c 2-Adrenergic receptor levels in obstructive and spastic Raynaud's syndrome James M. Edwards, M.D. , Edward S. Phinney, M.D., Lloyd M. Taylor, Jr., M.D., Edward J. Keenan, Ph.D., and John M. Porter, M.D., Portland, Ore.

The present study examines the hypothesis that alterations in the activity of ~2-adrenergic receptors (A2R) may underlie the clinical vasospasm seen in patients with Raynaud's syndrome. Platelets were isolated from 13 normal subjects, from 50 patients with va- sospastic Raynaud's syndrome, and from 20 patients with obstructive Raynaud's syn- drome and A2R levels measured. Binding capacity as determined in femtomoles per mil- ligram of protein (fmol/mg of protein) and affinity were measured by Scatchard plot analysis. In a separate experiment normal human platelets were incubated with either buffer, normal serum, or serum from patients with spastic Raynaud's syndrome and A2R levels were measured. A2R levels in normal subjects averaged 112 --- 18 fmol/mg; in the patients with spastic Raynaud's syndrome, 191 -+ 14 fmol/mg, p < 0.01; and in the patients with obstructive Raynaud's syndrome, 164 - 31 fmol/mg, p > 0.05 (ns). Of the patients with spastic Raynaud's syndrome, 26% had values that were less than the mean value of the normal subjects (69 --- 7 fmol/mg, p < 0.05). The A2R levels decreased after incubation with serum from patients who had spastic Kaynaud's syndrome by 17.4 - 3.1 fmol/mg (p < 0.05). These results indicate that most patients with vasospastic Kaynaud's syndrome have increased platelet A=R levels, which may constitute a primary patho- physiologic abnormality underlying this condition. The presence of subnormal A2R levels in a portion of the patients and the finding of a decrease in measurable A~R levels after incubation in serum from patients with spastic Raynaud's syndrome suggests the possi- bility of receptor modulation as a mechanism for increased cellular receptor synthesis. (J VAsc SuRG 1987;5:38-45.)

The pathophysiologic mechanisms producing clinical vasospasm in patients with Raynaud's syn- drome remain undefined despite a half century of suggestions, ranging from altered autonomic nervous system function to an intrinsic vascular wall defect. Ideally the clinical treatment of any disorder is de- veloped on the basis of an accurate understanding of its pathophysiology. Until this is understood, treat- ment, and often the measure of success of treatment, can be only empiric. In recent years there has been a large increase in our knowledge of the cellular mechanism(s) of vasospasm. It is clear that contrac- tion in smooth muscle, as in striated muscle, is ini- tiated by a rise in Wtoplasmic calcium, with the cal-

From the Division of Vascular Surgery, Departments of Surgery and Pharmacology, School of Medicine, The Oregon Health Sciences University.

Presented at the Fortieth Annual Meeting of the Society for Vas- cular Surgery, New Orleans, La., June 9-10~ 1986.

Supported by grant RR-00334, Clinical Research Center Branch, Division Research Resources, National Institutes of Health.

Reprint requests: John M. Porter, M.D., Division of Vascular Surgery, Oregon Health Sciences University, 3181 Sam Jackson Park Rd., Portland, OR 97201.

38

cium source being either extra- or intracellular- stores. 1 The calcium messenger system that mediates smooth muscle contraction also has a central role in the secretion of exocrinc, endocrine, and neurocr~,~=- substances, as well as being intimately involved in the metabolic functions of the cell. 2

One mechanism by which calcium exerts its ef-" fects is by binding to calmodulin, which appears to be a nearly universal intracellular calcium receptor protein. Calcium binding to calmodulin has different ~ effects in different systems and can in certain settings stimulate adenylate cyclase (increases cyclic adenosine monophosphate [cAMP]) or phosphodiesterase (de-' creases cAMP), as well as activate other protein ki- nasesY However, to date, the control mechanism(s) regulating the calcium shifts that appear responsible" for inducing vasospasm arc almost totally unknown. Although it is clear that the autonomic nervous sys- tem has a role in the control ofvasospasm, the precise" function of the autonomic nervous system in the pathophysiology of Raynaud's syndrome remains un- known, despite years of speculation.

In the past decade a complex body of data h~s

Volume 5 Number 1 lanuary 1987 ~2-Adrenergic receptor levels in Raynaud's syndrome 39

Table I. Diseases associated with patients having Raynaud's syndrome

Type of No associated Connective Raynaud's syndrome disease tissue disease Atherosclerosis

Vibrating too~s

Vasospastic (n = 50) 35 15 0 0 Mild obstruction (n = 11) 0 8 1 2 Severe obstruction (n = 9) 0 8 1 0 All patients (n = 70) 3--5 3~ 2

confirmed the existence of two types of c~-adrenergic receptors, termed oq and %. Originally researchers thought that al-adrenergic receptors were postsyn- aptic and stimulatory and %-adrenergic receptors were presynaptic and inhibitory. 4 %-Receptors are now known to exist both pre- and postsynaptically ~ d may also function in different tissues in either an inhibitory or facilitative role. Considerable evi- dence indicates that the o~2-adrenergic receptors may be important mediators of sympathetic activity, s,6

Fortuitously a pure population of oL2-adrenergic receptors exists on human platclet membranes and is thus available for analysis. 6 Although the precise re- lationship between platclet adrenergic receptor ac- tivity and that of the arterial wall has not been es-

tablished, clear linear relationships have been estab- lished between blood cell and peripheral receptors in other biologic systems, including human ~-adren- crgic receptors.

A pilot study of platelet e~2-adrenergic receptors from our laboratory revealed an increase in the num-

'ber of receptors in patients with spastic Raynaud's syndrome compared with both normal subjects and patients with obstructive Raynaud's syndrome. 7 The T~esent study extends these observations to a larger patient population with fnrther characterization of obstructive Raynaud's syndrome and presents the

"data from the preliminary studies concerning the in- fluence of sera from patients with vasospastic Ray- naud's syndrome on normal platelet c~2-adrenergic receptor levels.

METHODS

Platelcts were isolated from the blood of 70 pa- tients with Raynaud's syndrome and 13 normal sub- jects. The patients were selected randomly from more ",~an 500 patients participating in our clinic. All pa- tients tested had a typical history of Raynaud's syn- drome and an abnormal digital pressurc response to • ischemic cooling, which we considcr the most reliable vascular laboratory diagnostic test for Raynaud's syn- drome.8,9 Each patient underwent digital photopleth- '/smography with measuremcnt of multiple digital blood pressures. Patients were classified as having

Table II. Types of connective tissue diseases seen in patients with Raynaud's syndrome

No. of patients

Undifferentiated connective 4 tissue disease

Mixed connective tissue disease 8 Scleroderma 7 Other (less than 3 patients per t2

disease)

vasospastic Raynaud's syndrome if the room tem- perature (23 ° C), plethysmography, and digital blood pressures were normal (n = 50 patients). Patients were classified as having mild obstructive Raynaud's syndrome if digital pressures were 25 to 50 mm Hg less than brachial pressure in multiple fingers (n = 11) and as having severe obstructive Raynand's syn- drome if digital prcssures were greater than 50 mm Hg less than brachial pressure or if pulsatile digital flow was absent (n = 9). All patients classified as hav- ing obstructive Raynaud's syndrome had abnormal digital photoplethysmographic wavcforms.

In the vasospasm group 15 patients had or were suspected of having a connective tissue disease. In the mild obstruction group eight patients had con- nective tissue disease, whereas one patient had digital athcrosclerosis, and two patients had digital artery obstruction associated with the long-term use of vi- brating tools. In the severe obstruction group cight patients had conncctive tissue disease and the re- maining patient had atherosclerotic obstruction of the digital and palmar arteries (Table I). The most frequently observed connective tissue diseases in- cluded mixed connective tissue disease, scleroderma, and undifferentiated connective tissue disease (Ta- ble II).

The control subjects were healthy laboratory em- ployccs, resident physicians, or nurses. None of these people had a history of Raynaud's syndrome, and all had normal results of vascular laboratory tests, in- cluding digital photoplethysmography and digital pressure response to ischemic cooling. None of the patients or normal subjects was taldng prescrip-

40 Edwards et al.

Journal o f VASCULAR

SURGERY

..j

o

o

o

,l=

\

250

200

150

100

50

~- om I Denotes error . p<.01 compared to normal T standard

* * * p<.05 compared to normal - - ~ N.S.

i I

I

n=l 3 n = 5( Normal Spastic

I

n=11] Mild

Obstruction

n=Ol ;evere

Obstruction

Fig. 1. c~2-Adrenergic receptor levels in patients with Raynaud's syndrome.

tion medications at the time blood samples were obtained.

Platelet %-adrenergic receptor levels were mea- sured in the following manner. Blood (30 to 40 ml) was obtained from a peripheral vein and mixed with 4 to 5 ml of 3.2% sodium citrate. The blood was centrifuged at 400 x g for 10 minutes at 20 ° C to obtain platelet-rich plasma. Platelets were separated from the plasma by centrifugation at 16,000 x g for 10 minutes at 4 ° C.

The platelcts were washed once with 20 ml of adrcnergic receptor assay buffer (75 rnmol/L Tris- HCI containing i mmol/L EDTA; pH, 7.5) and the platelets were lysed after being resuspended in ice- cold, double-distilled water for 10 minutes. Lysed cells were then homogenized with 10 strokes of a Dounce homogenizer and the platelct membranes were isolated by centrifugation at 39,000 x g for 10 minutes at 4 ° C. The membranes were resuspcnded in adrenergic receptor assay buffer and stored frozen at - 7 0 ° C if the remainder of the assay was not performed the same day.

The membrane fractions were thawed at room temperature before radioreceptor assay and the pro- tcin concentration of the membrane suspension ad- justed to I to 2 mg/ml by dilution with assay buffer. Protein determinations were conducted according to the method of Lowry ct al. 1° The assay of ~2-adren- ergic receptors in platelet membranes was conducted with methods described previously. H Approximately 200 ~g of membrane protein (0.1 ml) was added to tubes containing 5.0 to 25 nmol/L tritium-dihy- droergocryptine (3H-DHE, New England Nuclear,

specific activity 30.7 Ci/mmol). Binding specificity was established by parallel incubations of tubes con- taining unlabeled clonidine (i0 -s tool/L). Equilib- rium binding conditions were established by incu- bation for 30 minutes at 25 ° C. The binding reaction was terminated by dilution with 4 ml of washing buffer (75 mmol/L Tris-HC1, 12.5 mmol/L MgCI2, and 1.5 mmol/L EDTA) and rapid filtration across" glass fiber filters (Whatman GF/C, Whatman Lab- oratory Products, Inc.). The filters were rapidly washed twice with ice-cold washing buffer to remove "~ loosely bound 3H-DHE. Radioactivity associated with membranes retained by the filters was quanti- tared by scintillation counting with Ready-So.~ HP/b (Beckman) as a scintillation fluid.

The affinity and capacity of 3H-DHE binding were determined by Scatehard plot analysis u and to-" tal binding capacity was corrected for nonspccific binding. Specific binding of 3H-DHE to human platelet membranes ranged from 50% to 70% at I0' umol/L 3H-DHE, and the equilibrium dissociation constant (K~,) of aH-DHE binding was approxi- mately 5 nmol/L. Assays of c~2-adrcnergic receptors were controlled by inclusion of rat brain cortex mem- brane preparations with each assay. This tissue has been demonstrated previously to contain significant levels of o~2-adrenergic receptors) 3 The high affinity and specific aH-DHE binding were expressed as fem- tomoles per milligram of platelet membrane protein.

The serum incubation studies were done with normal human platelets obtained from the Red Cross (single donor). Each unit was divided into equal al iquots and centrifuged at 400 x g to remove red

Volume 5 Number i January 1987 a2-Adrenergic receptor levels in Raynaud's syndrome 41

¢1) , , , j

o

"o

2 5 0 - 1- )< .001

2 0 0 -

150 -

100 -

5 0 -

=3;

Spast ic Raynaud's with A - 2 levels

greater than mean of normals

T

n=l 3

Normal

p = . O S

Spast ic Raynaud's with A - 2 levels

less than mean of normals

Fig. 2. (x2-Adrenergic receptor levels in patients with spas- tic Raynaud's syndrome.

cells. The platelet-rich plasma was then centrifuged at 12,000 rpm for 10 minutes at zero to 4 ° C and the plasma removed by aspiration. The platelet pellet was washed once with 20 ml of incubation buffer.

The pellet containing intact platelets was then resuspended in either buffer (as control), normal se- rum (1 ml, single donor), or serum from patients with vasospastic Raynaud's syndrome (1 ml, single donor) and incubated for 90 minutes (at 4 ° C). In each experiment the same normal platelets were used for all incubations, and the normal and vasospastic sera used were from different subjects. Simultaneous d ,plicates were used for each incubation and the mean value of the duplicates calculated. After incu- bation the mixture was centrifuged at 12,000 rpm for 10 minutes at zero to 4 ° C and washed once with 10 ml of incubation buffer. The intact platelets were subsequently assayed for ~x2-adrenergic receptors with the procedures described previously for isolated platelet membranes.

The Mann-Whitney test was used for statistical evaluation of o~2-adrenergic receptor levels and a chi- square test of differences was used for evaluation of the incubation experiment.

R E S U L T S

Levels of a2-adrenergic receptors in the platelets from normal subjects averaged 112 _+ 18 fmol/mg of protein. There was a significant elevation in oL2- adrenergic receptors in all patients with Raynaud's syndrome (190 _+ 12 fmol/mg of protein, p < 0.01) as well as in the subgroups with spastic Raynaud's

1 0 0

0 ~ 7 5 0.

~" 50 o ~

"0 E ~ 2 5

o

GO

Z~

! o 0

0 0

0

O0

1 2 3 4

Experiment Number

Fig. 3. Effect of serum from normal subjects and serum from patients with spastic Raynaud's syndrome on normal platelets. Open triangles = incubation with buffer; closed circles = incubation with normal serum; open circles = incubation with serum from patients with spastic Ray- naud's syndrome.

syndrome (191 _+ 14 fmol/mg of protein, p < 0.01) and mild obstructive Raynaud's syndrome (208 _+ 35 fmol/mg of protein, p < 0.05) (Fig. 1). Thirteen patients (26%) in the vasospasm group had levels of oL2-adrenergic receptors (69 - 7 fmol/mg) that were lower than the mean value of normal subjects. This difference bordered on statistical significance (p = 0.08) (Fig. 2). In the group with obstructive Ray- naud's syndrome the value was 164 ___ 31 fmol/mg of protein; this did not differ significantly from the value of either normal subjects or from that of pa- tients with spastic Raynaud's syndrome (p > 0.2).

Incubation of normal platelets with serum from normal subjccts did not affect the level of 0~2-adren- ergic receptors compared with incubation with buffer alone (mean absolute difference, - 2 . 0 _ 1.4 fmol/ mg of protein In = 5]). Incubation of normal plate- lets with serum from patients with vasospastic Ray- naud's syndrome resulted in an absolute decrease in measured 0~2-adrenergic receptor values of - 17.4 ___ 3.1 fmol/mg of protein (range -0 .3 to -33 .5 [n = 11]) (Fig. 3), which was statistically different from that seen with normal subjects (p < 0.01). The mean difference in 9 of 11 patients tested was - 2 1 fmol/mg of protein whereas that in the other two patients was - 0.45 fmol/mg of protein.

D I S C U S S I O N

Abundant evidence indicates that peripheral va- sospastic attacks in patients with Raynaud's syn-

42 Edwards et al.

Journal of VASCULAR

SURGERY

drome are produced by digital artery closure with transient cessation of digital blood flow. These epi- sodes occur in two distinct groups of patients: one has unobstructed digital arteries with normal or near normal blood flow between attacks; the second group has significant fixed digital artery obstruction with decreased digital blood flow between attacks. 14 These two groups have been described as having va- sospastic and obstructive Raynaud's syndrome, re- spectively. To explain the mechanism for attacks in the vasospasm group it is necessary to hypothesize an abnormal arterial response to cold, mediated ei- ther locally is or by the central nervous system (CNS), probably through autonomic outflow? 6 It has been suggested that attacks in the obstruction group re- quire only a normal vasoconstrictive response to cold to overcome the decreased intraluminal pressure and produce arterial closure. 17 This theory is unproved and patients with obstruction are probably a diverse group, some or all of whom have both obstruction and an intrinsically abnormal arterial response to cold.

The purpose of the present study was to inves- tigate the hypothesis that increased o~2-adrenergic re- ceptor levels may be involved in the pathogenesis of digital vasospastic attacks in patients with vasospastic Raynaud's syndrome as well as possibly in some pa- tients with obstructive Raynaud's syndrome. It is well known that CNS stimulation, such as ice on the fore- head, emotional stress, immersing the opposite hand in cold water, and performance of mental arithmetic can trigger a Raynaud's attack. Further evidence that the CNS may be involved results from the observa- tion that there is almost always at least temporary improvement in Raynaud's syndrome after regional sympathcctomy. The recurrence of Raynaud's syn- drome after sympathectomy may represent a dener- vation hypersensitivity reaction. Impressive addi- tional evidence of CNS participation in the Raynaud response has been obtained from the observations of Lafferty et al?8 that hand blood flow in patients with Raynaud's syndrome can be thermally entrained by temperature stimuli applied to the opposite hand, and that the entrainment pattern is distinctly different from that seen in normal subjects.

Recent evidence has suggested that there may be abnormalities in the sympathetic neuromuscular end plate 19,2° or alterations in adrenergic receptor func- tion in patients with Raynaud's syndrome. ~6m Ja- micson, Ludbrook, and Wilson 19 have suggested the possibility of abnormal adrenergic receptors that be- come hypersensitive when exposed to cold. Intrinsic abnormalities of arterial wall elasticity or contractility have also been proposed as contributory causes, and

both of these may be under partial ncurogenic con- trol. 22 As shown by the present study an increased arterial wall level of ~2-adrcnergic receptors in pa- tients with Raynaud's syndrome may bc the common erector pathway underlying file participation of the CNS in the production of vasospastic attacks.

Circulating lymphocytes possess a pure mem- brane [~-adrenergic receptor population. A linear re- lationship has been shown between myocardial and lymphocyte [3-adrenergic receptor density. 2a This link has been used to study the effect of therapeutic or experimental intervention on a remote site by mea- surement of accessible blood elements. 24 Although this relationship has not yet been shown in oti-ad- renergic receptors, it appears reasonable to assume that patients with spastic Raynaud's syndrome ha: ':~ increased arterial wall levels of ot2-adrenergic recep- tors paralleling the observed increase in platelet re- ceptor levels.

Measurements of c~2-adrenergic receptors have been made previously in human platelets mad normal values have been established. 25 The physiologic role of the o~2-adrenergic receptor on the platelet mem- brane is unknown, although some interesting obser- vations have been described. Stimulation of ot2-ad- renergic receptors in the platelet inhibits adenylate cyclasc, which results in a decrease in intracellular cAMP. Any decrease in intraplatelet cAMP greatly enhances platelet adhesion and aggregation, proba- bly by mechanisms that increase the platelet cyto- plasmic calcium concentration. In addition, there may be stimulation of the Na+/K + ATPase pump, which results in an increase in calcium efflux from the cell. 26 Interestingly, in patients with phcochr~ mocytoma, platelet ag-adrenergic receptor levels are decreased by 50% before operation and then return to normal levels after surgery, 2r'2a suggesting that cat- echolamines may have a role in the regulation of a2- adrenergic receptor activity.

The role of the o~2-adrencrgic receptor in the cir- culatory system is slowly being elucidated. In the coronary circulation of dogs o~2-adrenergic receptors provide control of vasoconstriction in the small re- sistive vessels, an effect which can be blocked by cal- cium channel antagonists, z9'3° The mechanism by which calcium channel blockers antagonize the va- soconstriction is not known, but this observation may explain the usefulness, in at least some patients, of the calcium channel blockers in the treatment of Raynaud's syndrome. In addition, (x2-adrenergic re- ceptors have been observed to participate in the con- trol of peripheral resistance in human beings? 1'32 This appears to occur through o~2-adrenergic receptor7 mediated stimulation of arterial smooth muscle.33 (~',

Volume 5 Number 1 lanuan' 1987 a2-Adrenergic receptor levels in Raynaud's syndrome 43

Adrenergic receptors are also found in the CNS. In rats injection o f oL2 agonists into the hypothalamus results in feeding in satiated animals, a response blocked by the local injection o f ~2 antagonists. 34

Although (x2-adrenergic receptor stimulation can induce contraction of arterial smooth muscle, the ac- tual mechanism of the contraction is not yet known, but it may involve decreases in intracellular cAMP. Calcium-mediated smooth muscle contraction in- duced by a2-adrenergic receptor stimulation has not been shown, but it is clear that smooth muscle con- traction may be initiated by a rise in cytoplasmic calcium. An increase in cytoplasmic cAMP stimulates calcium uptake into intracellular stores in platelets, an action which prevents platelet activation. 3"~ If c-adrenergic receptor stimulation inhibits adenylate cyclase in arterial smooth muscle cells as it does in the platelet, the resultant decrease in intracellular cAMP would increase available cytoplasmic calcium with subsequent smooth muscle contraction in a manner analogous to receptor-induced platelet acti- vation. I f platelet activation proceeds to aggregation, several vasoactive substances will be released from platelet granules. Thus a2-adrenergic receptor stim- Nation may produce arterial spasm both directly as well as through platelet stimulation.

Abundant precedence exists in biologic systems for humoral control o f cellular membrane receptor activity. The results of the present study indicate that patients with Raynaud's syndrome have an increased number of a2-adrenergic receptors on the platelet membrane and probably also the arterial wall. Whether this represents a real increase in receptor ~' ~es or a decrease in receptor blocking remains to be determined. _tim intriguing possibility is that there is a substance variably present in the sera o f some pa- tients with vasospastic Raynand's syndrome that binds to receptors and decreases the assayed levels o f oL2-adrenergic receptors. Chronic blockage may result in a feedback stimulus for the cellular synthesis o f a greatly increased number o f receptors.

As humoral blocking substances are rarely if ever present in constant concentrations, increased ~2-ad- renergic receptor effects would occur during phases of decreased serum-blocking substance concentra- tion. The interesting subgroup of patients who have vasospastic Raynaud's syndrome with e~2-adrenergic receptor levels distinctly below normal may be those with the blocking substance present. The widespread, but not universal, presence of an apparent blocking substance in Raynaud's sera as shown by the incu- bation studies supports this view.

The high incidence o f autoimmune connective ~ssue diseases occurring in patients with Raynaud's

syndrome suggests that the control factor(s) in the incubation experiments described in the present study (if this is what we are seeing) may be an an- tibody or antibody/antigen complex. I f correct, this hypothesis may lead to a unified theory o f the patho- genesis o f vasospastic Raynaud's syndrome.

We gratefially acknoMedge the excellent assistance of Ms. Debra Corbin, R.T., who performed the adrenergic receptor assays.

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structure of smooth muscle. Circ Res 1985;57:498-507. 2. Rasmussen H. The calcium messenger system (Part 1). N

Engl l Med 1986;314:1094-1101. 3. Rasmussen H. The calcium messenger system (Part 2). N

Engl J Med 1986;314:1164-70. 4. Hoffinan BB, Lefkowitz RJ. Alpha-adrenergic receptor sub-

types. N Engl J Med 1980;302:1390-6. 5. Langer SZ. Presynaptic regulation of catecholamine release.

Biochem Pharmacol 1974;23:1793-1800. 6, Motulsk3, HI, Insel PA. Adrenergic receptors in man. N Engl

J Med I982;307:18-29. 7. Keenan EJ, Porter JM. c~2-Adrenergic receptors in platelets

from patients with Raynaud's syndrome. Surgery 1983;94: 204-9.

8. Nielsen SL, Lassen NA. Measurements of digital blood pres- sure after local cooling. J Appl Physiol 1977;43:907-10.

9. Holmgren-Gates K, Tyburczy J& Zupan T, Baur GM, Porter JM. The noninvasive quantification of digital vasospasm. Bruit 1984;8:34-7.

10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with Folin phenol reagent. I Biol Chem I95 I; 193:265-75.

11. Newman KD, William LT, Bishopric NH, Letkowitz RJ. Identification of alpha-adrenergic receptors in hmnan platelets by 3H-dihydroergocryptine binding. J Clin Invest 1978;61: 395-402.

12. Scatchard G. The attraction of proteins for small molecules and ions. Ann b,rg Acad Sci 1949;51:660-72.

13. Greenberg DA, U'Prichard DC, Snyder SH. Alpha-nora& renergic receptor binding in mammalian brain: Differential labeling of agonist and antagonist states. Lifh Sci 1976;19: 69-76.

I4. Porter JM, Edwards JM. Raynaud's syndrome. Drug Therapy 1985 ;May: 72-90.

15. Lewis T. Experiments relating to the peripheral mechanism involved in spastic arrest of the circulation in the fingers, a variety of Raynaud's disease. Heart 1929; 15: 7-101.

16~ Coffman JD, Cohen AS. Total and capillary fingertip blood flow in Raynaud's phenomenon. N Engl J Med 1971;285: 259-63.

17. Hiral M. Cold sensitivity of the hand in arterial occlusive disease. Surgery, 1979;85:140-6.

18. Lafferty K, De Trafford JC~ Roberts VC, Cotton LT. Ray- naud's phenomenon and thermal entrainment: An objective test. Br Med J 1983;286:90-2.

19. Jamieson GG, Ludbrook J, Wilson A. Cold hypersensitivity in Raynaud's phenomenon. Circulation 1971;44:254-64.

20. Krakenbuhl B, Nielsen SL, Lassen NA. Closure of digital arteries in high vascular tone states as demonstrated by mea-

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surement of systolic blood pressure in the fingers. Scand J Clin Lab Invest 1977;37:71-6.

2I. Rosch J, Porter JM, Gralino BJ. Cryodynamic hand angi- ography in the diagnosis and management of Raynaud's syn- drome. Circulation 1977;55:80%14.

22. Sumner DS, Strandness DE Jr. An abnormal finger pulse associated with cold sensitivity. Ann Surg 1972; 175:294-8.

23. Brodde OE, Kretsh R, Ikezono K, Zerkowski HR, Reide- meister JC. Human beta-adrenoreceptors: Relation of myo- cardial and lymphocyte beta-adrenoreceptor density. Science 1986;231:1584-5.

24. Mann JJ, Brown RP, Halper Jp, Sweeney JA, Kocsis JH, Stokes PE, Blezikian JP. Reduced sensitivity of lymphocyte beta-adrenergic receptors in patients with endogenous de- pression and psychomotor agitation. N Engt J Med 1985; 313:715-20.

25. Brodde OE, AnlaufM, Graben N, Bock KD. Age-dependent decrease of alpha-2 adrenergic receptor number in human platelets. Eur J Pharmacol 1982;81:345-47.

26. Goldman CK, Marino L, Leibowitz SF. Postsynaptic alpha- 2-noradrenergic receptors mediate feeding induced by para- ventricular nucleus injection ofnorepinephrine and clonidine. Eur J Pharmacol 1985;115:I1-9.

27. Pfeifer MA, Malpass T, Stratton J, et al. Variations in cate- cholamines fail to alter human platelet alpha-2-adrenergic re- ceptor number or affinity for [3H]yohimbine or [3H]dihydro- ergocryptine. J Clin Invest 1984;74:1063-72.

28. Karliner IS, Motulsky HJ, Insel PA. Apparent "down-regu- lation" of human platelet alpha-2 adrenergic receptors is due to retained agonist. Mol Phannacol 1982;2i:36-43.

29. Heusch G, Deussen A, Schipke J, Thamer V. Alpha-1 and alpha-2-adrenoreceptor-mediated vasoconstriction of large and small canine coronary arteries in vivo. J Cardiovasc Phar- macol 1984;6:961-8.

30. Kakjhana M, Noda H, Ohtsuka S, Sugishita Y, Ito I. Sig- nificant role of alpha-2 adrenoceptors in coronary artery spasm. Jpn Circ J 1985;49:108-18.

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DISCUSSION

Dr. Larry H. Hollier (Rochester, Minn.). I have en- joyed reading this manuscript, but I think this study pro- vides more questions than answers.

The principal finding of this study is that most patients with Raynaud's syndrome caused by vasospasm have an increased number of a2-adrenergic binding sitcs on the platelet membrane. The investigators propose that the in- creased number of binding sites on platelets may reflect a generalized increase in the number of cl2-adrenergic recep- tors, including the population of receptors in vascular smooth muscle. The "local fault" in Raynaud's syndrome is therefore due to an increase in local adrenergic respon- siveness.

The theory is an interesting one and receives indirect support from recent studies at our institution. Both cq- and a:-adrenergie receptors are present on the postjunc- tional membrane of human digital arteries, and when stim- ulated, induce vasoconstriction, as demonstrated by Dr. John Cooke at Mayo Clinic. The dose response character- istics of these adrenergic receptors are similar to those in the canine saphenous vcin. In the canine tissue we have shown that the usual vasodilation with warming or vaso- constriction with cooling can be blocked or even reversed by c~2-adrenergic antagonists.

Changes in temperature appear to affect the affinity of the a2-adrenergic receptors to a greater degree than the cq-adrenergic receptor. I f the postjunctional c~2-adren-

ergic receptors in human digital arteries respond similarly to temperature changes, one would expect that an increased number of these receptors would induce greater thermal sensitivity.

Although the hypothesis forwarded by Dr. Porter and his colleagues is an intriguing one and deserves further investigation, I am concerned that their evaluation of the data is somewhat flawed. The patients with vasospasm a~ pear to fall into a bimodal distribution, with one group having significantly elevated c~2 binding levels and the other having significantly depressed a2 levels. There are no cri- teria placing the patient in one of these two groups before the data are collected. Therefore, the statistical analysis is invalid. However, the presence of this bimodal distribution remains an interesting observation.

Is it possible that in the group with lower a2-adrenergic levels, affinity of the c~2-adrenergic receptors is altered to a greater degree by temperature changes?

It would be interesting to observe what changes in affinity might occur, for instance, as measured by radioli- gand binding techniques, with changes in temperature.

Dr. David S. Sumner (Springfield, Ill.). The origin of Raynand's syndrome has intrigued clinical investigators for more than a century. In the past ideas about the origin have centered about two primary areas--either abnormal autonomic nerve function or intrinsic arterial wail defect.

The authors have presented data that may lead to a unifying theory of Raynaud's syndrome that may adc~ ~

Volume 5 Number 1 Januat T 1987 a2-Adrenergic receptor levels in Raynaud's syndrome 45

quately account for more of the previous physiologic ob- servations, that is, that these patients may have a serum substance blocking the et2-adrenergic receptors, leading to feedback cellular synthesis of additional receptors. I f the blocking substance proves to be an antibody or antigen- antibody complex, this could explain the high association between Raynaud's syndrome and autoimmune diseases.

How certain are you that the blocking substance is not simply norepinephrine? How do you plan to characterize the blocking substance? How do you explain the elevation of the ~2 receptors in the mild obstructive group? Do you have any data concerning the receptor levels on artery or vein tissue and how does this relate to that found on the platelet membrane?

Finally, we have observed in our laboratory that pa- tients with Raynaud's syndrome respond to sympathetic stimuli with arteriolar constriction; whereas direct cold e,,posure causes a more proximal digital artery constriction. I wonder whether this is compatible with your theories.

Dr. Edwards (closing). Dr. Hollier, despite the bi-

modal distribution, we could show that there is a significant increase in c~2-adrenergic receptor levels for the whole pa- tient population. The fact that a bimodal distribution exists intrigues us and we are conjecturing that there may be more of this blocking substance in the serum in some of these patients than in others; this would cause lower levels of u2-adrenergic receptors to be measured. We have not done any temperature-dependent binding or affinity stud- ies; that awaits further investigation.

Dr. Sumner, we do not think that this is just a cate- cholamine-dependent response. If we do these studies in membrane fragments, we can see no effect of incubation with sera. We can only see this effect in whole membranes, and we think that this may be due to either internalizaion of the receptor or stereospecific binding. We believe that patients who have had mild obstruction also have vaso- spasm and we think that they also will have elevated ~2 receptor levels. We have not yet measured tissue levels of o~2-adrenergic receptors.