Scandinavian Journal of Clinical and Laboratory Investigation

ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20

Adrenergic receptors are a fallible index of adrenergic denervation hypersensitivity A. Dejgaard, S. B. Liggett, N. J. Ch Ristensen, P.E. Cryer & J. Hilsted To cite this article: A. Dejgaard, S. B. Liggett, N. J. Ch Ristensen, P.E. Cryer & J. Hilsted (1991) Adrenergic receptors are a fallible index of adrenergic denervation hypersensitivity, Scandinavian Journal of Clinical and Laboratory Investigation, 51:8, 659-666, DOI: 10.3109/00365519109104578 To link to this article: http://dx.doi.org/10.3109/00365519109104578

Published online: 08 Jul 2009.

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Date: 29 March 2016, At: 00:02

Scand J Clin Lab Invest 1991; 51: 659-666

Adrenergic receptors are a fallible index of adrenergic denervation hypersensitivity

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A . D E J G A A R D , * S . B. L I G G E T T , ? N. J . CHRISTENSEN,$ P. E. C R Y E R f & J . HILSTED$( §Medical Department F, Glostrup Hospital; *Hvidare Hospital; $Department of Medicine and Endocrinology, Herlev Hospital; VDepartment of Internal Medicine and Endocrinology, Hvidovre Hospital, Copenhagen, Denmark; and ?Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, St. Louis, Missouri, USA

Dejgaard A, Liggett SB, Christensen NJ, Cryer PE, Hilsted J. Adrenergic receptors are a fallible index of adrenergic denervation hypersensitivity. Scand J Clin Lab Invest 1991: 51: 659-666. In view of evidence that neither interindividual nor induced intra-individual variations of adrenergic receptor status are related to metabolic or haemodynamic sensitivity to adrenaline in vivo, we took an alternative approach to assessment of the relevance of adrenergic receptor measurement by measuring these in a group of subjects with well-documented adrenergic denervation hypersensitivity, patients with diabetic autonomic neuropathy. Mononuclear leukocyte p2adrenergic receptor densities (and binding affinities), measured with '2sI-labelled pinodolol, and isoproterenol-stimulated cyclic AMP accumulation, in samples from patients with insulin-dependent diabetes mellitus (IDDM) with diabetic autonomic neuropathy (n=8), were no different from those in samples from patients with IDDM without neuropathy (n=8), or from non-diabetic subjects (n=8). In addition, platelet a,-adrenergic receptor densities (and binding affinities), measured with 3H-labelled yohimbine, and adrenaline-induced suppression of cyclic AMP contents did not differ among the three groups. Thus, in contrast to idiopathic autonomic failure, there is no generalized increase in adrenergic receptors in autonomic failure due to diabetic autonomic neuropathy. Regardless of the mechanism of adrenergic denervation hypersensitivity in such patients, these data provide further evidence that measurements of cellular adrenergic receptors (and adenylate cyclase) in vitro are a fallible index of sensitivity to catecholamines in vivo. Key words: catecholamines; diabetic autonomic neuropathy D r J . Hilsted, Department of Internal Medicine and Endocrinology, Hvidovre Hospital, KettegHrd Alle 30, D K-2650 Hvidovre, Denmark

Altered catecholamine-mediated or haemodynamic states can be the result of altered release of catecholamines, altered sensitivity to catecholamines, or both. Measurements of adrenergic receptors are ofteaused to attempt

to dissect the mechanisms of altered sensitivity. For example, decreased adrenergic receptor densities have been used to explain decreased sensitivity to catecholamines in patients with phaeochromocytomas [ 1-31, and increased 659

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adrenergic receptor densities have been used to explain adrenergic denervation hypersensitivity to catecholamines in patients with idiopathic autonomic failure [1,6-9). Measurements of adrenergic receptors on readily accessible circulating cells (mononuclear leukocytes and platelets) are frequently used to infer adrenergic receptor status on relatively inaccessible extravascular catecholamine target tissues. This practice is supported by evidence that adrenergic receptor density on circulating cells does reflect that on several target tissues, albeit in a subtype-specific fashion. For example, mononuclear leukocyte (MNL) p2adrenergic receptor density has been found to correlate with oz-adrenergic receptor densities on myocardial [lo], lung 1111, skeletal muscle [12], and myornetrial [13] membranes but not with PI (?B3)-adrenergic receptor density on adipocytes [ 121, al-adrenergic receptor density on lung membranes 1111, o r a2-adrenergic receptor density on platelets 1121. Furthermore, platelet a2-adrenergic receptor density has been found to be unrelated to lung a,-adrenergic receptor density [ l l ] or adipocyte o r skeletal muscle f3-adrenergic receptor densities [ 121. Despite the appeal of adrenergic receptor measurements, neither interindividual [ 121 nor induced intra-individual [ 141 variations in receptor densities, affinities or linked adenylate cyclase activities have been found to correlate with metabolic or haemodynamic sensitivity t o catecholamines in vivo. For example, in 22 normal humans Liggett et al., [I21 found no relationships between adrenergic receptor densities (on MNL, platelets, skeletal muscle membranes or adipocytes) and sensitivity to adrenaline in vivo. Furthermore, the same group [14] found no effect of triiodothyronine-

induced increments in P-adrenergic receptor densities on sensitivity t o adrenaline in vivo; this is documented on skeletal muscle membranes and adipocytes but probably occurs also on myocardium [lo], lung [ll] and MNL 112, 151. These findings suggest either that relatively small differences in tissue adrenergic receptor densities d o not alter cellular sensitivity to catecholamines, or that local or systemic regulatory compensation occurs [ 12, 141. Given this lack of relationship between adrenergic receptor status and sensitivity to catecholamines, we decided to take an alternative approach to assessment of the relevance of adrenergic receptor measurements by measuring these in a group of subjects with well-documented adrenergic denervation hypersensitivitypatients with diabetic autonomic neuropathy 1161. MATERlALS AND METHODS Study population Sixteen patients with insulin-dependent diabetes mellitus (IDDM) and eight healthy subjects volunteered for the study after giving informed consent. Eight of the patients with IDDM had autonomic neuropathy, as assessed by beat-to-beat variation in heart rate during hyperventilation [17] and by the plasma noradrenaline response to standing, and eight patients with IDDM had no signs of neuropathy (Table I). The investigation was approved by the local Ethical Committee. Four of the patients with neuropathy took diurectics for hypertension; these were discontinued 24 h prior to the smdy. None of the patients without neuropathy took any drugs apart from insulin, and the healthy subjects took no drugs at all.

T A H II.. ~ Clinical data (mean+sEM) on patient material studicd (insulin-dependent diabetes mellitus, IDDM)

Age (years)

Duration of diabetes (years)

(n=8)

48+5

IDDM N o neuropathy (n=8)

44+5

Rctinopathy (normal/ background/ HbAlC proliferative) (YO)

Vibration sense (V)

Beat-to heat variation (beats m i n - ' )

Serum creatinine (pmol I ')

25f5

4hk2

2f0.4

Y2f10

6/2/0

Y.Sf0.3

22f4

9f1

1324

0/2/6

7.8f0.3

IDDM Autonomic neuropathy

19+2

661

Adrenergic receptors in diabetic neuropathy

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Protocol The subjects met in the laboratory on two occasions with an interval of up t o 4 months. The experimental protocol was identical on these two occasions. After an overnight fast and tobacco abstention for at least 10 h, the subjects met in the laboratory without having taken their morning dose of insulin. A cannula was inserted into a cubital vein and the subjects subsequently rested supine on a couch. After 30 rnin of supine rest 200 ml of blood was drawn for blood cell membrane adrenergic receptor characterization on the first occasion, and an identical volume of blood was drawn on the second occasion for the determination of intact blood cell cyclic A M P accumulation. Subsequently, the subjects stood for 10 min. Blood samples (10 ml) were drawn for the determination of plasma catecholamines in the supine and in the standing position, and heart rate and blood pressure were registered.

methylxanthine for 15 min, then exposed to moI I-' isoproterenol for saline or lo-' to 10 min at room temperature. The reaction was terminated by boiling for 5 min. The aliquots were kept at -20°C until determination of cyclic AMP. Platelets were pelleted from plasma, washed three times with PBS, and either lysed and membranes prepared for ligand binding or used for intact cyclic A M P studies as described Plasma noradrenaline (ng mP)

0.

0.

Analytical procedures B2-Adrenergic receptor binding in MNL membranes was performed using '251-labelled pindolol as a P2-adrenergic receptor binding ligand, and a2-adrenergic receptor binding in platelet membranes was assessed using 'Hlabelled yohimbine, using methods previously described 111,121. Briefly, blood anticoagulated with sodium citrate was centrifuged at 400g for 15 min at room temperature. Plasma was removed to 1 cm of buffy coat and saved for the preparation of platelets. Twenty-five ml of phosphate buffered saline (PBS) was added to the remaining blood and then 10 ml Histopaque (Sigma 1077-1) was subfused with a spinal needle. After centrifugation at 400g for 30 min, the white blood cell layer was removed, and the cells were washed twice in PBS. MNL were then either lysed and membranes prepared for ligand binding or resuspended in PBS for whole-cell CAMP experiments as described previously [ 121.Receptor bindingwas performed using eight concentrations (5-300 pmol I-') of [ 12sI]-pindolol. Membranes were incubated in triplicate with ['2sI]-pindolol in the absence (total binding) or presence (non-specific binding) of 1 pmol I-' propranolol for 30 min at 37°C as described previously [12]. For cyclic AMP studies, 50 pl MNL suspension were preincubated with 0.1 mmol 1-' 3-isobutyl-l-

Plasma adrenaline (ng mV)

0.21 0.1

Systollc blood pressure (mmHg)

Heart rate (beats mind) T

901 80

T

70 Healthy Subjects

IDDM

no neuropathy

IDDM autonomic neuropathy

FIG 1. Plasma noradrenaline, plasma adrenaline, systolic blood pressure and heart rate in the supine position (clear bars) and in the standing position (shaded bars) in healthy subjects and in insulindependent diabetic patients with or without autonomic neuropathy. For details, see text.

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previously [ 11, 12, 181. a2-Adrenergic receptor binding was performed using eight concentrations of ['HI-yohimbine (0.5-25 nmol I-'). Membranes were incubated in triplicate with ['HI-yohimbine in the absence (total binding) or presence (non-specific binding) of 10 nmol I-' phentolamine. For cyclic AMP experiments, the effect of adrenaline o n inhibiting P G E , stimulation of CAMP in intact platelets was studied. Aliquots of 50 pl were preincubated at room temperature with 0.1 mmol I-' 3-isobutyl-I-methylxanthine for 15 min, then incubatedwith lOpmoll-' P G E l (Sigma-P5515), 1.0 pmol I-' propranolol chloride (to block any 0-adrenergic responses) and saline or adrenaline moI I-' for 10 min at room at IO-' to temperature. The incubation was terminated by boiling for 5 min, and the aliquots were kept at -20°C until measurement of CAMP. Adrenaline-induced inhibition of platelet cyclic AMP accumulation was also performed as described previously [12,14]. Plasma catecholamines were measured by an enzymatic single isotope derivative method [19]. Cyclic A M P was measured by radio-immunoassay [20].

A p-value (two-tailed) less than 0.05 was considered significant.

RESULTS Plasma noradrenaline, blood pressure and heart rate responses to standing Blood pressure decreased significantly in response to standing in the patients with autonomic neuropathy, whereas no significant changes were found in the patients without neuropathy or in the healthy subjects (Fig. 1). Heart rate increased significantly and similarly in the three groups in response to standing. Supine resting plasma noradrenaline was significantly lower in the patients with autonomic neuropathy compared with the other groups (p

Adrenergic receptors are a fallible index of adrenergic denervation hypersensitivity.

In view of evidence that neither interindividual nor induced intra-individual variations of adrenergic receptor status are related to metabolic or hae...
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