Resistance to multiple hormones in patients with pseudohypoparathyroidism: Association with deficient activity of guanine nucleotide regulatory protein

Resistance to Multiple Hormones in Patients with

Pseudohypoparathyroidism

Association with Deficient Activity of

Guanine Nucleotide Regulatory Protein

MICHAEL A. LEVINE, M.D.

ROBERT W. DOWNS, Jr., M.D.

Bethesda, Maryfand

ARNOLD M. MOSES, M.D.

Syracuse, New York

NEIL A. BRESLAU, M.D.

Dallas, Texas

STEPHEN J. MARX, M.D.

ROZ D. LASKER, M.D.

RENE E. RIZZOLI, M.D.

GERALD D. AURBACH, M.D.

ALLEN M. SPIEGEL, M.D.

Bethesda, Maryland

From the Metabolic Diseases Branch, National institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National institutes of Health, Bethesda, Maryland; the State University Hospital, Syracuse, New York (AMM); and the Department of Internal Medicine, University of Texas Health Science Center, Dallas, Texas (NAB). This work was supported in part by Dallas General Clinical Research Centers grant no. MOl-RR-00633, by National Institutes of Health grant no. lROlAM26253-01 (NAB), and by RR-229 General Clinical Research Centers Program, Division of Research Resources, National Institutes of Health (AMM). This work was presented in part at the annual meeting of the American Federation for Clinical Research, 1961, San Francisco, California [l] and at the Seventh International Conference on Calcium Regulating Hormones, 1981, Key- stone, Colorado [2]. Requests for reprints should be addressed to Dr. Allen M. Spiegel, National Institutes of Health, Building 10, Room 9C101, Bethesda, Maryland 20205. Manuscript accepted July 15, 1982.

Pseudohypoparathyroidism type I is characterized by resistance (defined as a deficient urinary CAMP response) to parathyroid hormone and, in most cases, hypocaicemia and hyperphosphatemia. Many patients with pseudohypoparathyroidisrn type I show a peculiar somatic phenotype termed Aibright’s hereditary osteodystrophy, but patients without this feature who show identical parathyroid hormone resistance have been described. Parathyroid hormone resistance in pseudohypoparathyroidism type I has been attributed to a defective parathyroid hormone receptor-adenyiate cyciase complex. Recent studies have demonstrated deficient activity of the guanine nucieotide regulatory protein (G unit) of adenyiate cyciase in many patients with pseudohypoparathyroidism. Since the G unit is common to ail tissues, as opposed to hormone receptors, which are tissue specific, a defective G unit shouid lead to resistance to multiple hormones that act by stimulating adenyiate cyciase. To test this hypothesis, we studied erythrocyte G unit activity and clinical endocrine function in 29 patients with pseudohypopara- thyroid&m type I. Thirteen patients had deficient erythrocyte G unit activity (43 f 9 percent of control [mean f 1 SD]); 16 patients had normal G unit activity (92 f 6 percent of control) (p <O.OOl). Pa- tients with deficient erythrocyte G unit activity had significantly greater (p <O.OOl) basal and thyrotropin-releasing hormone- stimulated thyrotropin levels than patients with normal erythrocyte G unit activity or normal control subjects (15.0 f 6.5 and 54.3 f 22.7; 4.5 f 2.0 and 19.5 f 6.6; 2.0 f 1.1 and 16.5 f 6.7 @J/ml, respectively). in the absence of goiter or antithyroid antibody, an elevated thyrotropin level in patients with deficient erythrocyte G unit activity is consistent with thyroid resistance to thyrotropin. Furthermore, patients with deficient erythrocyte G unit activity had significantly lower (p <0.02) integrated plasma CAMP increases to giucagon stimulation than either patients with normal erythrocyte G unit activity or normal subjects (5.1 f 2.2 versus 6.6 f 3.9 versus 6.6 f 3.6 PM X minutes), consistent with impaired hepatic cyciase responsiveness to giucagon. Clinical evidence of gonadai dysfunction was common in patients with deficient erythrocyte G unit activity, but was not observed in patients with normal erythrocyte G unit activity. These observations suggest that patients with pseudohypoparathyroidism and deficient erythrocyte G unit activity have a disorder that is generalized to cyciase-dependent tissues, and not limited to parathyroid hormone-sensitive tissues. Moreover, it appears that patients with pseudohypoparathyroidism and normal erythrocyte G unit activity may have a defect limited to parathyroid

April 1983 The American Journal of Medicine Volume 74 545

HORMONE RESISTANCE IN PSEUDOHYPOPARATHYROIDISM-LEVINE ET AL

hormone-sensitive tissues. These data support the hypothesis that a deficiency of G units is the basis for multiple hormone resistance in pseudohypoparathyroidism.

In 1942, Fuller Albright and his associates [3] described the clinical syndrome of pseudohypoparathyroidism, an uncommon metabolic disorder characterized by the biochemical features of hypoparathyroidism (hypo- calcemia and hyperphosphatemia) with apparently normal parathyroid hormone secretion [4,5]. The hallmark of this disorder as originally described by Al- bright is resistance of the target organs, bone and kidney, to the action of parathyroid hormone. In addition to the biochemical abnormalities, the patients described by Albright exhibited a unique somatic appearance (subsequently referred to as Albright’s hereditary osteodystrophy [AHO]) encompassing multiple devel- opmental defects: (1) round face, short neck, and stocky build; (2) one or more short metacarpals or metatarsals; and (3) foci of subcutaneous ossification. Later case reports described patients with identical biochemical evidence of parathyroid hormone resistance but without Albright’s hereditary osteodystrophy [6].

Characterization of the molecular basis for pseudohypoparathyroidism commenced when Chase and Aurbach showed that 3’,5’-cyclic adenosine monophosphate (CAMP) mediates the actions of parathyroid hormone on kidney and bone, and that parathyroid hormone infusion in man leads to a significant increase in urinary excretion of CAMP [7-g]. Chase, Melson, and Aurbach [9] applied these biochemical observations to the study of patients with pseudohypoparathyroidism, and found that these patients showed a markedly blunted urinary CAMP response to intravenous infusion of parathyroid hormone in comparison with normal subjects and patients with other forms of hypoparathyroidism. This observation suggested that pseudohypoparathyroidism type I” is caused by a defect in the plasma membrane-bound hormone-receptor adenylate cyclase complex that produces CAMP.

Although hypoparathyroidism is the most commonly recognized endocrine deficiency in pseudohypoparathyroidism, other clinical abnormalities in patients with pseudohypoparathyroidism, such as hypothyroidism [ 11,121 and hypogonadism [ 131, suggested that hormone resistance in the syndrome may be more generalized than appreciated heretofore. This led us to speculate that a defect in a component of the adenylate cyclase complex common to all tissues, such as the guanine nucleotide regulatory unit (G unit), might ac-

+ To distinguish it from pseudohypoparathyroidism type II [lo], a much rarer entity in which urinary CAMP response to parathyroid hormone infusion is intact but the phosphaturic response is blunted.

count for hormone resistance in pseudohypoparathyroidism. Recent studies from this laboratory [ 141, as

well as by Farfel et al [ 151, have demonstrated a deficiency of G unit activity in erythrocytes from patients ,with pseudohypoparathyroidism. In this paper, we report detailed biochemical and clinical studies in a large group of patients with pseudohypoparathyroidism. Our findings suggest that in pseudohypoparathyroidism, deficiency

of G unit activity in erythrocyte membranes correlates with decreased hormone responsiveness in several tissues, and indicates a generalized disorder of the adenylate cyclase complex.

PATIENTS AND METHODS

Subjects. Twenty-nine patients were studied. The diagnosis of pseudohypoparathyroidism was suspected on the basis of biochemical hypoparathyroidism (depressed serum calcium level, elevated serum phosphate level), elevated serum parathyroid hormone level, and/or a habitus suggestive of Albright’s hereditary osteodystrophy. Pseudohypoparathy- roidism type I was confirmed by documenting a markedly blunted urinary CAMP response to a standard intravenous infusion of parathyroid hormone. All patients were personally examined by at least one of the Authors (MAL, AMS, AMM, NAB), and underwent a radiographic bone survey (including hands and feet). The essential criterion used to characterize the (+)AljO phenotype was brachydactyly, unilateral or bi- lateral, involving hands or feet. Brachydactyly was diagnosed on the basis of standard radiographic criteria [ 161. Radio- graphs were reviewed by a radiologist who interpreted each study without knowledge of the patient’s clinical or biochemical status. Palpable or radio-opaque heterotopic subcutaneous ossifications were considered confirmatory of Albright’s hereditary osteodystrophy, and were not found in the absence of brachydactyly. While we recognize that se- lection of brachydactyly as the essential criterion for diag- nosing the (+)AHO phenotype is arbitrary, we felt that other features of the AH0 phenotype, e.g., short stature, round face, obesity, or intellectual impairment, are relatively non- kpecific and therefore, by themselves, inadequate evidence to establish a diagnosis of (+)AHO. Patients showing neither brachydactyly nor subcutaneous ossifications were considered to represent the (-)AHO variant of pseudohypoparathyroidism.

The control group consisted of 15 normal volunteer inpa- tients (nine females, six males), one patient each with post- surgical and idiopathic hypoparathyroidism, and two patients with primary hyperparathyroidism.

Testing was conducted in a metabolic unit of the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases 01 in Clinical Research Centers located in Dallas (NAB) and Syracuse (AMM). The study protocol was approved by the National Institutes of Health Clinical Research Com- mittee. Informed consent was obtained from patients and volunteers for each study. All patients had blood samples drawn for measurement of erythrocyte G unit activity and serum parathyroid hormone levels, but it was not possible to perform all other tests in each patient.

546 April 1983 The American Journal of Medlclne Volume 74

Study Protocols. The parathyroid hormone infusion protocol was similar to that described by Chase et al [9]. Subjects were given 250 U of bovine parathyroid extract (Parathor- mone TM, Eli Lilly Company, Indianapolis, Indiana) intravenously, and urine samples were collected during a period of diuresis maintained by oral intake of water (250 ml hourly). Urine was discarded at -60 minutes and collected at time 0, +30, -t-60, +120, and +240 minutes. Urinary CAMP is expressed as nmol/dl glomerular filtrate (calculated by multiplying urinary CAMP/urine creatinine in nmol/mg by serum creatinine in mg/dl).

Glucagon infusion was performed after an overnight fast. A 21gauge butterfly cannula was inserted in a superficial vein and patency maintained with an injection of a 100 U/ml so- lution of heparin into the tubing. Subjects were then kept supine for 30 minutes before baseline blood samples were drawn. After a bolus injection of 500 pg of glucagon (Eli Lilly Company), further blood samples for blood glucose and plasma CAMP levels were obtained at five, 15, 30, and 60 minutes. All patients were euthyroid (either spontaneously or when receiving thyroid hormone replacement therapy) at the time of glucagon infusion.

Thyroid function (thyroxine, triiodothyronine, basal thyrotropin) was evaluated in all patients. A standard thyrotropin- releasing hormone test (500 pg intravenously) was also performed in most patients with measurement of thyrotropin, triiodothyronine, and prolactin up to 180 minutes after thyrotropin-releasing hormone injection. Two patients who were taking Synthroid before study were switched to triiodothyronine (50 pug per day) four weeks before study. Triiodothy- ronine was then discontinued 14 days before thyrotropin- releasing hormone infusion.

Adrenal responsiveness to ACTH was assessed by mea- suring serum cortisol at 0, 30, and 60 minutes following infusion of 250 pg of synthetic ACTH l-24 (Cortrosyn, Or- ganon, Inc., West Orange, New Jersey). Gonadal function was evaluated clinically (physical examination, history of menstrual function and fertility). Assays. Parathyroid hormone concentration in plasma was measured by a radioimmunoassay specific for mid-region parathyroid hormone antigens [ 171. Parathyroid hormone concentrations in plasma of normal volunteers are less than 0.24 ng eq/ml plasma. CAMP was measured in urine by radioimmunoassay [ 181 with the Squibb gammaflo [ 191. Blood for plasma CAMP assay was collected into chilled tubes containing K-EDTA, and CAMP measurements were determined by radioimmunoassay as previously described [20].

Radioimmunoassays of thyrotropin, prolactin, cortisol, and thyroid hormones were performed by Hazleton Laboratories (Vienna, Virginia). Thyrotropin [ 211, prolactin [ 221, and cortisol [ 231 were measured by radioimmunoassay as previously described. Thyroxine was measured by radioimmunoassay; percentage free thyroxine was measured by equi- librium dialysis; and triiodothyronine levels were determined by radioimmunoassay [24-261. Antibodies to thyroglobulin were measured by the tanned-red-cell technique, and an- timicrosomal antibodies were detected by a hemagglutination inhibition method [ 271. Measurement of Erythrocyte G Unit Activity. Blood was obtained from patients and control subjects by venipuncture

and anticoagulated with acid citrate dextrose. Blood samples drawn in Dallas and Syracuse were shipped on ice to Be- thesda and erythrocyte membranes prepared w-thin 48 hours of collection. Previous studies [ 141 have shown that red cell membrane G unit activity is stable in blood samples stored for up to one week at 4’C. Erythrocyte membranes were prepared by hypotonic lysis [28] in 5 mM.sodium phosphate (pH 8.0) at OOC, and stored in liquid nitrogen. Activity was unchanged for up to three months under these conditions.

Assays of G unit activity were performed as previously described [ 141, and were based on activation of adenylate cyclase in detergent-solubilized turkey erythrocyte membranes by G units in detergent (Lubrol 12A9) extracts of human erythrocyte membranes. In brief, extracts containing G units were preincubated with guanosine 5’-@y-thio)tri- phosphate (GTP-Y-S), a nonhydrolyzable guanosine tri- phosphate (GTP) analog, in order to “activate” G units prior to addition to turkey erythrocyte membranes. Turkey erythrocyte membranes show negligible GTP-y-S-stimulated adenylate cyclase activity unless fl-adrenergic agonists are added. Addition of GTP-y-S-treated human erythrocyte G units (4.0 pg of protein) to an excess of catalytic units from solubilized turkey erythrocyte membranes (40 pg of protein) caused formation of an active adenylate cyclase complex, and resultant CAMP production was proportional to added human erythrocyte G units. Results of assays were expressed as a percentage of the activity of a standard membrane preparation consisting of pooled erythrocytes from five normal persons, and represent the mean of at least three determinations. In seven patients, the G unit was assayed in membranes prepared from two separate blood samples drawn at least 10 weeks apart. No significant differences were found between separate membrane preparations in a given person. Acetylcholinesterase Assay. Human erythrocyte membrane acetylcholinesterase activity was assayed with the Ellman reagent as previously described [29]. Membrane protein was determined by the method of Lowry et al [ 301. Statistics. Data were analyzed with the Student t test for unpaired samples. The 95 percent confidence interval was used for testing significance. Unless otherwise stated, results are expressed as mean f 1 SD. Plasma CAMP responses to glucagon were analyzed by determining the mean response for each time point and by determining the area under the curve above baseline (integrated response from 0 to 60 minutes).

RESULTS

Fifteen of our patients (13 females) showed features

characteristic of Albright’s osteodystrophy ((+)AHO), and 14 patients (12 females) had normal somatic (-)AHO phenotypes (Table I). The age of onset of symptoms (early adolescence), duration of treatment (mean 15 years), and age at time of study ((+)AHO:

mean 30 years; (-)AHO: mean 29 years) were similar for both groups. Twelve of 15 (+)AHO patients and 10 of 14 (-)AHO patients were taking vitamin D.

Serum calcium levels were similar in (+)AHO and

(-)AHO groups (4.10 f 0.36 versus 3.97 f 0.48

HORMONE RESISTANCE IN PSEUDOHYPOPARATHYROlDlSM-LEVINE ET AL

April 1983 The American Journal of Medicine Volume 74 547


TABLE I Characteristics of Patients with Pseudohypoparathyroidism

Somatic Features7

Serum Calcium*

Parathyroid Hormone

Urinary CAMP

Rsspollss to Parathyiold Hormone

Erythrocyte G Unit Actlvlly

(control = -1.00) Patient’ Age Sex

All1 27 Blll 21 Bll 42 Cl1 25 Dll 60 Fll 28 F12 28 HI12 22 HI15 19 HI17 16 HI18 14 Ill11 36 JJl 31 Kll 29 Lll 24 Nil 32 011 36 Qlll 21 Call2 23 Qll 44 Ql2 46 RI1 42 Sll 25 Ull 19 VI1 24 XI1 28 YII 10 Zlll 28 Zll 54

F F F F M F F F F F F F F F M F F F F F M F F F F F M F F

B, 0, S B, 0, S B, 0, S B, 0, S B, S B, 0, S B, 0, S B, 0, S B, 0, S B, 0, S B, 0, S B, 0, S B, S B, S B, S

S S

1 N N

i

I ND5 ND5

1

i

i

i

f

f

!

i

i

f 1

0.41 0.36 0.45 0.22 0.98 0.47 0.40 0.36 0.54 0.39 0.47 0.55 0.51 0.49 1.00 0.80 0.96 1.04 0.96 0.87 0.88 0.86 0.80 0.84 1.00 0.99 1.01 1.03 0.99

l Patient code: Letter refers to family: roman numeral refers to generation; arabic numeral refers to birth order. t Somatic features: B = brachydactyly; 0 = subcutaneous ossification; S = short stature. t Refers to serum calcium level prior to treatment with vitamin D and/or calcium. N = normocalcemic. 5 Urinary CAMP measurement not done; no phosphaturia following parathyroid hormone infusion in Dr. F. Albright’s clinic [3]. 1 = decreased; t = increased.

meq/liter). Plasma parathyroid hormone levels were above 0.24 ng/ml in each member of both groups. Two (+)AHO patients (Bll-l, Bl-1) were spontaneously normocalcemic, and had not taken either vitamin D or calcium. Both of these patients, however, showed a markedly blunted urinary CAMP response to parathyroid hormone (see Endocrine Studies section). Biochemical Studies. Erythrocyte G unit activity: Erythrocyte G unit activity was determined in all subjects (Table I, Figure 1). Patients who lacked features of Al- bright’s hereditary osteodystrophy showed normal G unit activity, whereas in 13 of 15 (+)AHO patients there was approximately a 50 percent reduction in G unit activity (0.92 f 0.08 versus 0.43 f 0.03) (p <O.OOl). There was no significant difference between the G unit activity in (-)AHO patients and control subjects.

Erythrocyte membrane acetylcholinesterase activity

was similar in the groups with (+)AHO and (-)AHO phenotypes (2.98 f 0.69 versus 2.46 f 0.35 mol/ liter/min/pg membrane). There was no correlation between individual values of acetylcholinesterase activity and G unit activity in either patients with pseudohypoparathyroidism or normal subjects. Endocrine Studies. Based on the results of the G unit assay, patients with pseudohypoparathyroidism were divided into two groups, one showing normal erythrocyte G unit activity, and the other showing deficient erythrocyte G unit activity. Sixteen patients were thus clas- sified as having normal erythrocyte G unit activity (12 females, four males), 14 of whom were (-)AHO, and two of whom were (+)AHO. l The group with deficient

l These two patients were indistinguishable from the (-_)AHO patients with respect to the endocrine tests to be described.

548 April 1983 The American Journal of Medlclne Volume 74


Figure 1. Erythrocyte membrane G unit activity in control, i-AHO, and - AH0 subjects. G unit activity was measured by adding solubilized human erythrocyte membranes containing G units to solubilized turkey erythrocfle membranes containing catalytic units (see Methods section); resultant adenylate cyclase activity is expressed as percentage of a pooled standard of normal human erythrocyte membranes. AH0 = A/bright’s hereditary osteodystrophy.

A

A

0

I 1

CONTROL +AHO -AHO n=9 n = 15 n = 14

*SIGNIFICANTLY DIFFERENT FROM CONTROL, -AHO P<O.OOl 1

erythrocyte G unit activity consisted of 13 patients with pseudohypoparathyroidism (all females), all of whom satisfied the criteria for (+)AHO.

Urinary CAMP response to parathyrold hormone infusion: The time course and magnitude of the changes in urinary CAMP following parathyroid hormone infusion were similar in both the group with deficient erythrocyte G unit activity and the group with normal erythrocyte G unit activity (Figure 2). Patients with pseudohypoparathyroidism type I showed a markedly blunted (less than fivefold above baseline) peak response to parathyroid hormone infusion. The two patients (BI-1, 811-l) who were normocalcemic without therapy (a mother and daughter) repeatedly showed a twofold and a l.Bfold peak urinary CAMP response, respectively. Normal subjects, in comparison, showed a brisk urinary CAMP response, with peak values gen- erally 40 to 50 times basal.

Thyroid function: None of the patients with pseudohypoparathyroidism who had normal erythrocyte G unit activity and four of the 15 patients with deficient erythrocyte G unit activity had at some point received thyroid hormone replacement for documented hypothyroidism. In the six months immediately prior to study (see Methods section), only two patients were

0 30 60 120 180

MINUTES

Figure 2. Results of parathyroid hormone infusion (se: k&hods section) in normal subjects (A - A), patients with pseudohypoparathyroidism and deficient erythrocyte G unit activity (0 - 0), and patients with pseudohypoparathy- rokiism andnormal erythrocyte G unit activity (0 - - - 0). The hatched area represents the 15-minute time period over which the parathyroid hormone (250 U) was infused. Each point represents the mean f SEM. GF = glomerular filtrate.

April 1983 The American Journal of Medklne Volume 74 549


TABLE II Basal Thyroid Parameters

Patients with Pwudohypopsrathyroldkm

Normal Dsfkislll G Unit G Unit Acllvily Activity

(n = 16) (n = 13)

i::ee T4 0 0 (0) (0) 9 6 (69) (62)

i:sH 0 0 (0) (0) 11 9 (65) (69)

Goiter 1 (6) 0 (0)

Antithyroid antibodies 1 (6) 1 (6)

Normal values: T4 (5.5 to 11.5 pg/dl), free T, (1.0 to 2.3 ng/dl), TI (54 to 216 ng/dl), TSH (0.6 to 6.0 pU/ml), all 95 percent confidence limits. Numbers in parentheses refer to percent of patients affected. 1 = decreased; T4 = thyroxine; Ts = triiodothyronine; t = increased; TSH = thyrotropin.

receiving thyroid hormone replacement. No patient exhibited clinical features of gross hypothyroidism.

Basal thyroxine, free thyroxine, triiodothyronine, and thyrotropin levels were normal in all patients with normal erythrocyte G unit activity who were examined, although one patient showed positive antithyroid antibodies (al-l) and one (r%2) showed diffuse goiter. Approximately 50 percent of the patients with pseudohypoparathyroidism who had deficient erythrocyte G unit activity, however, showed biochemical evidence of mild primary hypothyroidism (Table II). A more distinct separation of the two groups of patients with pseudohypoparathyroidism was noted by analysis of the results of a thyrotropin-releasing hormone infusion (Figure 3). The time course for thyrotropin response was similar in all patients and control subjects, with peak thyrotropin values at 20 to 30 minutes after thyrotro-

100

80

Peak

PHP (Low G Unit) PHP (nl G Unit)

Figure 3. Basal thyrotropin (TSH) and peak thyrotropin levels following intravenous administration of 500 pg of thyrotropin-releasing hormone. Hatched areas represent normal range for basal thyrotropin and peak thyrotropin levels after thyrotropin-releasing hormone infusion. The peak thyrotropin levels foC lowing thyrotropin-releasing hormone infusion in the two patients with Albright’s hereditary osteodystrophy and normal erythrocyte G unit activity (Di- 1 and LI- 1) were 25 ptJl ml and 13.5 pUf ml, respectively. PHP = pseudohypoparathyroidism; nl = normal.

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HORMONE RESISTANCE IN PSEUDOHYPOPARATHYROlDISM-LEVINE ET AL

TABLE III Basal and Peak Prolactin Levels after lhyrotropln-Releasing Hormone Stlmulatlon’

Basal prolactin

@g/ml) Peak prolactin

Normal Volunteers

(n = 5)

11 f3

48 f 20

Patients with Pseudohypoparathyroklidism

Normal Deficient G Unit G Unit Activity Activity

(n = 11) (n = 12)

9.6 f 4.4 12.8 f 10

68.1 f 54 42.6 f 33

(n&ml)

l All values exDressed as the mean f SE. None of the comparisons is statistically significant at p (0.05.

pin-releasing hormone infusion. Basal and peak thyrotropin values were similar in the normal subjects and patients with normal erythrocyte G unit activity (2.0 f 1.1 @/ml and 16.5 f 6.7 pU/ml versus 4.5 f 2.0 &j/ml and 19.5 f 6.6 @/ml, respectively), but patients with deficient erythrocyte G unit activity exhibited elevated basal and excessive peak thyrotropin responses to thyrotropin-releasing hormone (basal 15.0 f 6.5 @J/ml [p <O.OOl]; peak 54.3 f 22.7 @J/ml [p <O.OOl]).

The increase in serum triiodothyronine above baseline at 180 minutes after thyrotropin-releasing hormone infusion was similar in control subjects (53.6 f 23.6 ng/ml) and patients with normal erythrocyte G unit activity (45.0 f 16.8 ng/ml). In patients with deficient erythrocyte G unit activity, the triiodothyronine increase at 180 minutes (27.5 f 26 ng/ml) was significantly (p <O.OOl) less than in either the patients with normal erythrocyte G unit activity or normal control subjects, despite the greater increase in serum thyrotropin.

Prolactin levels: The mean basal and peak serum prolactin levels after thyrotropin-releasing hormone infusion in patients with deficient erythrocyte G unit activity and patients with normal erythrocyte G unit activity were not significantly different from the levels in control subjects (Table III). The peak prolactin responses to thyrotropin-releasing hormone in patients with deficient erythrocy-te G unit activity ranged from 16 to 87 ng/ml, and no patient showed a deficient response.

Hepatic responsiveness to glucagon: The plasma CAMP response to a single 500 pg dose of glucagon [31] was examined in 14 normal subjects, 11 patients with deficient erythrocyte G unit activity, and 14 patients with normal erythrocyte G unit activity. Glucagon caused a rapid increase in plasma CAMP, with the peak values reached between five and 15 minutes after the injection (Figure 4, top). The pattern and duration of response

0 5 15 30 60

TIME (mini

20

2 18

i

A

a 0

2 t

Y A

A

: AA

t

a a

t

:* 1:

00 0

,oo 0

000

0

I

CONTROL NGU DGU ” = 15 ” = 14 n = 11

J

Figure 4. Top, plasma CAMP response following intravenous administration of glucagon (500 pg) in 11 patients with deficient erythrocyte G unit activity (O), 14 patients with normal etythmcyte G unit activity (0 ), and 15 control subjects (A). AsterMs &note time points at which values in patients with deficient erythrocyte G unit activity were significantly different (*p < 0.05; l l p < 0.0 1) from values in patients with nom~l erythrocyte G unit activity and normal control subjects. Each point represents the mean f SEM. Bottom, plasma CAMP in response to gkagon. Group with deficient erythrocyte G unit activity (DGU) showed significantly lower response than group with normal erythrocyte G unit activity (NGU) (p < 0.02) or control group (p < 0.02). The data are expressed as the integrated response from 0 to 60 minutes following glucagon injection.

were qualitatively similar in all these groups, and a decline toward basal values was observed at 60 minutes, the last time point measured.

Basal plasma CAMP values were similar in all three groups; there were no significant differences in either

April 1983 The American Journal of Medlclne Volume 74 551


peak CAMP values or integrated plasma CAMP responses between the control subjects and the patients with normal erythrocyte G unit activity. The group of patients with pseudohypoparathyroidism who had deficient erythrocyte G unit activity, however, showed a smaller mean increase in the plasma concentration of CAMP in response to glucagon than was found in the control group or the patients with normal erythrocyte G unit activity. The integrated plasma CAMP response to glucagon (area under the response curve, Figure 4, bottom) was significantly lower (p <O.Ol) in patients with deficient erythrocyte G unit activity (5.1 f 2.2 pM X minutes) than in patients with normal erythrocyte G unit activity (8.6 f 3.4 @I X minutes) or control subjects (8.6 f 3.6 pM X minutes). In addition, similar degrees of blunting of the plasma CAMP response to glucagon were noted in the patients with deficient erythrocyte G unit activity when either peak response or summed plasma CAMP values (5, 15; 5, 15,30; or 5, 15, 30, 60 minutes) were compared with the values in the normal group or the group with normal erythrocyte G unit activity (data not shown).

apparently arrested at the Tanner 11-111 stage. Two women gave a history of previously normal menstrual function (including one successful pregnancy each), but one had subsequently undergone a hysterectomy (for unknown reasons) and the second was amenorrheic. The remaining two women gave a history of normal menstrual function.

Adrenal reserve: Early morning serum cortisol concentrations were normal in all patients with pseudohypoparathyroidism who were tested. Adrenal reserve, as assessed by maximal corticotropin stimulation, was also normal (30 minute cortisol value > 20 pg/dl or increase of 10 pgldl over basal).

COMMENTS

The increase in plasma CAMP was accompanied by an elevation of the blood glucose level in each patient. The increase in the blood glucose level (peak minus basal) was similar in each group (patients with deficient erythrocyte G unit activity: 47 f 2.8 mg/dl; patients with normal erythrocyte G unit activity: 45 f 3.1 mg/dl; control subjects: 57 f 5.3 mgldl [mean f SE]) despite the significant difference in plasma CAMP response to glucagon.

Pseudohypoparathyroidism type I is characterized by functional hypoparathyroidism, apparently normal parathyroid hormone secretion, and a deficient urinary CAMP response to exogenous parathyroid hormone. Albright’s original description of this condition impli- cated parathyroid hormone resistance as the basis of this disorder, and early biochemical investigations suggested that the molecular defect in pseudohypoparathyroidism type I resides in the parathyroid hormone receptor-adenylate cyclase complex.

Gonadal function: Patients with normal erythrocyte G unit activity displayed age- and sex-appropriate development of secondary sexual characteristics. All female patients with normal erythrocyte G unit activity described normal onset of menarche and puberty, menstruated normally, and offered no history suggestive of infertility. One of three postpubertal males with normal erythrocyte G unit activity had fathered a child.

Patients with pseudohypoparathyroidism who had deficient erythrocyte G unit activity exhibited a spectrum of gonadal dysfunction. Five of 13 female patients with deficient erythrocyte G unit activity menstruated erra- tically, if at all, and could be considered oligomenorrheic and (probably) anovulatory. Two of these women gave histories of infertility, and showed a weakly positive withdrawal bleeding response to a progestational agent. Development of secondary sexual characteristics in this group of patients has apparently progressed to the Tanner IV-V stage, and there has been no evidence of hirsutism, excessive androgenization, or ovarian en- largement. Primary amenorrhea was diagnosed in four patients who showed incomplete development of secondary sexual characteristics, and sexual maturation

Recent studies [reviewed in 321 have shown that the receptor-adenylate cyclase complex consists of at least three separable plasma membrane components- hormone receptor, catalytic unit, and a guanine nucleotide regulatory protein (G unit). The receptor binds hormones, the catalytic moiety forms CAMP, and the G unit acts as a coupling factor, enabling hormone- bound receptors to activate the catalytic unit. While hormone receptors are target cell specific, the G unit appears to be common to the adenylate cyclase complex of most tissues [32], Thus, one would expect abnormal receptors to lead to hormone resistance limited to a specific agonist; a defective G unit, in contrast, could cause hormone resistance generalized to all tissues in which hormone action is dependent upon activation of adenylate cyclase.

Previous reports [ 1 l- 131 of resistance to hormones other than parathyroid hormone in patients with pseudohypoparathyroidism led us to speculate that in some patients with pseudohypoparathyroidism, hormone resistance might be caused by a generalized, postre- ceptor defect such as an abnormality in the guanine nucleotide regulatory subunit of adenylate cyclase. In the present study, we were able to define two distinct groups of patients with pseudohypoparathyroidism, based on results of erythrocyte G unit assays: (1) a group of 16 patients who showed normal G unit activity; and (2) a group of 13 patients who showed an approxi- mate 50 percent reduction in G unit activity. In order to determine if the reduced erythrocyte G unit activity was


a specific defect or a manifestation of a generalized membrane abnormality, we measured acetylcholinesterase activity and found it similar in the group with normal erythrocyte G unit activity, the group with deficient erythrocyte G unit activity, and the control group. In addition, there were no differences in the major protein bands of the red cell membrane as seen on polyacrylamide gel electrophoresis. We then studied the hormone responsiveness of multiple target organs in which adenylate cyclase plays a role in mediating the physiologic response, and correlated these results to patient erythrocyte G unit activity.

Abnormal thyroid function was found in 12 of 13 patients with deficient erythrocyte G unit activity. Basal and/or peak thyrotropin-releasing hormone-stimulated thyrotropin levels were elevated, characteristic of primary hypothyroidism. Other reports of decreased thyrotropin secretion in pseudohypoparathyroidism [33] have not been confirmed upon testing with new, more highly sensitive thyrotropin radioimmunoassays. The presence of low-titer thyroid autoantibodies in only one patient, and the absence of other associated features of autoimmune disease exclude a diagnosis of autoimmune thyroiditis in these patients. The absence of goiter in association with increased plasma thyrotropin makes a diagnosis of a congenital defect of iodide metabolism unlikely, and is presumptive evidence of a failure of the thyroid gland to respond appropriately to a high concentration of thyrotropin [34]. Plasma thyrotropin in Patient BI-1 has been assayed in the past and was biologically active [ 111. These findings suggest that a deficiency of G unit activity in thyroid plasma membranes is the cause of thyrotropin resistance. Al- though most cases of hypothyroidism recognized in pseudohypoparathyroidism are mild, some patients may progress to frank clinical hypothyroidism and, as with four of our patients, require thyroid hormone replacement.

All of our patients with normal erythrocyte G unit activity showed normal thyrotropin response to thyrotropin-releasing hormone infusion and normal thyroid hormone secretion. Although others [ 151 have reported hypothyroidism in association with pseudohypoparathyroidism and normal erythrocyte G unit activity, the presence of antithyroid antibodies in the plasma of several of these patients [35] suggests an autoimmune basis for the hypothyroidism.

Further evidence of multihormonal resistance in pseudohypoparathyroidism is found in the defective hepatic responsiveness to glucagon noted in the low-G unit group. Thyroid status has been noted to modulate hepatic responsiveness to glucagon [36-381 but, as we noted in the Methods section, all patients were euthyroid (either spontaneously or during replacement therapy) at the time of this test. Patients with pseu-

dohypoparathyroidism who had normal erythrocyte G unit activity showed normal hepatic adenylate cyclase responsiveness to glucagon, whereas the group of patients with deficient erythrocyte G unit activity demonstrated glucagon resistance, as evidenced by a significantly lower mean plasma CAMP response. Although the mean values in the patients with deficient erythrocyte G unit activity and the patients with normal erythrocyte G unit activity were significantly different, considerable overlap was apparent amongst the plasma CAMP responses seen in individual patients with pseudohypoparathyroidism. This may have been due in part to the near-maximal stimulating dose of glucagon employed [ 3 1,381; it is possible that a lower glucagon dose would have afforded greater opportunity to distinguish deficient from normal responses. The distal effect of hepatic CAMP generation, glucose release, was normal in both groups, and indicates that despite the presumably reduced adenylate cyclase activity in patients with deficient erythrocyte G unit activity, sufficient intracellular CAMP does accumulate to transduce the hormone signal and express the distal physiologic effect (i.e., hyperglycemia). Similar dynamics have been identified in the lipocyte and adrenocortical cell, and would explain the normal adrenal cortisol response following ACTH stimulation in the patients with pseudohypoparathyroidism who had deficient erythrocyte G unit activity. Measurement of a more proximal effect of ACTH action, such as CAMP generation, could presumably be required to demonstrate ACTH resistance. Unfortunately, ACTH, at doses used in standard clinical tests, does not significantly increase peripheral plasma CAMP [ 391.

Gonadal dysfunction was common in patients with deficient erythrocyte G unit activity and pseudohypoparathyroidism. Sexual immaturity, amenorrhea, or oligomenorrhea were found in 10 of 13 women (see Results section), and only two women (BI- 1, Hll-2) had borne a child. In at least one previously reported case [ 131, this was apparently due to gonadotropin resistance (elevated basal serum luteinizing hormone and follicle-stimulating hormone levels and exaggerated gonadotropin response to luteinizing-releasing hormone). This case is extremely interesting because gonadotropin resistance, moderate glucagon resistance, and thyrotropin resistance were documented in addition to the characteristic parathyroid hormone resistance of pseudohypoparathyroidism (personal communication). This patient exhibited the dysrnorphic features of Albright’s hereditary osteodystrophy. and erythrocyte G unit activity was found to be markedly reduced when measured in our laboratory (unpublished observation). In the majority of women that we studied, basal gonadotropin levels were not elevated (data not shown); more extensive studies will be necessary to

HORMONE RESISTANCE IN PSEUDOHYPOPARATHYRO~DISM-LEVINE ET AL

April 1983 The Amerlcan Journal of Medicine Volume 74 553


define the basis for gonadal dysfunction in these patients. Patients who showed normal erythrocyte G unit activity all exhibited normal gonadal function, consistent with endocrine resistance limited to parathyroid hormone target tissues.

Deficiency of prolactin secretion (basal and in response to secretagogues such as thyrotropin-releasing hormone and chlorpromazine) has been noted in some patients with pseudohypoparathyroidism [ 15,351. These reports described patients with normal as well as deficient erythrocyte G unit activity, and included patients both with or without Albright’s hereditary osteodystrophy. We found no evidence for reduced prolactin levels (basally or in response to thyrotropin-releasing hormone) in patients with pseudohypoparathyroidism either with normal or diminished G unit activity. Although the reason(s) for this discrepancy is not apparent, it should be noted that the role of CAMP in mediating prolactin secretion is controversial, and current observations point to a major role of intracellular calcium as the second messenger [40]. Thus, it is not clear that a deficiency of G units or a decreased accumulation of cyclic nucleotides would impair prolactin reserve or secretion.

Our studies clearly show that pseudohypoparathyroidism type I is a heterogenous disorder composed of at least two groups of individuals: patients who show normal erythrocyte G unit activity and those who show deficient etythrocyte G unit activity. In our patient group, all but two patients with the (+)AHO phenotype showed deficient erythrocyte G unit activity (DI-1, LI-1), and all (-)AHO patients showed normal erythrocyte G unit activity. Another group of investigators has also reported that some persons with the (+)AHO phenotype may show normal erythrocyte G unit activity [lb]. These observations suggest that deficient G unit activity per se may not be the cause of Albright’s hereditary osteodystrophy. At present, the precise basis for the AH0 phenotype remains unknown.

Deficient erythrocyte G unit activity in patients with Albright’s hereditary osteodystrophy correlates well with the apparent presence of hormone resistance in multiple adenylate cyclasedependent tissues, and implicates this defect as the cause of a generalized disorder of adenylate cyclase. The limited nature of the endocrine perturbation in patients with normal erythrocyte G unit activity implicates abnormal parathyroid hormone or altered parathyroid hormone-receptor interactions as the basis for this disorder, but at present there is no direct evidence to support this hypothesis. It also remains possible that some patients with pseudohypoparathyroidism and normal erythrocyte G unit activity will be found to have multiple hormone resistance, and will prove to have another defect (e.g., increased

phosphodiesterase activity) leading to generalized hormone resistance [ 411.

Further support for the hypothesis that a deficiency of G unit activity could account for generalized hormone resistance in pseudohypoparathyroidism comes from studies of other accessible tissues. Thus, reduced G unit activity has been found in platelet [42] and cultured fibroblast [43] membranes from patients with pseudohypoparathyroidism who also show reduced erythrocyte membrane G units. We have recently found that cultured fibroblasts from patients with pseudohypoparathyroidism who show reduced erythrocyte G units and resistance to multiple hormones accumulate significantly less CAMP in response to a submaximal dose of prostaglandin Et than do control fibroblasts (Levine et al, unpublished observation). The latter observation is consistent with the notion that a reduction in functional G units may impair the ability of various tissues to respond (in terms of CAMP production and, in some cases, more distal physiologic effects) to,a variety of agents whose mechanism of action involves adenylate cyclase activation.

While deficient G unit activity helps explain resistance to multiple hormones in some patients with pseudohypoparathyroidism, it is not clear how an approximately 50 percent reduction in G unit activity im- pairs target organ response. Indeed, the clinical studies discussed earlier suggest considerable variability in the apparent resistance of different target organs to their tropic hormones. In part, this variability is a function of the index of responsiveness one chooses to measure; as previously discussed, proximal responses (e.g., plasma CAMP) are more likely to show a defect than distal responses (e.g., blood glucose response to glucagon, plasma cortisol response to ACTH). In some tissues, however, even distal responsiveness is commonly impaired (phosphaturic response to parathyroid hormone, thyroid hormone secretion in response to thyrotropin). Moreover, distal responses and overt clinical expression may be influenced or modified by other factors; for example, two of our patients (811-1, BI-1) were normocalcemic despite blunted urinary CAMP responses to parathyroid hormone infusion.

The same quantitative defect in G unit activity could also have different consequences with regard to ability to generate CAMP in various tissues. We still do not know the stoichiometry of the components of the adenylate cyclase complex, nor how it may vary in different tissues. Differences in membrane lipid composition and intracellular phosphodiesterase activity may also play important modulating roles.

Furthermore, an identical quantitative defect in adenylate cyclase activity may lead to different physiologic consequences in various tissues despite similarly


HORMONE RESISTANCE IN PSEUOOHYPOPARATHYROlDISM-LEVINE ET AL

reduced concentrations of CAMP, as different concentrations of CAMP may be required from tissue to tissue to activate specific protein kinases. Protein ki- nase activation, and not CAMP accumulation per se, is the rate-limiting step in expression of physiologic activity [44]. Deficiency of G unit activity may not be of sufficient magnitude to impair uniformly distal responses in diverse tissues, or even in one tissue (e.g., apparently normal renal l-cu-hydroxylase activity despite impaired phosphaturia in some patients with normocalcemic pseudohypoparathyroidism).

In summary, we have shown that many patients with pseudohypoparathyroidism manifest reduced erythrocyte G unit activity and are resistant to multiple hormones. We believe that this relationship is not coinci-

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We wish to thank Dr. Arthur C. Santora, II, for carefully reviewing this manuscript, the staff of the 8-West Clinical Center Research Unit (National Institute of Ar- thritis, Diabetes, and Digestive and Kidney Diseases) for assistance with inpatient testing, and Mrs. Lillian Perry for excellent secretarial assistance.

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Documents

Resistance to multiple hormones in patients with pseudohypoparathyroidism: Association with deficient activity of guanine nucleotide regulatory protein