolished after desensitization of the P2X-purinoceptors with , -m ATP. These results are not novel to this study but do reinforce the conclusion that at lower frequency of neuronal activity the neuromuscular transmission in the guinea pig vas deferens is mainly purinergic.

However, when stimulated at higher frequencies, e.g., 16, 32, and 64 Hz, the sympathetic nerves of the guinea pig vas deferens begin to release NE along with ATP during the initial 10 s of stimulation. The addition of NE to the transmitter mixture corresponds to the appearance of a prazosin-sensitive component of the twitch contraction. The relative participation of ATP and NE in the cocktail of cotransmitters and the amplitude of the resulting smooth muscle contraction vary with the increasing frequency of nerve stimulation due to the greater amount of NE. These results suggest that the sympathetic nerves of the guinea pig vas deferens are capable of changing the composition of the neurochemical message as a function of frequency of stimulation.

The release of NE from the sympathetic nerves of the guinea pig vas deferens appears to be regulated by a subset of prejunctional ₂-adrenoceptors that have little influence on the release of ATP (Driessen et al., 1993; Todorov et al., 1995; Westfall et al., 1996). These ₂-adrenoceptors adhere to the general rule regarding the relationship between frequency of nerve stimulation and negative feedback mediated by autoreceptors (Langer, 1997) and effectively reduce the release of NE at low but not at high frequencies of nerve stimulation. The results from our experiments with antagonists of the ₂-adrenoceptors demonstrate that a more effective autoinhibition at lower frequencies of EFS limits the relative amount of NE that is released together with ATP. The neurochemical message released under these conditions contains mainly ATP and evokes relatively low amplitude purinergic contractions. As the frequency of nerve stimulation increases, the ₂-adrenoceptor inhibition is overcome. This enables increasing recruitment of NE into the neurochemical message, resulting in an increase in amplitude of the contractile response of the guinea pig vas deferens. Based on this evidence we suggest that the prejunctional ₂-adrenoceptors are an essential element of the mechanism for encoding neuronal information. Their physiological role appears to include that of a regulator of the ultimate cotransmitter composition.

The results from contraction experiments reported here are consistent with those of previous studies suggesting that the relative contribution of ATP and NE to the neurogenic contraction of smooth muscle in the rodent vas deferens and blood vessels may vary (Stjarne and Astrand 1985; Kennedy et al., 1986; Evans and Cunnane 1992). In the present study we demonstrate directly that the changeable contribution of cotransmitters to the neurogenic twitch contraction of the guinea pig vas deferens reflects the relative proportions of ATP and NE in the cocktail released from the sympathetic nerves. ATP, which is released in relatively constant amounts, triggers the smooth muscle twitch contractions. The amplitude of these responses, however, appears to be controlled by the frequency-dependent recruitment of NE into the cotransmitter cocktail, which facilitates the purinergic contractions. Synergistic postjunctional interactions of ATP and NE in the rodent vas deferens, evidence for which was previously reported (Kazic and Milosavljevic, 1980; Huidobro-Toro and Parada, 1988; Kishi et al., 1990; Fujita et al., 1996), appear to involve both facilitation of ATP-induced contractions by NE and facilitation of NE-induced contractions by ATP. The results reported here demonstrate that when coreleased with ATP, NE contributes to the neurogenic twitch contraction and this action is mediated via ₁-adrenoceptors. The fact that the purinergic and the prazosin-sensitive, adrenergic components of the twitch contractions are abolished after desensitization of the P2X-purinoceptors with -m ATP suggests that NE does not produce contractions on its own but rather facilitates the contractions triggered by ATP. We conclude therefore that the postjunctional facilitation of ATP by NE is the physiologically relevant mechanism that controls the amplitude of the neurogenic twitch contraction in the guinea pig vas deferens.

This evidence suggests that the target smooth muscle cells are competent to recognize, distinguish between, and respond appropriately to neurochemical messages with different cotransmitter compositions. The cotransmitter interactions at postjunctional sites appear to be an essential element of the mechanism for decoding neuronal information.

We conclude therefore, that the sympathetic nerves of the guinea pig vas deferens encode information by releasing cotransmitters in different proportions. The selective, ₂-adrenoceptor-mediated prejunctional modulation of the differentially released cotransmitters appears to be an essential element of the mechanism for translation of information from a bioelectrical code (frequency of nerve activity) to a neurochemical code. Postjunctional neurotransmitter interactions seem to be involved in the process of decoding the informational content of these neurochemical messages by the target cells. It seems possible that the mechanism involving both differential release of cotransmitters and their cooperative postjunctional action may be a general feature of the neurochemical transmission throughout the nervous system.