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Muscarinic receptors on human eccrine sweat gland in aging and Alzheimer's disease
Muscarinic Receptors on Human Eccrine Sweat Gland in Aging and Alzheimer' s Disease Helena I. Ryer, Susan E. Katz, and Michael Serby
Age and Alzheimer's disease-related changes have been reported in the peripheral cholinergic system controlling sweating. We present (1) evidence of a high affinity muscarinic receptor localized on human eccrine sweat gland and (2) muscarinic receptor concentrations on eccrine gland samples from 22 patients with probable Alzheimer's disease of various degrees of dementia, 8 age-matched controls, and 11 young controls. Muscarinic receptors were measured using autoradiography with 3H-NMS as ligand. We found no evidence of changes related to aging or Alzheimer's disease in the overall concentration of receptors or in the amount of gland showing binding. Nor was there any correlation between the degree of dementia as measured by the Global Deterioration Scale or the Mini-Mental State Exam and 3H-NMS binding. In conclusion, we find no evidence that previously reported sweat gland functional changes associated with aging and Alzheimer's disease are reflected in changes in eccrine gland muscarinic receptor density. Key Words: Alzheimer's, aging, sweat gland, peripheral nervous system, muscarinic receptors
Introduction Alzheimer's disease (AD) is a neurodegenerative disorder that is the major cause of dementia in the elderly. Research efforts to find a diagnostic test for the disease have not yielded a clinically accurate and specific marker but have resulted in growing evidence of disease-related changes in tissues other than the brain. Biochemical abnormalities have been reported in a variety of nonneural tissues including fibroblasts, lymphocytes, red blood cells and platelets (Scott 1993). Amyloid deposits have now been reported in
From the Department of Psychiatry, State University of New York Health Science Center and VA Medical Center, Syracuse, NY (HIR); the Department of Dermatology, Albert Einstein College of Medicine, New York, NY (SEK); and the Department of Psychiatry, Mount Sinai School of Medicine, New York, NY
(MS). Address reprint requests to Helena I. Ryer, Ph.D., Clinical Communications (145 Bldg/B-2), Clinical Research & Development, Wyeth-Ayerst Research, P.O. Box 8299, Philadelphia, PA 19101. Received June 21, 1993; revised April 15, 1994.
@ 1995 Society of Biological Psychiatry
tissues outside of the brain such as skin, muscle, and intestine (Joachim et al 1989). In seeking a diagnostic marker, several factors recommend the evaluation of the peripheral neural system controlling sweating. The cholinergic system has been repeatedly shown to undergo a severe degeneration in AD (Coyle et al 1983) that is strongly linked to the degree of dementia (Perry et al 1978). Unlike most of the peripheral tissues examined so far in AD, the human eccrine sweat gland is directly innervated by cholinergic fibers (Uno 1977) and numerous pharmacological studies have established the cholinergic nature of the sweat response in humans (Sato et al 1989). The eccrine sweat gland also displays a well-documented dependence on its innervation for its functional response. Denervation of sweat glands results in loss of sweat response to cholinergic stimulation. This has been shown in instances of complete local denervation caused by postganglionic sympathectomy (Randall and Kimura 1955) as well as in instances of peripheral diabetic neuropathy 0006-3223/95/$09.50 SSDI 0006-3223(94)00 116-K
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(Kennedy et al 1984b; Low et al 1983; Levy et al 1991). Thus, the eccrine sweat gland would be expected to show altered function if degenerative changes were occurring in its cholinergic innervation during the course of AD. Both spontaneous and cholinergically stimulated sweating decreases with age (Silver et al 1964; Lamb et al 1983; Kenney and Fowler 1988) and in AD (Lamb et al 1983; Weinreb et al 1986). This evidence, combined with the basic nature of sweat gland functional regulation, as outlined above, argue for further investigation of this system in these populations. One of the levels at which cholinergic changes in sweat gland function could be regulated is at the level of the eccrine gland cholinergic receptor. Muscarinic receptor binding studies in human eccrine sweat gland have been limited to our own initial demonstration of specific binding of 3H-N-Methyl-scopolamine (NMS) using receptor autoradiography (Ryer et al 1986). In the following studies we provide further evidence of the muscarinic nature of cholinergic receptor binding on the human eccrine sweat gland and evaluate whether there are age- or AD-related changes in receptor concentration.
Methods
Subjects Subjects recruited for this study consisted of 22 people who met NINCDS-ADRDA criteria (McKhann et al 1984) for a diagnosis of probable AD (mean age, 68 _+ 10 years), 8 age-matched controls (mean age, 63 ~ 6 years) and 11 young controls (mean age, 32 +_ 7 years). There was no significant difference in age between the AD and agematched control group (t = 1.57, p = 0.13). Degree of dementia was determined for all probable AD subjects on the Global Deterioration Scale (GDS, Reisberg et al 1982) and for a subset of patients (n = 8) by the Mini-Mental State Exam (MMSE, Folstein et al 1975). AD subjects ranged in severity of dementia from stage 3 (mild cognitive decline) to stage 7 (very severe cognitive decline). Normal controls were recruited through hospital staff and family members. Exclusion criteria included current medications of a cholinergic or adrenergic nature, insulin-dependent diabetes or the presence of peripheral neuropathy. All procedures were approved by the Institutional Review Boards at the respective institutions.
Tiss!2e Single point comparisons in the concentration of muscarinic receptor binding sites between AD, age-matched, and young control groups were done on 4 mm punch biopsies obtained from the palm of the hand and immediately frozen in liquid nitrogen. Because of the greater quantity of tissue
H.I. Ryer et al
needed for the saturation and competition studies for characterization of the receptor; these studies could not be run on the small amount of biopsied tissue obtained from control and AD subjects. Instead characterization studies were performed on autopsied blocks of skin approximately 1 cm across taken from the sole of the foot. No analysis of postmortem receptor degradation was done for eccrine gland. In brain, muscarinic receptors have been reported to be fairly stable under refrigerated conditions for up to 96 hr (Whitehouse et al 1984). All skin samples were approximately 0.5 cm in depth and included all dermal layers. Frozen cross sections (16 Ixm) of skin samples were cut on a cryostat at -20°C and thaw mounted onto gelatin-coated slides. Slidemounted tissue was stored at -70°C until assay.
ReceptorAssay Skin slices were incubated for 1 hr at room temperature in 50 mMol/L potassium phosphate buffer (pH 7.4) under the following conditions. (1) For saturation experiments slices were incubated with a range of [N-methyl-3H] scopolamine (85 Ci/mmole, New England Nuclear, Boston MA) concentrations (0.008 nMol/L - 0.6 nMol/L) in the absence or presence of 1 ~Mol/L atropine sulfate to define specific binding. (2) For competition experiments slices were incubated with 0.24 nMol/L 3H-NMS in the absence or presence of unlabeled muscarinic agonists and antagonists. Muscarinic competitors analyzed were carbachol and atropine obtained from Sigma (St. Louis, MO) and oxotremorine sesquifumarate and pirenzepine from Research Biochemicals Incorporated (Natick, MA). (3) For assays on individual subjects, slices were incubated with 0.75 nMol/L 3H-NMS in the absence or presence of 1 p~Mol/L atropine sulfate. Following incubation, in all experiments, tissue was rinsed for l0 min in ice-cold buffer, dipped in bidistilled water, and then quickly air-dried. Slides were placed in x-ray cassettes along with plastic tritium standards (Amersham, Arlington Heights, IL) and exposed to tritium sensitive Ultrofilm (LKB Instruments, Gaithersburg, MD) for 2-8 months. Film was developed in Kodak D-19 developer and tissue sections were stained with cresyl violet.
AutoradiogramAnalysis Autoradiograms were analyzed by computer using an MicroComputer Imaging Device (MCID) image analysis system (Imaging Research, Ontario, Canada). Autoradiograms were superimposed over stained tissue sections. Areas of eccrine sweat gland were outlined on the tissue section and densitometry measurements were made from corresponding areas on the autoradiogram. Nonspecific binding was subtracted from all measures. Optical density measurements were converted to fmoles/mg tissue values by comparison with tritium plastic standards. These values were
Sweat Gland Receptors in Alzheimer' s
not corrected for the absorption effects of gland tissue. Therefore these measures are semiquantitative and although they accurately express relative differences in receptor concentrations they do not reflect true fmoles/mg tissue values. Muscarinic receptor binding on eccrine sweat gland was far from homogeneous. Within individuals there was often considerable difference in the density of binding from gland to gland. For example, in young controls the average SD for samples from individual subjects was 12.5 fmoles/mg or 32%. We felt changes in receptor concentration could occur via a change (1) in the concentration of receptors on glandular tissue or (2) in the amount of gland that showed receptor binding. Thus it was possible that some glands might lose binding completely, whereas others maintained receptor levels or even increased binding as a compensatory reaction to lost innervation. In order to distinguish between these possibilities we examined receptor binding both from the perspective of (1) the overall average density of binding shown on gland (fmoles/mg) and (2) the total amount of binding divided by the amount of gland present in the sample (fmoles/mg/gland). Gland and binding areas were measured in screen pixels.
BIOLPSYCHIATRY 1995;37:259-264
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morine, and carbachol were less than one but the resolution of the assay was not sufficient to allow for the accurate determination of different agonist affinity constants. As shown in Figure 2, there was no statistical differences in the overall average concentration of muscarinic binding sites on eccrine sweat gland between probable AD and age-matched controls (t = 0.69, p = 0.50) or between agematched and young controls (t = -0.98, p = 0.34). In Figure 3 the total binding in a slice was divided by the area of gland present. This was to evaluate the amount of change in gland showing muscarinic receptor binding in any of the groups. There was no statistical difference in fmoles/mg/gland area
Statistical Analysis For saturation and competition studies the apparent IC5o, Ka, Ki and Bm~ were determined with computer assisted analysis (EBDA/LIGAND, Biosoft, Cambridge, UK). Differences in muscarinic receptor concentration between subject groups were analyzed using independent t-tests. The relationship between age and receptor binding was evaluated using the Pearson correlation coefficient. The relationship between degree of dementia and concentration of muscarinic receptors was assessed by Spearman-rank order correlation. Differences were considered significant ifp --< 0.05. All statistical analysis was performed using the SYSTAT statistical software package (SYSTAT Inc., Evanston, IL).
Results 3H-NMS binding was restricted to areas of gland and was not seen on duct (Figure 1). Saturation studies revealed 3H-NMS specific binding to sweat gland to be saturable and of high affinity with a Kd of 0.07 nMol/L and a Bm= = 60 fmoles/mg tissue. Competition studies with well-characterized muscarinic receptor antagonists and agonists revealed a pharmacological profile typical of muscarinic receptors. The muscarinic antagonists atropine and pirenzepine blocked 3H-NMS binding with high affinity. The muscarinic agonists oxotremorine and carbachol showed an intermediate and low affinity, respectively, for the receptor. The calculated K, values were atropine, 0.001 txMol/L; pirenzepine, 0.02 IxMol/L; oxotremorine, 0.40 IxMol/L; and carbachol, 144 txMol/L. Hill coefficients for pirenzepine, oxotre-
Figure 1. Top: Cresyl violet stained section through eccrine gland (x81). Note the area of darkly staining duct running through the center of glandular tissue. Bottom: Corresponding autoradiogram of 3H-NMS binding to eccrine gland. Note the absence of binding in the area of duct.
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Figure 2. A scatterplot of the average concentration of specifically bound 3H-NMS to eccrine sweat gland. An average of 10 + 6.5 discrete areas of gland were sampled from each subject. between probable AD and age-matched controls (t = 0.40, p = 0.70) or between age-matched and young controls (t = -0.42, p = 0.68). Combining the control groups revealed no significant correlation between receptor binding and age (fmoles/mg r = 0.14, p = 0.58; fmoles/mg/gland area r = 0.04, p = 0.87. There was no correlation between either measure of receptor binding and the degree of dementia as measured by the GDS (fmoles/mg rs = -0.29, fmoles/mg/gland area r s = -0.33) or the MMSE (fmoles/mg rs = 0.24, fmoles/ mg/gland area rs = 0.33).
(Torres et al 1991). Muscarinic receptors appear to be highly localized to eccrine glandular tissue and are not seen on duct. This corresponds with the histochemical work showing acetylcholinesterase staining around human eccrine gland but not duct (Hurley et al 1953). The relative binding affinity of muscarinic antagonists and agonists for the human eccrine gland receptor reported here are typical of data for muscarinic receptors in brain (Cort6s and Palacios 1986; Cort6s et al 1986) and peripheral tissues (Sullivan and Turner 1990; Hootman and Ernst 1981; Dehaye et al 1984) including rat eccrine sweat gland (Torres et al 1991; Grant et al 1991). Recent work indicates that the muscarinic receptor on rat eccrine sweat gland is of the glandular M3 subtype and is linked to phospholipase C and increased phosphoinositide turnover (Torres et al 1991; Grant et al 1991). Both age (Silver et al 1964; Lamb et al 1983; Kenney and Fowler 1988) and AD (Lamb et a11983; Weinreb et a11986) related changes in pharmacologically induced sweating have been reported and are further supported by our own preliminary experiments (unpublished observations). We have not found evidence of substantive receptor changes related to age, a diagnosis of AD, or the degree of dementia, however. Our experiments revealed no change in either the concentration of receptors localized to gland or in the amount of gland showing binding. Although the dependence of human eccrine sweat gland functioning on its innervation has long been documented, the mechanism by which this hyposensitivity is effected remains unknown. Rat eccrine sweat gland function shows a similar dependence on its nervous innervation (Hayashi and 200
This study is an extension of our previous work showing specific binding of 3H-NMS to human eccrine sweat gland (Ryer et al 1986). The present data confirms the muscarinic nature of the human eccrine gland cholinergic receptor. As determined by the nonselective muscarinic antagonist Nmethylscopolamine, the eccrine gland muscarinic binding site is of a high affinity that is consistent with affinity constants found in a variety of central and peripheral tissues (Cort6s and Palacios 1986; Cort6s et al 1986; Sullivan and Turner 1990; Waelbroeck et al 1986; Siegel and Fischbach 1984; Hootman and Ernst 1981). Recent investigations of muscarinic receptors on rat eccrine sweat gland have reported disassociation constants of 0.16 nMol/L using a filtration assay with 3H-NMS as ligand (Grant et al 1991), and 0.37 nMol/L using autoradiography with 3H-QNB as ligand
0
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Figure 3. A scatterplot of the total amount of specific 3H-NMS binding for each subject divided by the area of gland present.
Sweat Gland Receptors in Alzheimer' s
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1995;37:259-264
Nakagawa 1963; Kennedy et al 1984a). Recent work in the rat indicates that denervation effects on sweat function do not appear to be regulated at the level of the muscarinic receptor or even at the level of the subsequent secondmessenger response. Denervated sweat glands in the footpad of the rat become completely unresponsive to cholinergic stimulation yet have the same number of muscarinic receptors and a similar phosphoinositide response to cholinergic stimulation as innervated glands (Grant et al 1991 ; Grant and Landis 1991). The evidence we have presented here suggests that this may well be the case in human eccrine sweat gland and that functional changes seen in aging and A D are not reflected in changes in muscarinic receptor levels. Further studies are needed to evaluate other presynaptic and postsynaptic aspects of cholinergically stimulated sweating in aging and AD. This includes seeking evidence
of degeneration of the cholinergic innervation of these glands and changes in physiological events further down the response chain from the interaction of acetylcholine with the muscarinic receptor. In summary, we have presented evidence that the cholinergic regulation of the human eccrine sweat gland is mediated through a muscarinic receptor of high affinity localized to glandular tissue. These studies also provide data on the range of receptor concentrations seen in the normal population. Further, we find no evidence that previously reported sweat gland functional changes associated with both aging and AD are reflected in changes in eccrine gland muscarinic receptor density. This work was supported by NINDS grant #NS-25512.
References Cortrs R, Palacios JM (1986): Muscarinic cholinergic receptor subtypes in the rat brain I. Quantitative autoradiographic studies. Brain Res 362:227-238. Cort6s R, Probst A, Tobler HJ, Palacios JM (1986): Muscarinic cholinergic receptor subtypes in the human brain II. Quantitative autoradiographic studies. Brain Res 362:239-253. Coyle JT, Price DL, Delong MR (1983): Alzheimer's disease: A disorder of cortical cholinergic innervation. Science 219:11841190. Dehaye, JP, Winard J, Poloczek P, Christophe J ( 1984): Characterization of muscarinic cholinergic receptors on rat pancreatic acini by N-[3H]-methylscopolamine binding. J Biol Chem 259:294-300. Folstein MF, Folstein SE, McHugh RP (1975): "MINI-MENTAL STATE" a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 12:189-198. Grant MP, Landis SC (1991): Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation. JNeurosci 11(12):3772-3782. Grant MP, Landis SC, Siegel RE (1991 ): The molecular and pharmacological properties of muscarinic cholinergic receptors expressed by rat sweat glands are unaltered by denervation. J Neurosci 11 (12):3763-3771. Hayashi H, Nakagawa T (1963): Functional activity of the sweat glands of the albino rat. J Invest Dermatol 41:365-367. Hootman SR, Ernst SA (1981): Characterization of muscarinic acetylcholine receptors in the avian salt gland. J Cell Biol 91:781-789.
Kennedy WR, Sakuta M, Sutherland D, Goetz FC (1984b): Quantitation of the sweating deficiency in diabetes mellitus. Ann Neurol 15:482-488. Kenney WL, Fowler SR (1988): Methylcholine-activated eccrine sweat gland density and output as a function of age. J Appl Physio165( 3 ): 1082-1086. Lamb K, Bradshaw CM, Szabadi E (1983): The responsiveness of human eccrine sweat glands to choline and carbachol: application to the study of peripheral cholinergic functioning in Alzheimer-type dementia. Eur J Clin Pharmaco124:55--62. Levy DM, Rowley DA, Abraham RR (1991 ): Changes in cholinergic sweat gland activation in diabetic neuropathy identified by computerized sweatspot analysis. Diabetologia 34:807-812. Low PA, Caskey PE, Tuck RR, Fealey RD, Dyck PJ (1983): Quantitative sudomotor axon reflex test in normal and neuropathic subjects. Ann Neuro114 :573-580. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Standlan EM (1984): Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of Department of Health and Human services task force on Alzheimer's disease. Neurology 19:939-944. Perry EK, Tomlinson BE, Blessed G, Bergmann K, Gibson PH, Perry RH (1978): Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. BrMedJ2:1457-1459.
Hurley HJ, Shelley WB, Koelle GB (1953): The distribution of cholinesterases in human skin, with special reference to eccrine and apocrine sweat glands. J Invest Dermatol 21 : 139-147. Joachim CL, Mori H, Selkoe DJ (1989): Amyloid [3-protein deposition in tissues other than brain in Alzheimer' s disease. Nature 341:226-230.
Randall WC, Kimura KK (1955): The pharmacology of sweating. Pharmacol Rev 7:365-397. Reisberg B, Ferris SH, De Leon MJ, Crook T (1982): The global deterioration scale for assessment of primary degenerative dementia. Am J Psychiatry 139:1136-1139. Ryer HI, Nock B, Serby M, Katz S, McEwen BS (1986): In vitro autoradiography of muscarinic acetylcholine receptors in human skin. Eur J Pharmacol 120:379-380. Sato K, Kang WH, Saga K, Sato KT (1989): Biology of sweat glands and their disorders. I. Normal sweat gland function. J
Kennedy WR, Sakuta M, Quick DC (1984a): Rodent eccrine sweat glands: a case of multiple efferent innervation. Neuroscience 11:741-749.
Scott RB (1993): Extraneuronal manifestations of Alzheimer's disease. J A m Geriatr Soc 41:268-276.
Am Acad Dermato120:537-563.
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Siegel RE, Fischbach GD (1984): Muscarinic receptors and responses in intact embryonic chick atrial and ventricular heart cells. Dev Biol 101:346-356.
Uno H (1977): Sympathetic innervation of the sweat glands and piloerector muscle of macaques and human beings. J Invest Dermato169:112-130.
Silver AF, Montagna W, Karacan I (1964): The effect of age on human eccrine sweating. In Montagna W (ed). Advances in Biology of Skin, vol. VI. Aging. New York: Pergamon Press, pp 129-150.
Waelbroeck M, Gillard M, Robbercht P, Christophe J (1986): Kinetic studies of [3H]-N-methylscopolamine binding to muscarinic receptors in the rat central nervous system: Evidence for the existence of three classes of binding sites. Mol Pharmacol 30:305-314.
Sullivan DM, Turner JT (1990): Characterization of the muscarinic cholinergic receptor in the opossum (Didelphis Virginiana, Kerr) submandibular gland: Differences in receptor density and subtype compared with higher mammalian species. Comp Biochem Physio197C( 1):65-70. Torres NE, Zollman PJ, Low PA (1991): Characterization of muscarinic receptor subtype of rat eccrine sweat gland by autoradiography. Brain Res 550:129-132.
Weinreb HJ, Buskrik DR, Mirsen TR (1986): Microdermatoglyphic patterns in Alzheimer's disease. Neurology 36(4):226227. Whitehouse PJ, Lynch D, Kuhar MJ (1984): Effects of postmortem delay and temperature on neurotransmitter receptor binding in a rat model of the human autopsy process. JNeurochem 43:553-559.
Age and Alzheimer's disease-related changes have been reported in the peripheral cholinergic system controlling sweating. We present (1) evidence of a high affinity muscarinic receptor localized on human eccrine sweat gland and (2) muscarinic receptor concentrations on eccrine gland samples from 22 patients with probable Alzheimer's disease of various degrees of dementia, 8 age-matched controls, and 11 young controls. Muscarinic receptors were measured using autoradiography with 3H-NMS as ligand. We found no evidence of changes related to aging or Alzheimer's disease in the overall concentration of receptors or in the amount of gland showing binding. Nor was there any correlation between the degree of dementia as measured by the Global Deterioration Scale or the Mini-Mental State Exam and 3H-NMS binding. In conclusion, we find no evidence that previously reported sweat gland functional changes associated with aging and Alzheimer's disease are reflected in changes in eccrine gland muscarinic receptor density. Key Words: Alzheimer's, aging, sweat gland, peripheral nervous system, muscarinic receptors
Introduction Alzheimer's disease (AD) is a neurodegenerative disorder that is the major cause of dementia in the elderly. Research efforts to find a diagnostic test for the disease have not yielded a clinically accurate and specific marker but have resulted in growing evidence of disease-related changes in tissues other than the brain. Biochemical abnormalities have been reported in a variety of nonneural tissues including fibroblasts, lymphocytes, red blood cells and platelets (Scott 1993). Amyloid deposits have now been reported in
From the Department of Psychiatry, State University of New York Health Science Center and VA Medical Center, Syracuse, NY (HIR); the Department of Dermatology, Albert Einstein College of Medicine, New York, NY (SEK); and the Department of Psychiatry, Mount Sinai School of Medicine, New York, NY
(MS). Address reprint requests to Helena I. Ryer, Ph.D., Clinical Communications (145 Bldg/B-2), Clinical Research & Development, Wyeth-Ayerst Research, P.O. Box 8299, Philadelphia, PA 19101. Received June 21, 1993; revised April 15, 1994.
@ 1995 Society of Biological Psychiatry
tissues outside of the brain such as skin, muscle, and intestine (Joachim et al 1989). In seeking a diagnostic marker, several factors recommend the evaluation of the peripheral neural system controlling sweating. The cholinergic system has been repeatedly shown to undergo a severe degeneration in AD (Coyle et al 1983) that is strongly linked to the degree of dementia (Perry et al 1978). Unlike most of the peripheral tissues examined so far in AD, the human eccrine sweat gland is directly innervated by cholinergic fibers (Uno 1977) and numerous pharmacological studies have established the cholinergic nature of the sweat response in humans (Sato et al 1989). The eccrine sweat gland also displays a well-documented dependence on its innervation for its functional response. Denervation of sweat glands results in loss of sweat response to cholinergic stimulation. This has been shown in instances of complete local denervation caused by postganglionic sympathectomy (Randall and Kimura 1955) as well as in instances of peripheral diabetic neuropathy 0006-3223/95/$09.50 SSDI 0006-3223(94)00 116-K
260
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(Kennedy et al 1984b; Low et al 1983; Levy et al 1991). Thus, the eccrine sweat gland would be expected to show altered function if degenerative changes were occurring in its cholinergic innervation during the course of AD. Both spontaneous and cholinergically stimulated sweating decreases with age (Silver et al 1964; Lamb et al 1983; Kenney and Fowler 1988) and in AD (Lamb et al 1983; Weinreb et al 1986). This evidence, combined with the basic nature of sweat gland functional regulation, as outlined above, argue for further investigation of this system in these populations. One of the levels at which cholinergic changes in sweat gland function could be regulated is at the level of the eccrine gland cholinergic receptor. Muscarinic receptor binding studies in human eccrine sweat gland have been limited to our own initial demonstration of specific binding of 3H-N-Methyl-scopolamine (NMS) using receptor autoradiography (Ryer et al 1986). In the following studies we provide further evidence of the muscarinic nature of cholinergic receptor binding on the human eccrine sweat gland and evaluate whether there are age- or AD-related changes in receptor concentration.
Methods
Subjects Subjects recruited for this study consisted of 22 people who met NINCDS-ADRDA criteria (McKhann et al 1984) for a diagnosis of probable AD (mean age, 68 _+ 10 years), 8 age-matched controls (mean age, 63 ~ 6 years) and 11 young controls (mean age, 32 +_ 7 years). There was no significant difference in age between the AD and agematched control group (t = 1.57, p = 0.13). Degree of dementia was determined for all probable AD subjects on the Global Deterioration Scale (GDS, Reisberg et al 1982) and for a subset of patients (n = 8) by the Mini-Mental State Exam (MMSE, Folstein et al 1975). AD subjects ranged in severity of dementia from stage 3 (mild cognitive decline) to stage 7 (very severe cognitive decline). Normal controls were recruited through hospital staff and family members. Exclusion criteria included current medications of a cholinergic or adrenergic nature, insulin-dependent diabetes or the presence of peripheral neuropathy. All procedures were approved by the Institutional Review Boards at the respective institutions.
Tiss!2e Single point comparisons in the concentration of muscarinic receptor binding sites between AD, age-matched, and young control groups were done on 4 mm punch biopsies obtained from the palm of the hand and immediately frozen in liquid nitrogen. Because of the greater quantity of tissue
H.I. Ryer et al
needed for the saturation and competition studies for characterization of the receptor; these studies could not be run on the small amount of biopsied tissue obtained from control and AD subjects. Instead characterization studies were performed on autopsied blocks of skin approximately 1 cm across taken from the sole of the foot. No analysis of postmortem receptor degradation was done for eccrine gland. In brain, muscarinic receptors have been reported to be fairly stable under refrigerated conditions for up to 96 hr (Whitehouse et al 1984). All skin samples were approximately 0.5 cm in depth and included all dermal layers. Frozen cross sections (16 Ixm) of skin samples were cut on a cryostat at -20°C and thaw mounted onto gelatin-coated slides. Slidemounted tissue was stored at -70°C until assay.
ReceptorAssay Skin slices were incubated for 1 hr at room temperature in 50 mMol/L potassium phosphate buffer (pH 7.4) under the following conditions. (1) For saturation experiments slices were incubated with a range of [N-methyl-3H] scopolamine (85 Ci/mmole, New England Nuclear, Boston MA) concentrations (0.008 nMol/L - 0.6 nMol/L) in the absence or presence of 1 ~Mol/L atropine sulfate to define specific binding. (2) For competition experiments slices were incubated with 0.24 nMol/L 3H-NMS in the absence or presence of unlabeled muscarinic agonists and antagonists. Muscarinic competitors analyzed were carbachol and atropine obtained from Sigma (St. Louis, MO) and oxotremorine sesquifumarate and pirenzepine from Research Biochemicals Incorporated (Natick, MA). (3) For assays on individual subjects, slices were incubated with 0.75 nMol/L 3H-NMS in the absence or presence of 1 p~Mol/L atropine sulfate. Following incubation, in all experiments, tissue was rinsed for l0 min in ice-cold buffer, dipped in bidistilled water, and then quickly air-dried. Slides were placed in x-ray cassettes along with plastic tritium standards (Amersham, Arlington Heights, IL) and exposed to tritium sensitive Ultrofilm (LKB Instruments, Gaithersburg, MD) for 2-8 months. Film was developed in Kodak D-19 developer and tissue sections were stained with cresyl violet.
AutoradiogramAnalysis Autoradiograms were analyzed by computer using an MicroComputer Imaging Device (MCID) image analysis system (Imaging Research, Ontario, Canada). Autoradiograms were superimposed over stained tissue sections. Areas of eccrine sweat gland were outlined on the tissue section and densitometry measurements were made from corresponding areas on the autoradiogram. Nonspecific binding was subtracted from all measures. Optical density measurements were converted to fmoles/mg tissue values by comparison with tritium plastic standards. These values were
Sweat Gland Receptors in Alzheimer' s
not corrected for the absorption effects of gland tissue. Therefore these measures are semiquantitative and although they accurately express relative differences in receptor concentrations they do not reflect true fmoles/mg tissue values. Muscarinic receptor binding on eccrine sweat gland was far from homogeneous. Within individuals there was often considerable difference in the density of binding from gland to gland. For example, in young controls the average SD for samples from individual subjects was 12.5 fmoles/mg or 32%. We felt changes in receptor concentration could occur via a change (1) in the concentration of receptors on glandular tissue or (2) in the amount of gland that showed receptor binding. Thus it was possible that some glands might lose binding completely, whereas others maintained receptor levels or even increased binding as a compensatory reaction to lost innervation. In order to distinguish between these possibilities we examined receptor binding both from the perspective of (1) the overall average density of binding shown on gland (fmoles/mg) and (2) the total amount of binding divided by the amount of gland present in the sample (fmoles/mg/gland). Gland and binding areas were measured in screen pixels.
BIOLPSYCHIATRY 1995;37:259-264
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morine, and carbachol were less than one but the resolution of the assay was not sufficient to allow for the accurate determination of different agonist affinity constants. As shown in Figure 2, there was no statistical differences in the overall average concentration of muscarinic binding sites on eccrine sweat gland between probable AD and age-matched controls (t = 0.69, p = 0.50) or between agematched and young controls (t = -0.98, p = 0.34). In Figure 3 the total binding in a slice was divided by the area of gland present. This was to evaluate the amount of change in gland showing muscarinic receptor binding in any of the groups. There was no statistical difference in fmoles/mg/gland area
Statistical Analysis For saturation and competition studies the apparent IC5o, Ka, Ki and Bm~ were determined with computer assisted analysis (EBDA/LIGAND, Biosoft, Cambridge, UK). Differences in muscarinic receptor concentration between subject groups were analyzed using independent t-tests. The relationship between age and receptor binding was evaluated using the Pearson correlation coefficient. The relationship between degree of dementia and concentration of muscarinic receptors was assessed by Spearman-rank order correlation. Differences were considered significant ifp --< 0.05. All statistical analysis was performed using the SYSTAT statistical software package (SYSTAT Inc., Evanston, IL).
Results 3H-NMS binding was restricted to areas of gland and was not seen on duct (Figure 1). Saturation studies revealed 3H-NMS specific binding to sweat gland to be saturable and of high affinity with a Kd of 0.07 nMol/L and a Bm= = 60 fmoles/mg tissue. Competition studies with well-characterized muscarinic receptor antagonists and agonists revealed a pharmacological profile typical of muscarinic receptors. The muscarinic antagonists atropine and pirenzepine blocked 3H-NMS binding with high affinity. The muscarinic agonists oxotremorine and carbachol showed an intermediate and low affinity, respectively, for the receptor. The calculated K, values were atropine, 0.001 txMol/L; pirenzepine, 0.02 IxMol/L; oxotremorine, 0.40 IxMol/L; and carbachol, 144 txMol/L. Hill coefficients for pirenzepine, oxotre-
Figure 1. Top: Cresyl violet stained section through eccrine gland (x81). Note the area of darkly staining duct running through the center of glandular tissue. Bottom: Corresponding autoradiogram of 3H-NMS binding to eccrine gland. Note the absence of binding in the area of duct.
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Figure 2. A scatterplot of the average concentration of specifically bound 3H-NMS to eccrine sweat gland. An average of 10 + 6.5 discrete areas of gland were sampled from each subject. between probable AD and age-matched controls (t = 0.40, p = 0.70) or between age-matched and young controls (t = -0.42, p = 0.68). Combining the control groups revealed no significant correlation between receptor binding and age (fmoles/mg r = 0.14, p = 0.58; fmoles/mg/gland area r = 0.04, p = 0.87. There was no correlation between either measure of receptor binding and the degree of dementia as measured by the GDS (fmoles/mg rs = -0.29, fmoles/mg/gland area r s = -0.33) or the MMSE (fmoles/mg rs = 0.24, fmoles/ mg/gland area rs = 0.33).
(Torres et al 1991). Muscarinic receptors appear to be highly localized to eccrine glandular tissue and are not seen on duct. This corresponds with the histochemical work showing acetylcholinesterase staining around human eccrine gland but not duct (Hurley et al 1953). The relative binding affinity of muscarinic antagonists and agonists for the human eccrine gland receptor reported here are typical of data for muscarinic receptors in brain (Cort6s and Palacios 1986; Cort6s et al 1986) and peripheral tissues (Sullivan and Turner 1990; Hootman and Ernst 1981; Dehaye et al 1984) including rat eccrine sweat gland (Torres et al 1991; Grant et al 1991). Recent work indicates that the muscarinic receptor on rat eccrine sweat gland is of the glandular M3 subtype and is linked to phospholipase C and increased phosphoinositide turnover (Torres et al 1991; Grant et al 1991). Both age (Silver et al 1964; Lamb et al 1983; Kenney and Fowler 1988) and AD (Lamb et a11983; Weinreb et a11986) related changes in pharmacologically induced sweating have been reported and are further supported by our own preliminary experiments (unpublished observations). We have not found evidence of substantive receptor changes related to age, a diagnosis of AD, or the degree of dementia, however. Our experiments revealed no change in either the concentration of receptors localized to gland or in the amount of gland showing binding. Although the dependence of human eccrine sweat gland functioning on its innervation has long been documented, the mechanism by which this hyposensitivity is effected remains unknown. Rat eccrine sweat gland function shows a similar dependence on its nervous innervation (Hayashi and 200
This study is an extension of our previous work showing specific binding of 3H-NMS to human eccrine sweat gland (Ryer et al 1986). The present data confirms the muscarinic nature of the human eccrine gland cholinergic receptor. As determined by the nonselective muscarinic antagonist Nmethylscopolamine, the eccrine gland muscarinic binding site is of a high affinity that is consistent with affinity constants found in a variety of central and peripheral tissues (Cort6s and Palacios 1986; Cort6s et al 1986; Sullivan and Turner 1990; Waelbroeck et al 1986; Siegel and Fischbach 1984; Hootman and Ernst 1981). Recent investigations of muscarinic receptors on rat eccrine sweat gland have reported disassociation constants of 0.16 nMol/L using a filtration assay with 3H-NMS as ligand (Grant et al 1991), and 0.37 nMol/L using autoradiography with 3H-QNB as ligand
0
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Figure 3. A scatterplot of the total amount of specific 3H-NMS binding for each subject divided by the area of gland present.
Sweat Gland Receptors in Alzheimer' s
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Nakagawa 1963; Kennedy et al 1984a). Recent work in the rat indicates that denervation effects on sweat function do not appear to be regulated at the level of the muscarinic receptor or even at the level of the subsequent secondmessenger response. Denervated sweat glands in the footpad of the rat become completely unresponsive to cholinergic stimulation yet have the same number of muscarinic receptors and a similar phosphoinositide response to cholinergic stimulation as innervated glands (Grant et al 1991 ; Grant and Landis 1991). The evidence we have presented here suggests that this may well be the case in human eccrine sweat gland and that functional changes seen in aging and A D are not reflected in changes in muscarinic receptor levels. Further studies are needed to evaluate other presynaptic and postsynaptic aspects of cholinergically stimulated sweating in aging and AD. This includes seeking evidence
of degeneration of the cholinergic innervation of these glands and changes in physiological events further down the response chain from the interaction of acetylcholine with the muscarinic receptor. In summary, we have presented evidence that the cholinergic regulation of the human eccrine sweat gland is mediated through a muscarinic receptor of high affinity localized to glandular tissue. These studies also provide data on the range of receptor concentrations seen in the normal population. Further, we find no evidence that previously reported sweat gland functional changes associated with both aging and AD are reflected in changes in eccrine gland muscarinic receptor density. This work was supported by NINDS grant #NS-25512.
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Kennedy WR, Sakuta M, Sutherland D, Goetz FC (1984b): Quantitation of the sweating deficiency in diabetes mellitus. Ann Neurol 15:482-488. Kenney WL, Fowler SR (1988): Methylcholine-activated eccrine sweat gland density and output as a function of age. J Appl Physio165( 3 ): 1082-1086. Lamb K, Bradshaw CM, Szabadi E (1983): The responsiveness of human eccrine sweat glands to choline and carbachol: application to the study of peripheral cholinergic functioning in Alzheimer-type dementia. Eur J Clin Pharmaco124:55--62. Levy DM, Rowley DA, Abraham RR (1991 ): Changes in cholinergic sweat gland activation in diabetic neuropathy identified by computerized sweatspot analysis. Diabetologia 34:807-812. Low PA, Caskey PE, Tuck RR, Fealey RD, Dyck PJ (1983): Quantitative sudomotor axon reflex test in normal and neuropathic subjects. Ann Neuro114 :573-580. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Standlan EM (1984): Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of Department of Health and Human services task force on Alzheimer's disease. Neurology 19:939-944. Perry EK, Tomlinson BE, Blessed G, Bergmann K, Gibson PH, Perry RH (1978): Correlation of cholinergic abnormalities with senile plaques and mental test scores in senile dementia. BrMedJ2:1457-1459.
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