Introduction

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The cardiovascular system is richly supplied by neuropeptide-containing nerves (Wharton and Gulbenkian, 1987; Mione et al., 1990). These include small-caliber unmyelinated sensory fibers (C, and A fibers) sensitive to the neurotoxin capsaicin, that contribute to the regulation of cardiac performance and vascular tone via peripheral co-release of CGRP, SP and related neurokinins (Holzer, 1988; Maggi and Meli, 1988; Rubino and Burnstock, 1996). Because of the dual afferent and efferent function, the term sensory-motor nerves has been used to identify this neuronal population (Burnstock, 1990; Rubino and Burnstock, 1996). In both cardiac and perivascular sensory-motor neurotransmission, CGRP is the principal active transmitter that interacts with specific receptors in the target organ. In the myocardium, sensory-motor neurotransmission evokes cardioexcitatory effects, including positive chronotropism and inotropism, associated with coronary vasodilatation (Saito et al., 1987; Franco-Cereceda, 1991), the cardioexcitatory effects of CGRP being in several respects comparable to those of norepinephrine (Mantelli et al., 1991; Rubino and Burnstock, 1996). Vasodilator responses to sensory-motor stimulation have been shown in other vascular beds, including the mesenteric arterial tree (Kawasaki et al., 1988; Rubino and Burnstock, 1996).

Functional cross-talk between sympathetic and sensory-motor innervation has been shown in the heart and the mesenteric vascular bed. The sympathetic cotransmitters norepinephrine, neuropeptide Y and ATP modulate sensory-motor neurotransmission in isolated guinea pig atria and rat mesenteric vascular bed via prejunctional mechanisms (Kawasaki et al., 1990; Amerini et al., 1991). Long-term interactions have also been documented between these nerves. Previous investigations in our laboratory have shown that long-term sympathectomy by guanethidine in newborn rats evokes a selective increase of CGRP-like immunoreactivity in several tissues, a striking increase being observed in the atrial myocardium (Aberdeen et al., 1990, 1992). On the other hand, sensory-motor denervation by neonatal treatment with capsaicin increases perivascular sympathetic neurotransmission in the rat mesenteric arterial bed via both pre- and postjunctional mechanisms (Ralevic et al., 1995a). However, whether sensory-motor denervation has functionally expressed effects in the peripheral control of cardiac contractility remains to be established. Because sensory-motor nerves activate a variety of reflex responses leading to peripheral discharge of sympathetic and parasympathetic cardiac innervation, this information would be an important contribution to understanding of the autonomic control of cardiac function and would further elucidate the functional cross-talk between different components of the autonomic innervation in the heart.

The present study aimed therefore to evaluate the effects of sensory-motor denervation on cardiac sympathetic neurotransmission. Newborn rats were treated with capsaicin to cause permanent disruption of sensory-motor nerves (Holzer, 1991). Inotropic responses to EFS as well as to tyramine and exogenous norepinephrine were evaluated in atria isolated from rats treated with capsaicin or vehicle. The inotropism of CGRP was also compared in control and capsaicin-treated preparations.

	Materials and Methods

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Capsaicin treatment. Capsaicin treatment of newborn rats was performed as described previously in order to obtain sensory-motor denervation (Ralevic et al., 1995a). Sprague-Dawley rats were given s.c. injections of capsaicin (50 mg/kg b.wt.) on days 1, 2, 3, 5, 7 and 14 of life. Hypothermic anesthesia was used on the first few days of treatment by placing the pups in ice until movements and sensory perception were drastically reduced. Different litters were injected with vehicle (10% Tween 80, 10% ethanol, 80% saline). A third group of litters was used as age-matched controls. Tissue was obtained from these rats 12 to 14 weeks after capsaicin or vehicle injection. At the time of experimentation, body weight significantly differed among groups, being 607.3 ± 19.0 (n = 6), 550.7 ± 12.5 (n = 7) and 509.1 ± 13.8 g (n = 8) in control, Tween 80-treated and capsaicin-treated rats, respectively. Success of the capsaicin treatment was confirmed by lack of inotropic responses in vitro to capsaicin (1 µM).

Experimental preparation. The experimental model was as previously described (Rubino and Burnstock, 1995). Briefly, atria were isolated and mounted vertically in 10-ml organ baths containing Tyrode's solution of the following composition (mM): NaCl 115; KCl 4.7; CaCl₂ 1.8; MgSO₄ 1.2; KH₂PO₄ 1.2; NaHCO₃ 25; glucose 10, oxygenated with 95% O₂ and 5% CO₂ and kept at a constant temperature of 30°C, in order to reduce the metabolic need of the tissue without affecting the contractile function of the preparations, and in order to reduce the spontaneous atrial activity. Atria were stretched until the maximal spontaneous contractility was reached by applying a basal tension of 0.8 g. The atrial preparations were then electrically driven at a constant rate (4 Hz) throughout the experiment by punctuate electrodes connected with a pulse generator (model S9, Grass Instrument Co., Quincy, MA). Isometric contraction was recorded by an isometric force-displacement transducer (model FT 03C, Grass Instrument Co., Quincy, MA) and a D.C. preamplifier on a pen recorder (model 79D, Grass Instrument Co., Quincy, MA). Experiments were started after a period of equilibration of at least 60 min. Cumulative concentration-effect curves for the agonists tested were obtained at 20-min intervals. Because basal contractile tension did not significantly differ among control, capsaicin-treated and Tween 80-treated preparations, inotropic responses to EFS and to the agonists tested were evaluated as milligrams of increase above the basal contractile tension.

Sympathetic neurotransmission. Sympathetic neurotransmission was evaluated by EFS of the preparations. Trains of increasing numbers (2-32) of field pulses (20 Hz, 100-120 V, 1 ms) were applied at 3-min interval through two platinum plates parallel to the preparations and connected with a second pulse generator (model S45, Grass Instrument Co.). Atropine (1 µM) was present throughout the experiments in order to eliminate the parasympathetic response to EFS. Cardiac responses to EFS were almost abolished in the presence of the beta adrenoceptor blocker propranolol (1 µM) and were sensitive to 60-min treatment with guanethidine (50 µM), which confirmed that sympathetic neurotransmission was being evaluated.

Drugs. Rat CGRP, 8-methyl-N-vanillyl-6-nonenamide (capsaicin), ()norepinephrine (arterenol bitartrate) and tyramine hydrochloride were from Sigma Chemical Co. (St. Louis, MO). ()Propranolol (Inderal) was from ICI (Macclesfield, England), and guanethidine (Ismelin) was from Ciba Laboratories (Horsham, England). Reactive salts were from RBI.

Data evaluation. Data are reported as mean values ± S.E.; n refers to the number of animals from which experimental preparations were obtained. Analysis of data was performed on an IBM personal computer using FigP and GBStat softwares. The pD₂ values were calculated from the concentration-response curves constructed with the FigP software as log concentration of agonist giving 50% of the maximal effect. Group comparisons were made by applying repeated-measures analysis of variance according to Ludbrook (1994). Results were considered significantly different when P < .05.

	Results

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Cardiac responses to EFS and tyramine. Control preparations developed a basal tension of 255.2 ± 32.3 mg (n = 6). Basal tension in capsaicin- and Tween 80-treated preparations did not differ significantly, being 240.0 ± 25.8 (n = 9) and 219.6 ± 25.6 mg (n = 7), respectively. In the presence of atropine 1 µM, EFS evoked positive inotropic responses that increased with increasing number of field pulses in each experimental group. However, no significant difference was shown between control and capsaicin-treated preparations (fig. 1A). When the highest number of pulses (32) was applied, cardiac contractile tension increased by 412.3 ± 59.1 (n = 4), 377.5 ± 93.5 (n = 7) and 318.0 ± 66.6 mg (n = 4) in control, capsaicin-treated and Tween 80-treated preparations, respectively. In order to test calcium-independent outflow of catecholamines from sympathetic nerve terminals, we evaluated inotropic responses to the sympathomimetic agonist tyramine. Increasing concentrations of tyramine (0.3-10 µM) evoked positive inotropic responses that were not significantly different among control, capsaicin-treated and Tween 80-treated preparations (fig. 1B).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Positive inotropic responses of isolated atria to an increasing number (2-32) of field pulses (panel A) and to increasing concentrations of tyramine (panel B) in control (n = 4-6; ), capsaicin-treated (n = 7-8; ) and Tween 80-treated (n = 4; ) preparations. No significant differences were shown between experimental groups.

Cardiac responses to norepinephrine, CGRP and calcium. The sympathetic neurotransmitter norepinephrine, applied exogenously at concentrations of 1 to 300 nM, evoked similar increases of the contractile tension in control, capsaicin-treated and Tween 80-treated preparations (fig. 2A). Maximal inotropic responses developed in the presence of norepinephrine 300 nM were 239.0 ± 29.7 (n = 5), 273.7 ± 27.6 (n = 7) and 307.0 ± 86.1 mg (n = 4) increases of the basal contractile tension in control, capsaicin-treated and Tween 80-treated preparations, respectively. The pD₂ values were 7.49 ± 0.05 (n = 5), 7.26 ± 0.07 (n = 7) and 7.43 ± 0.07 (n = 4) in atria from control, capsaicin-treated and Tween 80-treated animals, respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Positive inotropic effect of increasing concentrations of norepinephrine (panel A), CGRP (panel B) and extracellular calcium (panel C) in control (n = 5-6; ), capsaicin-treated (n = 6-7; ) and Tween 80-treated (n = 4; ) atria. No significant differences were found between control and treated preparations.

	Discussion

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The high selectivity of capsaicin for sensory-motor nerves is now well established (Holzer, 1988, 1991; Maggi and Meli, 1988). The s.c. administration of high doses of capsaicin in newborn rats has been shown to cause, within 30 min, a permanent degeneration of unmyelinated primary sensory fibers (Jancso et al., 1977; Holzer, 1991). A drastic reduction in the content of CGRP- and SP-immunoreactivity has been shown in the cardiovascular system after neonatal administration of capsaicin (Wimalawansa, 1993). Therefore, in the present study, we administered capsaicin to newborn rats in order to obtain sensory-motor denervation.

The results of the present investigation show that sensory-motor denervation does not modify cardiac sympathetic neurotransmission. Cardiac responses to EFS and tyramine were unaffected by neonatal treatment with capsaicin, which indicates that mechanisms including both calcium-dependent exocytosis and calcium-independent release of the sympathetic neurotransmitter norepinephrine were maintained. These observations provide direct evidence for a lack of functional changes in the sympathetic innervation of the myocardium after sensory-motor denervation. The functional data of the present study are in line with results showing unchanged levels of norepinephrine in atria and superior cervical ganglion after capsaicin treatment of developing rats (Luthman et al., 1989). Lack of functionally expressed changes in sympathetic innervation were also shown in the rat tarsal muscle (Fike et al., 1992). In contrast, a significant increase of norepinephrine levels was shown in the vasculature of iris, cornea and oral cavity after sensory denervation (Terenghi et al., 1986; Luthman et al., 1989). Similarly, in the rat mesenteric arterial bed, sympathetic neurotransmission was enhanced by neonatal treatment with capsaicin as a consequence of increased tissue levels of norepinephrine (prejunctional changes) and hyper-responsiveness to the exogenous neurotransmitter (postjunctional changes; Ralevic et al., 1995a). In the present study of the heart, inotropic responses to norepinephrine did not differ between control and treated tissues, a result that indicates a lack of postjunctional changes in cardiac sympathetic neurotransmission because of sensory-motor denervation.

Long-term interactions between the peripheral terminals of sympathetic and sensory-motor nerves could be due to competition for a limited supply of neurotrophic factors between neuronal populations innervating a common target. Therefore, ablation of any of the two neuronal populations may result in marked terminal proliferation in the remaining neurons. However, although neonatal sympathectomy is consistently associated with increased levels of sensory-motor innervation, after sensory-motor denervation both an increase of and a lack of changes in sympathetic innervation have been described in different tissues. This discrepancy between no change and an increase in sympathetic innervation in the heart and tarsal muscle (this study; Fike et al., 1992) and in the vasculature (Terenghi et al., 1986; Luthman et al., 1989; Ralevic et al., 1995a) after sensory-motor denervation may be explained by the fact that an enrichment of sympathetic innervation may not be evidenced in tissues highly supplied with sympathetic nerves, such as myocardium and skeletal muscle, whereas it is observed in the relatively less densely innervated blood vessels.

Furthermore, the present results lead us to speculate that sympathectomy and sensory-motor denervation are associated with patterns of reinnervation not only based on the availability of neurotrophic factors in the target tissue but also determined by function. Our previous studies have shown hyperinnervation of atrial tissue by sensory-motor nerves after neonatal sympathectomy, which implies a larger release of CGRP within the myocardium. In these conditions, not only does the peripheral activity of capsaicin-sensitive nerves represent the local motor response to the activation of sensory-mediated reflexes, but it might also provide compensatory cardioexcitatory actions by peripheral release of CGRP when the tissue content of norepinephrine is reduced. The converse does not apply, because sympathetic nerves are unable to compensate for the lost efferent function provided by sensory-motor nerves and therefore do not increase. Capsaicin treatment might rather lead to a richer innervation of the myocardium by capsaicin-insensitive sensory fibers that could compensate for a variety of reflex responses normally supplied by sensory-motor nerves. In light of these observations, the present study adds new information on cardiac autonomic innervation and further support for the importance of the peripheral function of sensory-motor nerves in the autonomic control of the heart.

In addition to cardioexcitatory actions due to activation of sensory-motor nerves, direct effects of capsaicin have recently been described in the heart, primarily mediated by blocking of Ca⁺⁺ channels (D'Alonzo et al., 1995). The blocking of K⁺ ion conductance by capsaicin has also been shown in isolated cardiac myocytes (Castle, 1992). In the present investigation, however, neonatal treatment with capsaicin appeared to reduce the animals' growth without affecting the myocardial function; body weight was lower, whereas neither basal contractile tension nor calcium-evoked inotropism differed between control and capsaicin-treated preparations. Such a lack of direct actions of capsaicin on the cardiac contractile machinery rules out any possible misinterpretation of the present data as resulting from the effect of capsaicin on body weight.

It is well documented that sympathetic denervation results in enhanced responsiveness of most tissues, including the myocardium, to adrenoceptor agonists (Ishii et al., 1982; Cros and McNeil, 1987; Rice et al., 1987). The present study shows that inotropic responses of atrial myocardium to the sensory-motor transmitter CGRP were similar in control and capsaicin-treated preparations. Neither maximal responses nor pD₂ values were significantly different after neonatal capsaicin treatment. Similar results were obtained in the mesenteric arterial bed isolated from capsaicin-treated rats, where vasodilator responses to CGRP did not differ from those in control preparations (Ralevic et al., 1995b). The data the present investigation yielded are also consistent with a lack of supersensitivity to CGRP in other tissues after chronic treatment with capsaicin, as described by McEwan and co-workers (McEwan et al., 1993).

In summary, the present findings indicate that sensory-motor denervation by neonatal treatment with capsaicin does not affect cardiac sympathetic neurotransmission. Furthermore, they show that sensory-motor denervation is not associated with supersensitivity of the atrial myocardium to the sensory-motor neurotransmitter CGRP. These data will contribute to the understanding of the autonomic control of cardiac function.