Development and properties of the secretory response in rat sweat glands: Relationship to the induction of cholinergic function in sweat gland innervation

DEVELOPMENTAL

BIOLOGY

123,I79-I90

(1987)

Development and Properties of the Secretory Response in Rat Sweat Glands: Relationship to the Induction of Cholinergic Function in Sweat Gland Innervation LESLIE Department

of Neurobiology,

M.

STEVENS

Harvard

Medical

Received

February

AND

C.

STORY

School, 25 Shattuck 23, 1987; accepted

April

LANDIS’ Street,

Boston,

Massachusetts

02115

21, 1987

Previous studies suggest that the sympathetic innervation of the sweat glands in the rat is initially noradrenergic and during development undergoes a transition in neurotransmitter phenotype to become cholinergic. To characterize this system and its development further, we have examined the adrenergic and cholinergic components of the secretory response in adult and immature rats and have studied the onset of sweating in the plantar sweat glands of developing rats. Stimulation of the sciatic nerve in adult rats elicited a secretory response which was completely blocked by the cholinergic antagonist, atropine, and was unaffected by adrenergic antagonists, indicating that nerve-evoked secretion was cholinergic. In adult rats, the sweat glands were quite sensitive to cholinergic agonists. In addition to acetylcholine, the mature sweat gland innervation contains vasoactive intestinal peptide (VIP). In some rats, the injection of VIP alone elicited a secretory response which was blocked by atropine, suggesting that the response to VIP was mediated cholinergically. In contrast to cholinergic agonists, the glands responded relatively infrequently and with reduced volumes of sweat to the LY- and fi-adrenergic agonists 6-fluoronorepinephrine and isoproterenol. However, when VIP, which is a potent vasodilator, was simultaneously injected with adrenergic agonists, glands in many of the injected footpads exhibited a secretory response. The response to adrenergic agonists in combination with VIP was reduced by atropine and by phentolamine plus propranolol, but was blocked completely only by a combination of the three antagonists, indicating that both adrenergic and cholinergic mechanisms were involved. In immature rats, sweating evoked by nerve stimulation first appeared at 14 days of age in 25% of the rats tested. Both the percentage of rats sweating and the number of active glands increased rapidly. At 16 days, 50% of the rats tested exhibited some active glands, and by 21 days all rats tested exhibited a secretory response. In 16-day-old rats, nerve-evoked sweating was almost completely inhibited by local injection of 1 pM atropine, but was unaffected by phentolamine and propranolol in concentrations up to 10 PM. Similarly, the glands were sensitive to 10 PM muscarine, but they exhibited no secretory response to the (Yadrenergic agonists, clonidine and 6-fluoronorepinephrine, nor to the P-adrenergic agonist, isoproterenol, at concentrations up to 50 PM. The simultaneous injection of VIP with adrenergic agonists did not reveal an adrenergically mediated secretory response in 16-day-old animals. Thus, in immature as well as adult rats, the neurally evoked secretory process was mediated through a musearinic cholinergic mechanism. The onset of nerveand agonist-induced sweating in developing rats lagged behind the development of other cholinergic properties in the sweat glands, raising the possibility that the cholinergic responsiveness of the gland cells is induced by the release of acetylcholine from the gland innervation. Consistent with this hypothesis is the observation reported here that in adult animals in which the glands were sympathetically denervated at birth, the glands did not respond to either sciatic nerve stimulation or cholinergic agonists. 0 1987 Academic

Press, Inc.

evoked by nerve stimulation (Langley, 1922; Patton, 1948; Foster and Weiner, 1970). In contrast, adrenergic antagonists are ineffective in blocking the response to nerve stimulation, except at high concentrations, which also block the effects of cholinergic agonists (Foster and Weiner, 1970). In addition, Dale and Feldberg (1934) showed that acetylcholine appears in the venous effluent from the footpad after nerve stimulation, providing direct evidence for the secretion of acetylcholine. In the rat, the sympathetic innervation of the sweat glands appears to be cholinergic as well. Leblanc and Landis (1986) have demonstrated that choline acetyltransferase, the synthetic enzyme for acetylcholine, is present in the sweat gland innervation. Cholinergic agonists elicit sweating (Hayashi and Nakagawa, 1963;

INTRODUCTION

Although the majority of sympathetic neurons in vivo are noradrenergic, a subpopulation is cholinergic. The most thoroughly characterized cholinergic sympathetic innervation is that supplied to the eccrine sweat glands in the footpad of the cat. The sympathetic origin of this innervation was first demonstrated by Langley (1891, 1894) and later confirmed by Patton (1948). The sweating response exhibits a cholinergic pharmacology: sweat secretion can be elicited by cholinergic agonists, and muscarinic cholinergic antagonists block the sweating i Present address: Department of Pharmacology, Case Western serve Medical School, 2119 Abington Road, Cleveland, OH 44106.

Re-

179

0012-1606187 Copyright All rights

$3.00

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

180

DEVELOPMENTAL BIOLOGY

Weschler and Fisher, 1968; Quatrale and Laden, 1968; Matsumoto and Abe, 1976; Sato and Sato, 1978), and the cholinergic antagonist atropine inhibits spontaneous sweating (Hayashi and Nakagawa, 1963). However, it has not yet been directly demonstrated that atropine blocks nerve-evoked sweating in the rat. In addition to acetylcholine, immunoreactivity for vasoactive intestinal polypeptide (VIP), a peptide which has been associated with cholinergic function and localized in the sympathetic innervation of cat sweat glands (Lundberg et aL, 1979), is also present in the fiber plexus of rat sweat glands (Yodlowski et aZ., 1984). The sympathetic innervation of rat sweat glands is of particular interest because of its unusual developmental history. The sweat gland innervation initially displays noradrenergic properties. At 7 days after birth, the axons present in the developing glands exhibit catecholamine histofluorescence and immunoreactivity for the catecholamine synthetic enzymes, tyrosine hydroxylase and dopamine P-hydroxylase, and their terminals contain numerous small granular vesicles after potassium permanganate fixation (Siegel et al., 1982; Landis and Keefe, 1983). Unlike the adult innervation, these fibers do not contain detectable choline acetyltransferase activity or VIP-like immunoreactivity, and acetylcholinesterase activity can be demonstrated only around a few glands (Siegel et al., 1982; Landis and Keefe, 1983; Leblanc and Landis, 1986). During the second and third postnatal weeks, however, properties associated with cholinergic function develop in the sweat gland innervation. Choline aeetyltransferase activity first appears at 11 days and increases fourfold between Days 14 and 21 (Leblanc and Landis, 1986). At 14 days, both acetylcholinesterase activity and VIP-like immunoreactivity can be detected (Siegel et al, 1982; Landis and Keefe, 1983). Noradrenergic properties are concomitantly diminished; by 21 days the fibers no longer contain endogenous catecholamines, and tyrosine hydroxylase immunoreactivity and dopamine P-hydroxylase immunoreactivity are markedly reduced (Siegel et al., 1982; Landis and Keefe, 1983). These results suggest that the neurons which innervate the sweat glands undergo a transition during the first 3 weeks after birth in which their noradrenergic properties are diminished or lost and cholinergic traits are acquired. To characterize this system and its development further, we studied the onset of transmission in the developing sweat glands and characterized the roles of acetylcholine and catecholamines in generating the secretory response in young and adult animals. In adults, nerve-evoked sweating occurred through a cholinergic mechanism, although some glands were also responsive to adrenergic agonists. Cholinergically mediated sweating was first detectable in 14-day-old rats and increased

VOLUME

123, 1987

markedly during the third postnatal week. In developing animals, even though both catecholamines and acetylcholine are present in the innervation during this period, we were unable to detect an adrenergically mediated secretory response. The results presented here raise the possibility that the development of the secretory response in the gland cells may be dependent upon the development of cholinergic function in the gland innervation. MATERIALS

AND

METHODS

Animals In most cases, pregnant female rats were obtained from Charles River (CD strain; Wilmington, MA). The day of birth was counted as 0, and rats were assayed for sweating on Days 12, 14, 16, 18, 21, and 25. For some experiments, litters were obtained from Charles River which were born and shipped the same day, and in this case the day of arrival was counted as day 0. For studies of adult rats, Charles River CD rats between 6 weeks and 4 months of age were used. Some rats were sympathectomized as neonates by repeated injections of 100 mg/kg 6-hydroxydopamine (Sigma, St. Louis, MO) on Days l-7 and on Day 12. This treatment has been shown to eliminate the cholinergic sympathetic innervation of the sweat glands (Yodlowski et aZ., 1984), as well as the adrenergic sympathetic innervation of other peripheral tissues (Clark et ah, 1972; Finch et ak, 1973). Sweating Assay Sweating was assayed by making a mold of the plantar surface with a silicone elastic material (Syringe Elasticon, Kerr Co., Romulus, MI), as described by Kennedy et al. (1984). Base material (0.1 ml) was mixed with l-2 drops of hardener in a plastic dish with a spatula and then applied to the plantar surface with the spatula. The impression material is immiscible with water; as it hardens the sweat droplets form pores in the mold. Each pore represents the activity of an individual sweat gland (Kennedy et ah, 1984). The pores can be easily counted using a dissecting microscope, allowing quantitation of the sweating response. Nerve Stimulation

and Drug Testing

Developing rats were anesthetized with 0.45 g/kg subcutaneous ehloral hydrate dissolved in 0.9% NaCl. The sciatic nerve was exposed and fitted with a Silastic cuff (0.25 in inner diameter, 0.47 in outer diameter) in which two platinum electrodes were embedded (Sauter et al., 1983). Adult animals were anesthetized with 0.6 g/kg subcutaneous chloral hydrate and a cuff size of 0.30 X 0.65-in. or 0.40 X 0.85 in. was used. After fitting the

STEVENS

AND

LANDIS

Devekpnent

cuff, the nerve and cuff were coated with mineral or paraffin oil. In most experiments, the sciatic nerve was stimulated at 15 V at 5.5 Hz. This amplitude and frequency seemed optimal for eliciting sweating in the developing animals; occasionally other amplitudes and frequencies were used. The nerve was stimulated with alternating current pulses to prevent polarization of the electrodes. The voltage peaked at 15 V and then decreased exponentially, dropping to 5 V within 10 msec. At the beginning of an experiment, the nerve was stimulated for a 3-min period to allow the ducts to fill. The paw was then wiped dry, the impression material was applied, and the nerve was stimulated for another 2.5 min. After the material hardened, approximately 2-3 min after the cessation of stimulation, the mold was removed by grasping an end with forceps and peeling it off the plantar surface. The footpads to be tested were then injected with the drug of interest, and another coat of impression material was applied either immediately (for agonists) or 5 min postinjection at the beginning of a second 2.5-min period of nerve stimulation (for antagonists). In adult rats, agonists were tested without a period of nerve stimulation. Footpads were injected with a saline solution containing the drug(s), and sweating was assayed 2-3 min after injection. Four normal animals and two experimental animals which had been treated with 6-hydroxydopamine were injected subcutaneously with 3 mg/kg pilocarpine and sweating was assayed 20-min postinjection (Kennedy and Sakuta, 1984).

of Sweating

in the Rat

181

footpad, so that injected drugs were probably diluted by no more than a factor of 2. In adults, the dilution factor may have varied from 2 to as much as 5, depending on the footpad injected. Since we can only estimate the amount of dilution, the sensitivity of the glands to cholinergic agents provided an internal control for dilution. To test for an adrenergic component in nerve-evoked sweating, we used maximal concentrations of the (Y-and @-adrenergic antagonists, phentolamine and proprano101, that do not produce nonspecific effects. Dun and Karczmar (1980) found that in the superior cervical ganglion, phentolamine at concentrations above 10 pM and propranlol at concentrations of 5 pM and higher decrease the size of the compound action potential, presumably acting like local anesthetics. In addition, cu-adrenergic antagonists can have antimuscarinic effects at high concentrations (Kayaalp and McIsaac, 1969; Beddoe, et aZ., 1971). We injected phentolamine and propranolol at concentrations of 10 PM. Assuming a dilution factor of 5 to 2, the concentration reaching the gland cells was 2-5 PM, which was low enough to avoid nonspecific effects, but high enough to inhibit the secretory response induced by 50 pM 6-fluoronorepinephrine plus isoproterenol in the presence of VIP. We used the LY-and fiadrenergic agonists 6-fluoronorepinephrine and isoproterenol at a concentration of 50 PM. Since we saw effects in adult rats at 50 pM, we used this concentration for immature rats as well. RESULTS

Sweating in Adult Rats

All pharmacological agents tested were dissolved in 0.9% NaCl. Except in the two cases described above, drugs were administered locally by injecting volumes of 2-10 ~1 into individual footpads using a lo-p1 Hamilton syringe. Atropine, isoproterenol, propranolol, methacholine, and muscarine were obtained from Sigma (St. Louis, MO). 6-Fluoronorepinephrine was obtained from Research Biochemicals, Inc. (Wayland, MA). Clonidine (Sigma) and phentolamine (Ciba-Geigy Corp., Summit, NJ) were kind gifts from Dr. Randall Pittman. VIP, obtained from Boehringer-Mannheim (Indianapolis, IN), was dissolved in 1 mM acetic acid or 1 mM HCl at a concentration of 300 pM and stored at -20°C. Immediately prior to use, it was thawed and diluted 1 to 100 in 0.9% NaCl. Control injections of 1 mM HCl diluted 1 to 100 in saline had no secretory effect. The concentrations of drugs used in these studies varied from 0.1 to 50 pM. There was undoubtably dilution of the drug after injection, resulting in a lower local concentration in the glands. In immature animals, the size of the injection volume was relatively high compared to the size of the

The secretory response in the adult rat was cholinergic. A typical adult sweating pattern resulting from sciatic nerve stimulation is shown in the mold pictured in Fig. 1. All 11 plantar footpads exhibited many active glands, 20-40 in most pads. The cholinergic antagonist atropine inhibited sweat secretion. At a concentration of 1 pM, atropine completely abolished the secretory response induced by nerve stimulation, as shown in Fig. 1. In contrast, nerve-evoked secretion was unaffected by the (Y- and /3-adrenergic antagonists, phentolamine and propranolol, at concentrations up to 10 pM. The ability of several agonists to induce sweating in adult rats was tested by footpad injection. The response to 20 pM muscarine is shown in Fig. 1; all or most of the glands in the footpad were activated. At a concentration of 5 PM, 70% of the glands responded to muscarine. Sweating was also induced by local injections of the cholinergic agonists methacholine and pilocarpine and by systemic injection of pilocarpine (not shown). Since VIP is present in the gland innervation as well as acetylcholine (Yodlowski et al, 1984), we also tested the effect of VIP on sweat secretion. VIP produced a secretory re-

182

DEVELOPMENTAL BIOLOGY

b

VOLUME 123.1987

STEVENS

EFFECTS

AND LANDIS

OF MUSCARINE,

Drugs Muscarine VIP VIP + atropine 6-Fluoronorepinephrine + isoproterenol 6-Fluoronorepinephrine + isoproterenol + VIP 6-Fluoronorepinephrine + isoproterenol + VIP + phenotolamine + propranolol 6-Fluoronorepinephrine + isoproterenol + VIP + atropine 6-Fluoronorepinephrine + isoproterenol + VIP + atropine + phentolamine + propranolol

VIP,

Development

TABLE AND ADRENERGIC

of

Sweating

1 AGONISTS

No. footpads (No. rats) tested

183

in the Rat

ON SWEATING

IN ADULT

Percentage with glands

of footpads responding

RATS Percentage of glands

4 (3) 9 (6) 6 (3) 8 (7)

100 56 17 38

93 7 1 13

28 (14)

82

25

17 (10)

30

2

9 (7)

56

12

13 (7)

0

0

Note. The concentrations used were as follows: 20 pM muscarine, 3 pM VIP, 50 pM 6-fluoronorepinephrine, 50 &i isoproterenol, 10 pM phentolamine, 10 pM propranolol and 1 pM atropine. The footpads were injected with the test agents and sweating was assayed 2-3 min after injection. The nerve was not stimulated prior to injecting the drug(s) except in two footpads in which 6-fluoronorepinephrine and isoproterenol were tested and one footpad in which musearine was tested. The results from these footpads were consistent with the data from unstimulated footpads. The injections were carried out on the digital footpads. To calculate the percentage of glands responding, the number of glands activated was divided by the total number tested. The total number of glands tested was estimated by multiplying the number of footpads injected by the average number of glands per digital footpad which were activated by sciatic nerve stimulation. This number, 25, was arrived at by counting the number of glands which responded to nerve stimulation in the digital footpads in four adult rats and calculating the average number of glands activated per footpad.

sponse in 7% of the glands tested, which were distributed in 56% of the footpads tested (Table 1). The response to VIP was almost completely blocked by simultaneously injecting 1 piI4 atropine: only one gland responded out of the 6 footpads tested. This result suggests that VIP caused sweat secretion through the release of acetylcholine from the nerve fibers innervating the sweat glands, When footpads were injected with a combination of the cy-and P-adrenergic agonists, 6-fluoronorepinephrine and isoproterenol, both at a concentration of 50 PA& only 13% of the glands tested responded, and only 38% of the footpads tested contained sensitive glands (Table 1). Since some a-adrenergic agents produced vasoconstriction, which might decrease sweat production, we also tested the effect of injecting 3 &f VIP, which is a potent vasodilator (Said and Mutt, 1970), simultaneously with 50 &l isoproterenol and 6-fluoronorepinephrine. In the presence of VIP, the percentage of footpads with glands responsive to adrenergic agonists was 82, more

than a twofold increase over that seen with isoproterenol and 6-fluoronorepinephrine alone, and the percentage of responsive glands increased to 25. Atropine decreased the response to isoproterenol and 6-fluoronorepinephrine plus VIP, but it did not eliminate it completely; in the presence of 1 p&f atropine, the percentage of responsive glands was reduced from 25 to 12. Adrenergic antagonists were more effective. When phentolamine and propranolol, at concentrations of 10 PM each, were simultaneously injected with VIP and adrenergic agonists, the percentage of responsive glands was reduced to 2. When all three antagonists were used, atropine (1 pilf), phentolamine (10 p&f), and propranolol(l0 p&f), the response to isoproterenol and 6-fluoronorepinephrine plus VIP was completely abolished. No glands responded out of 13 footpads tested. These results indicate that the secretory response to adrenergic agonists in the presence of VIP was mediated through both adrenergic and cholinergic mechanisms. In general, the secretory response

FIG. 1. Sweating response in an adult rat. (a) The sciatic nerve was stimulated for a 3-min period to fill the ducts of the glands, then the elastic material was applied, and the nerve was stimulated for an additional 2.5 min. The activity of each gland is represented by a pore in the hardened mold. (b) After assaying the efficacy of nerve stimulation in eliciting sweating, the footpad designated by the arrow was injected with 1 pM atropine. Five-minutes postinjection the nerve was stimulated for an additional 2.5-min period and sweating was assayed. The secretory activity of the glands in the injected footpad was completely blocked. (c) On the unstimulated side, the footpad designated by the arrow was injected with 20 &fmuscarine and sweating was assayed. Many of the glands in the injected footpad responded with sweat secretion. X5.6.

184

DEVELOPMENTAL BIOLOGY

produced by adrenergic agonists was reduced in volume and was shorter in duration compared to that seen with either nerve stimulation or cholinergic agonists. Although the glands do exhibit a response to adrenergic agents, our evidence that nerve-evoked sweating was completely blocked by atropine and unaffected by adrenergic antagonists indicates that neurotransmission in the sweat glands of the adult did not involve an adrenergic component. This is consistent with a previous study in which it was demonstrated that the innervating fibers in the adult gland do not contain detectable catecholamines (Landis and Keefe, 1983). Development

of Sweating

When developing rats were assayed for sweat secretion in response to sciatic nerve stimulation, no sweating was detectable in any of the 10 rats tested at 12 days of age (Fig. 2). Nerve-evoked sweating was first detected at 14 days. At this age, 25% of the 24 rats tested had sweat glands which could be activated by nerve stimulation. This percentage rose sharply with increasing age. At 16 days 50% of the rats tested exhibited active glands, and by 18 days 94% of the rats responded to nerve stimulation. At 21 days and older, 100% of rats tested exhibited some active glands. In contrast to adult rats, in which stimulation of the sciatic nerve activates over 300 glands distributed among the 11 plantar footpads, only a few active glands were detected during nerve stimulation in immature animals. Figure 3 shows a mold taken from a l&day-old rat in which only 4 glands exhibited a detectable secretory response, The number of responsive glands increased rapidly, however, and by 21 days, in the representative mold shown in Fig. 3B, 66 sweat glands were activated by nerve stimulation. Eflects of Adrenergic and Cholinergic Sweating in Developing Rats

Antagonists

on

We wanted to identify the neurotransmitter(s) responsible for the sweating response in developing rats. We chose 16 days as an age at which a reasonable number of animals exhibited a response to nerve stimulation, but also as an age at which the innervation of many glands contains and presumably releases both catecholamines and acetylcholine (Landis and Keefe, 1983; Leblanc and Landis, 1986). To test the ability of adrenergic and cholinergic antagonists to inhibit secretory activity, functional glands were first identified by their response to nerve stimulation. Footpads with active glands were injected with a saline solution containing the antagonist to be tested, and 5 min postinjection the nerve was again stimulated and the sweating response was assayed. When footpads containing active glands

VOLUME 123, 1987

I2

14

I6

16 Age

21

25

(days)

FIG. 2. Onset of nerve-evoked sweating in developing rats. Rats at 12,14,16,18,21, and 25 days of age were tested for the ability of sciatic nerve stimulation to elicit a secretory response in the plantar sweat glands. The percentage of rats with active glands is plotted for each age group. The numbers in parentheses indicate the number positive/ number tested.

were injected with saline solution alone as a control, the secretory responses of 10% of the glands in these footpads were inhibited. Since the volume of the injection relative to the footpad was significant in immature rats, this effect may have been due to dilution of the transmitter released from the nerve or to tissue damage. For these experiments, then, we considered a 10% block as a baseline, and only percentages of inhibition higher than this were considered specific. As shown in Table 2, a local injection of 1 wCLM atropine was very effective in inhibiting nerve-evoked secretion. In 14 rats tested at this concentration, 27 out of 28 glands (96%)were completely blocked. Even at a concentration of 0.1 PM, atropine was effective in blocking 83% of the 38 glands tested. In contrast, at a concentration of 10 Frn, the (Y- and fladrenergic antagonists phentolamine and propranolol had no effect on the secretory response (Table 2). Eflects of Adrenergic and Cholinergic Sweating in Develop& Rats

Agonists on

The results described above indicate that there was not an adrenergic component in the nerve-evoked sweating response in developing rats, although catecholamines are present in and likely to be released from the innervating fibers at 16 days (Landis and Keefe, 1983). We also tested directly whether developing glands could sweat in response to adrenergic agonists. In these experiments, glands which were competent to sweat were identified by their response to nerve stimulation. Footpads with active glands were then injected with a solution containing the test agonist and sweat secretion was assayed. As a control, footpads containing active glands were injected with saline solution alone. In no

STEVENS

AND LANDIS

Development

of Sweating

in

FIG. 3. Pattern of nerve-evoked sweating in developing rats. (a) Mold taken from a 16-day-old rat. by the arrows) were activated by sciatic nerve stimulation. X7.5. (b) Mold taken from a X-day-old some active glands after sciatic nerve stimulation. X7.0.

case did saline alone evoke a secretory response. As shown in Table 3, neither the a-adrenergic agonists, clonidine and 6-fluoronorepinephrine, nor the ,&adrenergic agonist, isoproterenol, was able to elicit detectable secretory responses at concentrations up to 50 FM. Since VIP enhanced the response of glands in adult animals to adrenergic agonists, in some 16-day-old rats we simultaneously injected 3 PM VIP with adrenergic agonists, testing footpads both with and without glands which responded to nerve stimulation. VIP did not unmask an adrenergically mediated secretory response. In contrast, glands which responded to nerve stimulation were sensitive to the cholinergic agonist, muscarine. Every active gland tested responded to 20 PM muscarine (Table 3). In fact, at 50 PM, even though only 15 glands responded to nerve stimulation, the injection of muscarine into the footpads containing those glands revealed the presence of an additional 6 activatable glands. In general, however, cholinergic agonists mim-

185

the Rat

Only four glands in two footpads rat. Almost all of the footpads

(indicated contained

icked the pattern produced by nerve stimulation; when footpads which lacked nerve-activated glands were injected with 20 or 50 PM muscarine, sensitive glands were not detected. This was also true for 12-day-old rats, in which no glands were detectably responsive to nerve stimulation. In addition, the simultaneous injection of 3 PM VIP with muscarine did not alter the sweating pattern seen with the cholinergic agonist alone in 16day-old animals. Sweating in Glands Sympathetically .from Birth

Denervated

In order to examine the possible influence of innervation on the development of the secretory response, we studied several adult rats whose sweat glands had been sympathetically denervated by neonatal treatment with the neurotoxic catecholamine congener 6-hydroxydopamine (Yodlowski et aL, 1984). These glands develop in

186

DEVELOPMENTAL BIOLOGY

VOLUME 123, 1987

TABLE 2 EFFECTS OF ADRENERGIC AND CHOLINERGICANTAGONISTS ON SWEATING IN 16-DAY-OLD RATS Antagonists Atropine 1.0 jlM 0.1 WM

Phenotolamine 10.0 pM Propranolol 10.0 pM Phentolamine + propranolol 10.0 pM Saline

No. glands responding to nerve stimulation

Percentage glands blocked

No. rats

28 38

96 83

14 17

5

10

3

5

0

3

10

10 10

45

6 20

Note. The sciatic nerve was stimulated for a period of 3 min to fill the ducts of the glands, and sweating was then assayed while the nerve was stimulated for an additional 2.5 min. Footpads in which glands had responded to nerve stimulation were injected with a saline solution containing the antagonist or with saline alone as a control. Five minutes after the injection sweating was again assayed during a 2.5-min period of nerve stimulation. The percentage of inhibition was calculated by dividing the number of glands whose secretory response was blocked by the antagonist by the number of glands which had responded to the initial nerve stimulation. If the activity of a gland was decreased but not eliminated, (as judged by comparing the size of the pores it formed in the molds taken before and after injection of the antagonist), it was counted as 0.5 instead of 1.

the absence of sympathetic innervation, but are innervated by sensory fibers. The sensory innervation expresses immunoreactivity for the neuropeptide substance P, but does not exhibit choline acetyltransferase or acetylcholinesterase activity (Yodlowski et al., 1984; Leblanc and Landis, 1986). In two of these rats, stimulation of the sciatic nerve did not activate any sweat glands. The glands in these rats also did not respond to the local injection of 20 &lf muscarine. In addition, in two rats which were injected systemically with the cholinergic agonist pilocarpine, none of the plantar sweat

glands responded. In a fifth rat in which sympathectomy was incomplete, nerve stimulation activated two glands on one of the plantar footpads. When this footpad was injected with 20 PM muscarine, six glands responded. The injection of other footpads on the same paw did not reveal any additional sensitive glands, however. DISCUSSION

In these studies, we established that neurotransmission in the adult rat was cholinergic. Glands in the adult

TABLE 3 EFFECTS OF ADRENERGIC AND CHOLINERGICAGONISTS ON SWEATING IN 16-DAY-OLD RATS No. glands responding to nerve stimulation

Agonist

No. glands responding to agonist

No. rats

Muscarine 15

50 pM 20 pM 10 /,LM

11

21 6 4

7 3 7

16

0

10

17

0

9

16

0

10

15

0 0

10

6

Clonidine 50 /.LM

6-Fluoronorepinephrine 50 /LM

Isoproterenol 50 rM

6-Fluoronorepinephrine 50 fiM

Saline

+ isoproterenol 14

9

Note. The sciatic nerve was stimulated for 3 min to fill the ducts of the glands, and then sweating was assayed during a subsequent 2.5-min period of nerve stimulation. Footpads in which glands had responded to nerve stimulation were injected with a saline solution containing the agonist or with saline alone as a control. Sweating was assayed immediately after injection.

STEVENS

AND

LANDIS

Development of Sweating

in the Rat

187

secreted in response to cholinergic agonists, and some echolamines (Landis and Keefe, 1983), we found that 13% of the glands tested were capable of sweating in glands also responded to VIP through a cholinergically mediated pathway. Although nerve-evoked sweating in response to the adrenergic agonists 6-fluoronorepineThe response to adrenergic adults was cholinergic, some glands also secreted in re- phrine and isoproterenol. increased by simultaneously sponse to adrenergic agonists. In developing animals, a agents was significantly nerve-evoked secretory response was first detectable at injecting VIP, an effect which appeared to be mediated from nerve terminals, 14 days. Secretion in immature rats was mediated chol- by the release of acetylcholine inergically, and there was no detectable response to ad- since the enhanced secretory response disappeared when renergic agents. Chemical sympathectomy by treatment atropine was included. However, when adrenergic aninjected with the adrenof neonatal rats with 6-hydroxydopamine prevented the tagonists were simultaneously ergic agonists and VIP, over 90% of the secretory redevelopment of responsiveness to cholinergic agonists sponse was blocked. This result would suggest that most in the gland cells. of the sweating seen with 6-fluoronorepinephrine and In adult rats, nerve-evoked sweating was blocked by isoproterenol plus VIP is mediated through adrenergic the cholinergic muscarinic antagonist atropine but was mechanisms and that the primary effect of VIP is to unaffected by adrenergic antagonists. These observaenhance the secretory response to adrenergic agents. tions indicate that nerve-evoked secretion was mediated cholinergically and provide evidence for the release of Studies on primate sweat glands suggest a hypothesis which would explain these apparently contradictory reacetylcholine from the sympathetic fibers innervating sults. Sato and Sato (1981) have shown that /3-adrenergic the sweat glands. The documentation of cholinergic agonists stimulate the production of CAMP in the sweat transmission in this system, in addition to previous glands and that dibutyryl CAMP can elicit a secretory studies which have described other properties associated with cholinergic function in the sweat gland innervation response from the glands in vitro, suggesting that the (Landis and Keefe, 1983; Leblanc and Landis, 1986), es- response induced by P-adrenergic agonists is mediated stimulation of CAMP tablishes that in the rat, as in the cat (Langley, 1922; by CAMP. Isoproterenol-induced is potentiated by the cholinergic agonist methacholine, Dale and Feldberg, 1934; Patton, 1948; Foster and Weialthough methacholine itself does not stimulate adener, 1970), the sweat gland innervation is functionally nylate cyclase in the sweat gland (Sato and Sato, 1983). cholinergic. Glands in the adult responded readily to muscarinic Thus, by analogy, the enhancement by VIP of the adcholinergic agonists. In addition, VIP elicited sweat se- renergic secretory response may have been brought cretion from a small proportion of glands. Previous about by VIP-released acetylcholine, which potentiated studies of the action of VIP in peripheral tissues suggest the CAMP production induced by isoproterenol. The abolition of the VIP effect by atropine is consistent with at least two mechanisms by which VIP may have evoked secretion. (1) VIP may have induced the release of ace- this hypothesis. In addition, if the primary role of VIP tylcholine, an effect which has been documented in the (via the subsequently released acetylcholine) was to enenteric nervous system (Cohen and Landry, 1980; Yau hance the adrenergic stimulation of adenylate cyclase, et ah, 1985; Kusonoki et ah, 1986). (2) VIP may have en- then it would be predicted that the response to adrenhanced the binding of acetylcholine to muscarinic re- ergic agents plus VIP would be largely blocked by adceptors, an effect seen in the cat submandibular gland renergic antagonists, which it was. (Lundberg et ab, 1982b), where VIP potentiates the saWhen we examined the development of cholinergic livatory response to acetylcholine (Lundberg et al, 1980, transmission in the glands of immature rats, we found 1981a,b, 1982a). Since virtually all of the sweating in- that sweat secretion in response to nerve stimulation duced by VIP in our experiments was blocked by the was first detectable in 14-day-old rats, but that only 6 simultaneous injection of 1 ~Matropine, it seems likely of the 24 rats we tested at this age exhibited active that the primary effect of VIP was to release acetylchoglands. Over the next week of development, the incidence line from the fibers innervating the sweat glands. Thus, of sweating increased rapidly, so that by Day 21 100% with normal synaptic transmission in the glands, VIP of the rats tested secreted in response to nerve stimuis most likely to act by increasing acetylcholine release, lation, although the full complement of adult glands was although it may also potentiate the cholinergic response not functional until approximately 6-8 weeks after birth. by enhancing the binding of acetylcholine to the musIn a previous study, Matsumoto and Abe (1976) found carinic receptor (Lundberg et al., 1982b), and/or may act that sweating induced by the cholinergic agonist methaas a vasodilator (Said and Mutt, 1970; Lundberg et al., choline is undetectable in the plantar sweat glands of 1980, 1981a,b, 1982a). 16-day-old rats, present in only 6% of 18-day-old rats, Although the innervation of the majority of sweat and increases gradually until 100% of the rats exhibit a glands in the adult rat does not contain detectable cat- secretory response by 28 days. The earlier onset of

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sweating seen in the present study most likely results tribute a major component to the secretory response in from a difference in the techniques used to elicit sweatdeveloping rats. ing. Matsumoto and Abe (1976) used local injections of It is of interest that the response to adrenergic agomethacholine, a cholinergic agonist that is less potent nists did not appear until approximately 6 weeks of age, for muscarinic receptors than muscarine (Bebbington at least 3 weeks past the age when 100% of rats reand Brimblecombe, 1965; Kosterlitz, 1967), and most sponded to nerve stimulation. The adrenergic secretory likely did not test all of the footpads on each paw. In response may require the maturation of additional seour study we stimulated the sciatic nerve, which supplies cretory elements which become functional only after the sympathetic innervation to all of the plantar footpads, cholinergic pathway has been established. Since the adand then assayed for sweating in all of the footpads. We renergic response was present only in adult animals, by found that in developing rats, active glands were present which time catecholamines have disappeared from the in only a few footpads, so it is quite likely that if Matgland innervation (Landis and Keefe, 1983), it is unlikely sumoto and Abe (1976) tested only a few of the plantar that there is ever an adrenergic component in the normal footpads of each rat, they would have missed the re- secretory response of the gland cells. It is possible, howsponsive glands. Consistent with this explanation, at 28 ever, that during the period in which they are present days, the age at which Matsumoto and Abe (1976) first in the gland innervation, catecholamines interact with report 100% responsiveness, we found that all of the the gland cells in ways which were undetectable in our footpads contained active glands (data not shown). assay. Because the innervation of the sweat glands passes The onset of sweating corresponded roughly to the through a period in which both catecholamines and ace- appearance of cholinergic function in the nerve fibers, tylcholine are present (Landis and Keefe, 1983; Leblanc but was somewhat delayed. We were unable to elicit and Landis, 1986), we were particularly interested in nerve-evoked sweating in 12-day-old rats even though determining whether, during this period, we could detect choline acetyltransferase activity is detectable as early an adrenergically mediated secretory response. Al- as Day 11 in the gland innervation (Leblanc and Landis, though at 16 days the innervation of the sweat glands 1986). It is possible that at Day 12 insufficient acetylpresumably releases catecholamines upon stimulation, choline was released to elicit a secretory response. Howwe were unable to detect an adrenergic component in ever, muscarine was also ineffective in evoking sweating the nerve-evoked secretory response. The adrenergic in 12-day-old rats. Similarly, at 14 and 16 days, only a antagonists phentolamine and propranolol did not insmall fraction of the adult number of approximately 300 hibit neurally evoked sweating in concentrations up to glands was activated by nerve stimulation, and the few 10 phf, whereas 1 pM atropine blocked 96% of the re- additional glands revealed by cholinergic agonists were sponse. This result indicates that, as in the adult, nervein footpads containing glands which responded to nerve evoked sweat secretion in immature animals is cholistimulation. These results suggest that the relatively nergically mediated. Although adult animals did exhibit low incidence of sweating seen at younger ages may be some response to adrenergic agonists, in developing rats due not to an absence of the cholinergic transmitter, but we were unable to detect a secretory response to adrenrather to the insensitivity of the glands to cholinergic agents. In fact, our evidence that sweating did not occur ergic agents using both (Y- and fl-adrenergic agonists alone and in combination. We maximized the sensitivity before Day 14, several days after choline acetyltransof our assay by testing glands which were known to be ferase activity is detectable (Leblanc and Landis, 1986), indicates that acetylcholine is present in the gland incompetent to sweat and by filling the ducts of active begins and glands by stimulating the nerve prior to injecting the nervation before cholinergic transmission adrenergic agonist. In some cases we also simultaneously raises the possibility that the presence of acetylcholine injected VIP, which potentiated the secretory response in the gland innervation may be necessary to induce the secretory response in gland cells. Several lines of evito adrenergic agents in adults. The injection of VIP with 6-fluoronorepinephrine and isoproterenol did not elicit dence support this hypothesis. First, we have reported a detectable secretory response, however. Since few here that in adult animals in which the sweat glands glands exhibited a secretory response at 16 days and the had never been sympathetically innervated, the glands amount of sweat secretion was much less than in the were unresponsive to cholinergic agonists. Since these adult, we cannot rule out the possibility that the glands glands do receive some innervation from noncholinergic had a response to adrenergic agents which was small in sensory fibers (Yodlowski, et ah, 1984; Leblanc and Lanmagnitude relative to the cholinergic response, as in the dis, 1986), this result suggests that it is the presence of adult, and was consequently not detected in our assay. acetylcholine and not innervation per se which is imHowever, we can conclude that although catecholamines portant for the secretory response. Secondly, in animals were present in the gland innervation, they did not con- in which the appearance of cholinergic function in the

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sweat gland innervation was delayed by a single neonatal treatment with 6-hydroxydopamine, there was a concomitant delay in the onset of sweating evoked by both nerve stimulation and cholinergic agonists (Stevens and Landis, unpublished observations). At 21 days when 100% of control rats exhibited a secretory response to nerve stimulation, the percentage of animals with delayed innervation which exhibited active glands was only 27%, and the number of active glands per animal was very small. In these rats the glands were sympathetically innervated, but at 21 days the innervation was still predominantly noradrenergic. At 6 weeks and later, when the innervation was more cholinergic, almost all of the rats exhibited some sweating. Sweat glands also require cholinergic innervation in order to maintain their sensitivity to cholinergic agonists. Sweat glands of the rat (Hayashi and Nakagawa, 1963), mouse, (Kennedy et al., 1984), and man (Janowitz and Grossman, 1950) which have been denervated are unresponsive to cholinergic agonists. When denervated glands in the mouse are reinnervated by cholinergic sympathetic fibers, they recover their sensitivity to cholinergic agonists (Kennedy et ab, 1984; Kennedy and Sakuta, 1984). These results, then, suggest that the presence of acetylcholine in the gland innervation may be required for both the induction and maintenance of the sweating response in the rat. It is not clear which step in the secretory process might be influenced by acetylcholine. The lack of responsiveness of immature and uninnervated glands to cholinergic agonists may result from the absence of muscarinic receptors or from failure of a subsequent step in the secretory process. Several lines of evidence suggest that the insensitivity of the glands does not result from a lack of muscarinic receptors. First, preliminary studies using N-methylscopolamine as a ligand have shown the presence of muscarinic binding sites in the sweat glands of adult rats sympathectomized at birth by treatment with 6-hydroxydopamine (Grant, M. and Landis, S., unpublished observations). Despite the apparent presence of muscarinic receptors, we found that these rats did not sweat in response to the injection of cholinergic agonists. Second, in two other systems, embryonic chick heart (Galper et al., 1977; Galper et ak, 1984; Halvorsen and Nathanson, 1984) and neonatal rat pancreas (Doyle and Jamieson, 1978; Werlin and Grand, 1979), the onset of muscarinic function does not correspond to an increase in receptor density, but rather seems likely to be related to the appearance of a postreceptor step in the physiological response. These results suggest that in the developing sweat glands, the cholinergic sympathetic innervation may promote the maturation and maintenance of some element(s) of the postreceptor secretory process, possibly by interactingwith muscarinic receptors which are already present on the gland cells. Thus, the devel-

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opment of cholinergic function in the sweat gland innervation may be an essential step in the development of the secretory response in the rat. We wish to thank Alan Williard, whose generous assistance was crucial to the initiation of these experiments. This research was supported by a grant from the Dysautonomia Foundation and LJSPHS Grants NS 23678, NS 07112, and MH18012. L.S. was the recipient of a predoctoral graduate fellowship from the National Science Foundation. S.C.L. was an Established Investigator of the American Heart Association, supported in part by funds contributed by the Massachusetts Affiliate. REFERENCES BEBBINGTON, A., and BRIMBLECOMBE, R. W. (1965). Muscarinic receptors in the peripheral and central nervous systems. Adv. Drug Res. 2, 143-172. BEDDOE, F., NICHOLLS, P. J., and SMITH, H. J. (1971). Inhibition of the muscarinic receptor by dibenamine. B&hem Phamnucol. 20,33673376. CLARK, D. W. J., LAVERTY, R., and PHELAN, E. L. (1972). Long-lasting peripheral and central effects of 6-hydroxydopamine in rats. Brit. J. Phamnacol. 44,233-243. COPEN, M. L., and LANDRY, A. S. (1980). Vasoactive intestinal polypeptide: Increased tone, enhancement of acetylcholine release, and stimulation of adenylate cyclase in intestinal smooth muscle. Life Sci. 26, 811-822. DALE, H. H., and FELDBERG, W. (1934). The chemical transmission of secretory impulses to the sweat glands of the cat. J. Physiol. 82,121128. DOYLE, C. M., and JAMIESON, J. D. (1978). Development of secretagogue response in rat pancreatic acinar cells. Dev. Biol. 65,11-27. DUN, N. J., and KARCZMAR, A. G. (1980). A comparative study of the pharmacological properties of the positive potential recorded from the superior cervical ganglia of several species. J. PharmacoL Exp. Z’her. 215,455-460. FINCH, L., HAEUSLER, G., and THOENEN, H. (1973). A comparison of the effects of chemical sympathectomy by 6-hydroxydopamine in newborn and adult rats. Brit. J. Pharmacol. 47,249-260. FOSTER, K. G., and WEINER, J. S. (1970). Effects of cholinergic and adrenergic blocking agents on the activity of the eccrine sweat glands. J. Physiol. 210, 883-895. GALPER, J. B., DZIEKAN, L. C. and SMITH, T. W. (1984). The development of physiologic responsiveness to muscarinic agonists in chick embryo heart cell cultures. J. Biol. Chem. 259, 7382-7390. GALPER, J. B., KLEIN, W., and CATTERALL, W. A. (1977). Muscarinic acetylcholine receptors in developing chick heart. J. Biol. Chem. 252, 8692-8699. HALVORSEN, S. W., and NATHANSON, N. M. (1984). Ontogenesis of physiological responsiveness and guanine nucleotide sensitivity of cardiac muscarinic receptors during chick embryonic development. Biochemistry 23,5813-5821. HAYASHI, H., and NAKAGAWA, T. (1963). Functional activity of the sweat glands of the albino rat. J. Invest. Dewnatol. 41,365-367. JANOWITZ, H. D., and GROSSMAN, M. I. (1950). The response of the sweat glands to some locally acting agents in human subjects. J. Invest. Dermatol. 14,453-458. KAYAALP, S. O., and MCISAAC, R. J. (1969). Effects of adrenergic receptor blocking agents on the ganglionic transmission. Eur. J. Pharmacol. 7.264-269. KENNEDY, W. R., and SAKUTA, M. (1984). Collateral reinnervation of sweat glands. Ann Neural. 15.73-78. KENNEDY, W. R., SAKUTA, M., and QUICK, D. C. (1984). Rodent eccrine

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