Introduction

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Catecholamine biosynthesis is increased in adrenal medulla and sympathetic neurons when the sympathetic nervous system is stimulated (for reviews, see Zigmond et al., 1989; Kvetnansky and Sabban, 1993; Kumer and Vrana, 1996). Catecholamine biosynthesis is primarily controlled by the activity of TH (EC 1.14.16.2), which catalyzes the rate-limiting step in the biosynthetic pathway. Paradigms that stimulate the sympathetic nervous system rapidly activate TH enzymatic activity (Zigmond et al., 1989; Kumer and Vrana, 1996). This activation of TH is due to its phosphorylation on one or more serine sites, leading in most instances to a decrease in its K_m for the cofactor, tetrahydrobiopterin.

Numerous forms of acute stress, such as decapitation, electroconvulsive shock and subcutaneous formaldehyde injections, are associated with rapid activation of TH in the rat adrenal medulla (Masserano and Weiner, 1979, 1981; Masserano et al., 1981). This activation is blocked when the splanchnic nerve innervating the adrenal gland is transsected. Similarly, in the superior cervical ganglion, electrical stimulation of preganglionic fibers leads to activation of TH in the principal postganglionic nerves (Rittenhouse and Zigmond, 1990). It should be noted that not all forms of stress lead to activation of adrenal TH. For example, cold stress, which induces TH mRNA and TH protein via transsynaptic mechanisms, does not elicit activation of TH in the rat adrenal medulla (Fluharty et al., 1983). Nevertheless, most forms of stress and most drug treatments that stimulate the sympathetic nervous system activate TH and the prevailing evidence supports the hypothesis that this activation is mediated transsynaptically by neurotransmitters released from preganglionic nerve terminals.

The principal neurotransmitter of preganglionic nerves is acetylcholine. A number of early studies concluded that acetylcholine mediates the transsynaptic regulation of TH via its interaction with postsynaptic nicotinic cholinergic receptors (Zigmond et al., 1989). However, more recent studies have shown that multiple neurotransmitters are released from preganglionic nerve fibers and that stimulation of numerous postsynaptic receptors is associated with the activation of postganglionic TH in adrenal medulla and sympathetic ganglia (Zigmond et al., 1989; Kumer and Vrana, 1996).

In a previous report, we demonstrated that TH is activated in rat adrenal medulla by systemic administration of nicotine (Fossom et al., 1991b). Surprisingly, these effects of nicotine are not blocked by the administration of hexamethonium, a nicotinic receptor antagonist. Furthermore, even though nicotinic receptors are known to desensitize rapidly during chronic exposure to agonist, repeated injections of nicotine result in a sustained activation of adrenal TH. In contrast, hexamethonium blocks nicotine's effect on TH in adrenal glands in which the splanchnic nerve is transsected. In addition, activation of adrenal TH is observed in denervated adrenals after a single injection of nicotine but not after repeated injections of nicotine, suggesting that, as expected, nicotinic receptors desensitize in the denervated gland after repeated injections of the drug. These results have led us to hypothesize that adrenal TH is activated by systemically administered nicotine by two mechanisms: (1) direct interaction of nicotine with chromaffin cell nicotinic receptors; and (2) stimulation of the splanchnic nerve by nicotine, presumably due to its actions in the central nervous system, leading to the activation of adrenal TH via transsynaptic mechanisms. Because hexamethonium does not block the effect of nicotine in innervated glands, chromaffin cell receptors other than the nicotinic receptor must participate in this transsynaptic regulation of TH. However, which chromaffin cell receptors participate in this response remains unclear.

A number of non-nicotinic chromaffin cell receptors are likely candidates for mediating this transsynaptic response. These receptors include muscarinic cholinergic receptors and several noncholinergic receptors, including those for VIP, PACAP and adenosine. Agonists for these receptors stimulate appropriate second messenger systems in rat adrenal medulla, mediate the phosphorylation of TH and/or elicit catecholamine release under in situ conditions (Wakade and Wakade, 1983; Malhotra and Wakade, 1987b; Malhotra et al., 1989; Roskoski and Roskoski, 1989; Haycock and Wakade, 1992). In the present report, we test whether muscarinic cholinergic agonists activate TH in rat adrenal medulla in vivo, and we test whether this muscarinic receptor-mediated response participates in the transsynaptic activation of TH elicited by nicotine.

	Experimental Procedures

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Treatment of animals. Male Sprague-Dawley rats (150-200 g) were purchased from Charles River or Harlan animal facilities and permitted free access to food and water for 24 hr before treatment. Drugs were administered as follows: 0.5 to 20 mg/kg bethanechol s.c., 0.25 to 1.5 mg/kg carbachol s.c., 2.3 mg/kg nicotine s.c. (expressed as nicotine base, even though the drug was administered as the bitartrate salt), 15 mg/kg hexamethonium i.p. and 2 mg/kg atropine i.p. All drugs were dissolved in phosphate-buffered saline (10 mM potassium phosphate and 150 mM NaCl), buffered to pH 7.5 and administered in a volume of 1 ml/kg. Antagonists were injected 10 to 15 min before the injection of cholinergic agonists. Control rats were injected with an identical volume of phosphate-buffered saline, pH 7.5. At the appropriate time after drug administration, the rats were rapidly anesthetized using sodium pentobarbital at 150 mg/kg i.p. Adrenal glands were removed 3 to 5 min later and frozen rapidly on dry ice. Sodium pentobarbital was used to anesthetize the animals before death because it was shown to produce minimal stimulation of the adrenal gland by itself (Masserano and Weiner, 1979). All procedures and drug administrations were performed in accordance with the guidelines and approval of the University of Rochester Committee on Animal Resources.

Denervation of left adrenal gland. The left adrenal gland was denervated as described previously (Fossom et al., 1991b). Experiments were performed using these animals 4 to 6 days after surgery. Control experiments were run, which demonstrated that the effects of bethanechol on TH activity in the innervated and denervated glands were identical when administered 2, 4 or 6 days after surgery. To monitor whether the left adrenal gland was denervated, choline acetyltransferase activity was measured in the denervated and innervated glands. The results used for this study were obtained from animals in which 70% of the choline acetyltransferase activity was lost on the denervated side. This percentage of loss of choline acetyltransferase activity was shown previously to be associated with 80% loss of the transsynaptic activation of adrenal TH elicited by either nicotine administration or decapitation (Fossom et al., 1991b).

Enzyme assays. Adrenal glands were removed, rapidly frozen on dry ice and stored at 80°C. All subsequent procedures were performed at 4°C. Frozen adrenal glands were homogenized in 250 µl of 30 mM potassium phosphate (pH 6.8), 50 mM NaF and 10 mM EDTA, and the homogenate was centrifuged at 20,000 × g for 15 min. When appropriate, a 25-µl aliquot of the supernatant was removed for assaying choline acetyltransferase activity. The remainder of the supernatant was used for measuring TH activity.

Statistical analyses. The results were analyzed by one-way analysis of variance, using the computer program INSTAT. Comparisons between groups were made using the Student-Neuman-Keuls or Dunnett's multiple comparisons test, as noted in the figure and table legends. A level of P < .05 (two-tailed) was considered statistically significant.

	Results

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Effects of bethanechol and carbachol on adrenal TH activity. Different doses of either bethanechol or carbachol were administered subcutaneously to rats. Bethanechol, a selective muscarinic receptor agonist elicited an ~3-fold activation of adrenal TH at 10 or 20 mg/kg; 5 mg/kg bethanechol produced a variable 2- to 3-fold activation, whereas lower doses did not activate the enzyme (fig. 1A). A time course of the effect of bethanechol (10 mg/kg s.c.) on adrenal TH activity indicated that the enzyme was activated maximally 10 to 20 min after injection of the drug (fig. 2). Enzyme activity then decreased back to control activity by 1 hr after drug administration. Carbachol, an agonist that stimulates both nicotinic and muscarinic cholinergic receptors, elicited a 2- to 3-fold activation of adrenal TH at doses of 0 .5 mg/kg (fig. 1B). In subsequent experiments, we used 10 mg/kg bethanechol and 1.5 mg/kg carbachol to activate adrenal TH.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Dose-response curves for the effects of bethanechol or carbachol on TH activity in rat adrenal gland. Rats were administered subcutaneously different doses of (A) bethanechol or (B) carbachol, and adrenal glands were removed 20 min after the injection. TH activity was assayed using 0.1 mM 6 MPH4. The data represent the mean ± S.E.M. from 3 to 6 rats.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Time course for the effect of bethanechol on TH activity in rat adrenal gland. Rats were administered subcutaneously 10 mg/kg bethanechol, and adrenal glands were removed at different times after the injection. TH activity was assayed using 0.1 mM 6 MPH4. The data represent the mean ± S.E.M. from 3 or 4 animals.

Effects of cholinergic receptor antagonists on the activation of adrenal TH elicited by bethanechol or carbachol. Rats were injected with cholinergic receptor antagonists 10 to 15 min before the administration of either bethanechol or carbachol. The muscarinic receptor antagonist atropine (2 mg/kg i.p.) completely blocked the activation of TH elicited by either cholinergic agonist (table 2). In contrast, the nicotinic receptor antagonist hexamethonium (15 mg/kg i.p.) did not inhibit the activation of TH elicited by either muscarinic agonist. This dose of hexamethonium was previously shown to block effectively nicotinic receptor-mediated activation of adrenal TH in denervated adrenal glands (Fossom et al., 1991b). Neither atropine nor hexamethonium produced any effect on adrenal TH activity when administered alone (table 2).

Effect of bethanechol on TH activity in denervated adrenal glands. To determine whether the muscarinic receptor-mediated activation of adrenal TH occurred independent of transsynaptic influences, left adrenal glands were denervated by surgical transsection of the splanchnic nerve. Four to 6 days after surgery, the animals were administered saline or bethanechol, and adrenal TH activity was measured in both innervated (right adrenal) and denervated (left adrenal) glands 20 min after injection. To assess the effectiveness of the surgical denervation, choline acetyltransferase activity was measured in both adrenal glands. Based on previous studies (Fossom et al., 1991b), when choline acetyltransferase activity was diminished in the left adrenal gland by >70%, the gland was considered to be effectively denervated. Only data from animals that met this criteria were used for the results reported in figure 2. Bethanechol produced a 3-fold activation of TH in the innervated gland. However, surprisingly, bethanechol did not activate TH in the denervated gland (fig. 3).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Effect of splanchnic nerve transection on activation of adrenal TH by bethanechol. The left adrenal gland of each rat was denervated by surgically transsecting the splanchnic nerve. Four to 6 days after surgery, the rats were administered saline or bethanechol (10 mg/kg) subcutaneously, and adrenal glands were removed 20 min after the injection. TH activity was assayed using 0.1 mM 6 MPH4. The data represent the mean ± S.E.M. from 8 rats.

Effect of repeated injections of bethanechol on adrenal TH activity. In the next set of studies, we tested whether the muscarinic receptor-mediated activation of adrenal TH desensitized after prolonged exposure to agonist (table 3). When rats were repeatedly administered saline four times (one injection/hr), adrenal TH activity was unaffected. Hence, in agreement with previous studies (Fossom et al., 1991b), repeated subcutaneous injections were not stressful enough to activate adrenal TH. When rats were injected with saline 3 times (one injection/hr) and then administered bethanechol 1 hr after the third saline injection, adrenal TH activity was increased ~3-fold, as observed in naive rats injected once with bethanechol (figs. 1 and 2 and table 1). In contrast, when rats were injected with bethanechol three times (one injection/hr) and then challenged with a fourth injection of bethanechol (1 hr after the third injection), activation of TH was not observed (table 3). This result was in contrast to that which occurred with repeated administration of nicotine; TH was still activated after the fourth injection of nicotine (table 3). This latter result confirmed data obtained in our previous study, which used a slightly different treatment protocol (seven injections of nicotine, one injection every 30 min) (Fossom et al., 1991b).

Effect of cholinergic antagonists on adrenal TH activation elicited by nicotine. As reported in previous studies (Fossom et al., 1991b) and confirmed in table 5, nicotine increased adrenal TH activity 2-3-fold, and this activation was not blocked by either hexamethonium or atropine. The lack of effect of hexamethonium on this nicotinic response is thought to be due to the ability of nicotine to stimulate the splanchnic nerve, presumably via central mechanisms, leading to transsynaptic activation of adrenal TH mediated by multiple chromaffin cell receptors. To test whether the blockade of both nicotinic and muscarinic cholinergic receptors inhibited this transsynaptic activation of adrenal TH, rats were treated with both hexamethonium and atropine before the administration of nicotine. Even though these doses of hexamethonium and atropine totally block nicotinic and muscarinic receptor-mediated responses, respectively, in the adrenal gland (see (Fossom et al., 1991b) for hexamethonium and table 2 for atropine), the nicotine-mediated activation of TH was not inhibited in animals pretreated with both antagonists (table 5).

	Discussion

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The enzymatic activity of TH is highly regulated in neurons of the peripheral and central nervous systems (for reviews, see Zigmond et al., 1989; Kumer and Vrana, 1996). Short-term regulation occurs after acute stimulation of catecholaminergic neurons and is due to activation of preexisting enzyme molecules. Activation of TH by most extracellular signals is mediated by phosphorylation, consequent to activation of a number of different protein kinases. Three serine sites in the amino-terminal domain of the enzyme are substrates for these protein kinases. Serine 40 is phosphorylated by PKA and PKC and to a lesser degree, CamK; phosphorylation of this site leads to a decrease in the K_m for tetrahydrobiopterin. Serine 19 is phosphorylated by CamK; in the presence of the activator protein, 14:3:3, this phosphorylation results in an increased V_max. Serine 31 is phosphorylated by the extracellular regulated protein kinases, ERK1 and ERK2; these kinases are activated by a number of signals, including phorbol esters. Hence, agonist occupation of receptors linked to activation of either PKA, PKC, CamK, ERK1 or ERK2 can result in phosphorylation of distinct sites on TH, leading to enzyme activation and increased catecholamine biosynthesis.

In the adrenal medulla, TH is activated by stimuli that excite the sympathetic nervous system. In most cases, these effects on TH are dependent on intact innervation of the gland by the splanchnic nerve. Most evidence suggests that neurotransmitters released from the splanchnic nerve interact with adrenal chromaffin cell receptors, leading to activation of different protein kinases and consequent phosphorylation and activation of TH (see fig. 4 for diagram depicting this model). Agonists for numerous receptors stimulate the phosphorylation and/or activation of the enzyme in a number of cell culture or in situ model systems (for review, see Kumer and Vrana, 1996). These receptors include nicotinic and muscarinic acetylcholine receptors, VIP and PACAP receptors, adenosine A₂ receptors and bradykinin receptors. Even though agonist occupation of these receptors stimulates signaling pathways that regulate TH, it is not clear which of these receptors are effective in the rat adrenal medulla in vivo. Nor is it clear which, if any, of these receptors participate in the transsynaptic regulation of adrenal TH that occurs during stress or after nicotine administration.

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Schematic diagram of adrenal chromaffin cells innervated by the splanchnic nerve depicting a hypothetical model of some pertinent adrenal chromaffin cell receptors and protein kinases that regulate TH in rat adrenal medulla, along with splanchnic nerve neurotransmitters that may participate in transsynaptic regulation of TH. nAChR, nicotinic acetylcholine receptor; mAChR, muscarinic acetylcholine receptor; G, G protein; PLC, phospholipase C; AC, adenylyl cyclase; DAG, diacylglycerol; and IP3, inositol tris-phosphate.

Our previous results have provided evidence that agonist occupation of chromaffin cell nicotinic receptors leads to activation of TH in denervated adrenal glands (Fossom et al., 1991b). However, this nicotinic receptor-mediated pathway is not necessary for the transsynaptic activation of TH that occurs in innervated glands after nicotine administration. This conclusion is based on (1) the inability of hexamethonium to block the effect of nicotine and (2) the sustained activation of TH that occurs after repeated injections of nicotine in innervated but not denervated adrenal glands (Fossom et al., 1991b). It also appears that nicotinic receptors are not necessary for the transsynaptic stimulation of TH gene transcription rate elicited by nicotine because hexamethonium does not block this effect in innervated adrenal glands (Fossom et al., 1991a). These results lead to the hypothesis that chromaffin cell receptors other than the nicotinic acetylcholine receptor must participate in the transsynaptic regulation of TH activity. In the present report, we have tested whether muscarinic acetylcholine receptors are linked to adrenal TH activation and whether these receptors mediate the transsynaptic response elicited by nicotine.

Our results demonstrate that muscarinic agonists rapidly increase TH activity in rat adrenal gland and that this effect is blocked by the muscarinic receptor antagonist atropine but not by the nicotinic receptor antagonist hexamethonium. The muscarinic-mediated activation of TH is due to a decreased apparent K_m for pterin cofactor, similar to that observed when rat adrenal TH is activated by numerous other stimuli (Kumer and Vrana, 1996). These results agree with previous data demonstrating that muscarinic agonists activate TH in rat superior cervical ganglion (Ip et al., 1982). However, they differ with reports using isolated perfused rat adrenal gland and cultured bovine adrenal chromaffin cells (Pocotte et al., 1986; Haycock and Wakade, 1992). In these studies, muscarine phosphorylated TH on serine 31 but did not activate the enzyme. Our results using the rats with the denervated adrenal glands are consistent with these latter findings. Our studies show that the bethanechol-mediated activation of adrenal TH in vivo requires presynaptic innervation of the adrenal medulla. This result was unexpected and cannot yet be unequivocally explained by the available evidence. However, one interpretation that is consistent with the earlier work using in situ adrenal medulla model systems (Pocotte et al., 1986; Haycock and Wakade, 1992) is that agonist occupation of muscarinic receptors on adrenal chromaffin cells is sufficient to phosphorylate the enzyme on serine 31 but that a second signal derived from transsynaptic influences may be required to activate the enzyme. As pointed out by Haycock and Wakade (1992), phosphorylation of serine 40 is most strongly associated with enzyme activation in adrenal medulla. Hence, it is possible that activation of PKA and the consequent phosphorylation of serine 40 by a transsynaptic messenger along with activation of the muscarinic receptor are needed to observe activation of TH in rat adrenal by muscarinic agonists. Precisely how administration of muscarinic agonists leads to transsynaptic stimulation of the adrenal medulla remains unclear because bethanechol and carbachol are highly charged molecules and are not normally considered centrally active drugs. However, it is possible that these muscarinic agonists stimulate the splanchnic nerve via other undefined mechanisms. It should be noted that this interpretation is not consistent with results from studies using explanted rat superior cervical ganglion, in which TH is phosphorylated and activated in response to muscarinic agonists alone without the need for presynaptic input (Ip et al., 1982).

Another possible explanation for this lack of activation of TH by bethanechol in denervated adrenal glands is that signaling mechanisms required for TH activation are lost after denervation of the gland. This hypothesis seems less likely because bethanechol stimulates TH gene transcription rate in denervated adrenals (Tank et al., 1996), suggesting that muscarinic receptors and at least some signaling mechanisms linked to muscarinic receptor activation remain active in adrenal medulla from denervated glands. Furthermore, nicotine activates TH in denervated adrenals (Fossom et al., 1991b); hence, at least some mechanisms responsible for TH activation also remain functional in the denervated glands. Nevertheless, it is possible that signaling mechanisms specifically involved in TH activation in response to muscarinic agonist may be down-regulated after denervation.

Even though a single injection of bethanechol activates TH, this response is lost when the drug is administered repeatedly four times over a 3-hr period. This apparent desensitization requires at least 3 hr of exposure to bethanechol because no significant desensitization is observed after 1 or 2 hr of exposure to this drug (table 4). This result differs from the sustained increase in TH activity that occurs after repeated administration of nicotine for 3 hr (table 3 and Fossom et al., 1991b). Presumably, this loss in response to bethanechol is due to desensitization of muscarinic receptors or signaling pathways linked to this receptor; more work is required to differentiate between these possibilities. However, because of this loss of response, it is unlikely that muscarinic receptors play a significant role in the sustained transsynaptic activation of TH that occurs during repeated nicotine administration, when nicotinic receptors are desensitized. This conclusion is supported further by the cross-tolerance studies in table 3. Bethanechol still activates TH after nicotine is given repeatedly. If nicotine administration were stimulating muscarinic receptors by transsynaptic mechanisms (via the release of acetylcholine from preganglionic nerves) continuously for 3 hr, then the muscarinic response would be expected to desensitize at least partially. Because no desensitization is observed, it suggests that muscarinic receptors do not participate significantly in the sustained activation of TH during repeated nicotine administration. In addition, nicotine still activates TH when the muscarinic response is desensitized due to repeated injections of bethanechol, suggesting that muscarinic receptors are not necessary for the acute response to nicotine. Finally, the antagonist studies in table 5 also support the hypothesis that muscarinic receptors are not required for the transsynaptic response to nicotine. Even though atropine completely blocks the muscarinic response and hexamethonium completely blocks the nicotinic response (in denervated adrenal glands, see Fossom et al., 1991b), combined treatment with both atropine plus hexamethonium does not block the response to nicotine. This latter result agrees with findings by Wakade and coworkers (Malhotra and Wakade, 1987a; Malhotra and Wakade, 1987b) who measured the effects of cholinergic and noncholinergic agonists and antagonists on catecholamine release from rat adrenal medulla and indicates that noncholinergic receptors participate in the transsynaptic activation of adrenal TH.

In summary, our results support the hypothesis that bethanechol and carbachol activate adrenal TH by interacting with muscarinic acetylcholine receptors. This muscarinic receptor-mediated effect desensitizes after repeated injections of bethanechol and is dependent on presynaptic innervation of the adrenal gland. However, muscarinic receptors do not apparently participate in sustaining TH activation during repeated injections of nicotine, when nicotinic receptors are desensitized. Furthermore, neither nicotinic nor muscarinic cholinergic receptors are essential for the transsynaptic regulation of the enzyme by nicotine, implicating the involvement of noncholinergic chromaffin cell receptors in this transsynaptic regulation of TH activity. Presently, it is not clear to what extent cholinergic and noncholinergic adrenal chromaffin cell receptors participate in the regulation of TH activity under normal physiological conditions or when an animal is subjected to different types of acute stress. Even though we cannot state definitively that all stimuli that work via transsynaptic mechanisms activate TH by the same receptors and signaling pathways as nicotine administration, our results do suggest that multiple receptors, both cholinergic and noncholinergic, likely participate in the response to these stimuli.