Cytochemical Population Analyses of Glycogen in Neutrophil Leukocytes of Chronic Myelocytic Leukaemia during Busulfan Treatment

Abstract A comparative investigation of glycogen content and glycogen synthesis in neutrophil leukocytes from patients with untreated chronic myelocytic leukaemia (CML) and CML in remission was undertaken using microspectrophotometry of the periodic acid-Schiff (PAS) reaction and autoradiography after glucose-H³ incorporation into glycogen of single cells. The glycogen content was low and the rate of glycogen synthesis high in neutrophils from untreated CML, indicating an increased glycogen turnover rate in these cells compared to normal neutrophils. Both glycogen content and rate of glycogen synthesis reached normal levels in CML neutrophils in busulfan induced remission. This normalization of glycogen metabolism was not observed until WBC approached normal values after several weeks of treatment. This indicates that the effect of busulfan on normalization acted at the level of the myeloid precursor cell. The mechanism of normalization may be due to busulfan partially or completely eradicating a cytochemically abnormal leukaemic cell population and thereby "unmasking" a cytochemically normal cell population.     

Thyroid hormone regulates ontogeny of beta adrenergic receptors and adenylate cyclase in rat heart and kidney: effects of propylthiouracil-induced perinatal hypothyroidism.

In mature animals, thyroid hormone is permissive for beta adrenergic receptor expression and adrenergic control of adenylate cyclase. To determine if endogenous thyroid hormones play a similar role in the development of receptors and transduction mechanisms, we administered propylthiouracil perinatally to rat dams and pups from gestational day 17 through postnatal day 5. Circulating thyroid hormones were completely suppressed through postnatal day 10 and then rose to only slightly subnormal values by the 3rd to 4th postnatal week. In the heart, hypothyroidism completely suppressed the initial development of beta adrenergic receptor binding sites, with recovery paralleling the return of thyroid hormone levels. In contrast, development of basal and isoproterenol-stimulated adenylate cyclase activity showed more lasting deficiencies with a delayed onset corresponding to general growth impairment; however, forskolin-stimulated adenylate cyclase developed in a nearly normal pattern. Effects on development of renal beta receptors and adenylate cyclase were of smaller magnitude and comprised only the delayed onset phase; receptor deficiencies appeared after 10 days and adverse effects on adenylate cyclase were limited to the isoproterenol-sensitive component, consisting of a shift of the ontogenetic peak to later ages. Endogenous thyroid hormones thus contribute two distinct factors to beta receptor/adenylate cyclase development: they are obligatory for cardiac beta receptor development, but also, in parallel with general effects on growth and development, serve to program the ontogeny of transduction factors linking the receptors to adenylate cyclase. The predominance of propylthiouracil effects on isoproterenol-stimulated adenylate cyclase but not on enzymatic responses to forskolin suggests that thyroid hormones may be controlling the development of regulatory G-proteins.     

N-Acetyl-beta-D-glucosaminidase activity in normal and chronic lymphocytic leukaemic lymphocytes.

N-Acetyl-beta-D-glucosaminidase (EC 3.2.1.30) activity was measured fluorimetrically in: (a) lymphocytes from 20 normal donors, (b) enriched B and T lymphocyte populations prepared by E rosette sedimentation from 8 normal subjects, (c) lymphocytes from 15 untreated B cell chronic lymphocytic leukaemic patients. The pH profiles and optima (4.7) were similar in all preparations. Normal B lymphocytes had higher activity than normal T lymphocytes and both these preparations and normal unfractionated lymphocytes had significantly higher activity than chronic lymphocytic leukaemic lymphocytes. The apparent Michaelis constants were similar in normal unfractionated, B enriched and T enriched lymphocytes, whereas a reduced affinity for the enzyme was observed in the leukaemic lymphocytes. The difference in enzyme content between normal and chronic lymphocytic leukaemic lymphocytes cannot therefore be explained on the basis of a high B cell percentage in patients with chronic lymphocytic leukaemia.     

Differential regulation of right and left ventricular beta-adrenergic receptors in newborn lambs with experimental cyanotic heart disease.

To determine whether chronic hypoxemia secondary to an intracardiac right-to-left shunt alters regulation of the myocardial beta-adrenergic receptor/adenylate cyclase system, we produced chronic hypoxemia in nine newborn lambs by creating right ventricular outflow obstruction and an atrial septal defect. Oxygen saturation was reduced to 65-74% for 2 wk. Eight lambs served as normoxemic controls. beta-receptor density (Bmax) and ligand affinity (KD) were determined with the radio-ligand [125I]iodocyanopindolol and adenylate cyclase activity determined during stimulation with isoproterenol, sodium fluoride (NaF), and forskolin. During chronic hypoxemia, Bmax decreased 45% (hypoxemic, 180.6 +/- 31.5 vs. control, 330.5 +/- 60.1 fmol/mg) in the left ventricle (exposed to hypoxemia alone) but was unchanged in the right ventricle (exposed to hypoxemia and pressure overload). KD was not different from control in either ventricle. Left ventricular isoproterenol-stimulated adenylate cyclase activity was decreased by 39% (30.0 +/- 4.3% increase vs. 44.1 +/- 9.5% increase) whereas right ventricular adenylate cyclase activity was unchanged. Stimulation of adenylate cyclase with NaF or forskolin was not different from control in either ventricle. Circulating epinephrine was increased fourfold whereas circulating and myocardial norepinephrine were unchanged. These data demonstrate a down-regulation of the left ventricular beta-adrenergic receptor/adenylate cyclase system during chronic hypoxemia secondary to an intracardiac right-to-left shunt.     

Retinol and retinyl esters in rabbit bone marrow and blood leukocytes

Skrede B, Olafsdottir AE, Blomhoff R, Norum KR. Retinol and retinyl esters in rabbit bone marrow and blood leukocytes. Scand J Clin Lab Invest 1993; 53: 515-519.

Due to the well documented effects of retinoids on growth and differentiation of some leukaemic cells in vivo and in vitro, we measured the amount of retinol and retinyl esters in bone marrow, blood leukocytes, and liver in rabbits fed large doses of retinyl palmitate. Both Chinchilla rabbits and Watanabe Heritable Hyperlipidaemic rabbits which lack functional low density lipoprotein receptors, were fed 26 μmoles (25.000 IU) of retinyl palmitate daily for 8 weeks. The animals stored retinoids in large amounts in the liver, whereas only minor amounts were stored in bone marrow. More than 97% of the retinoids in the liver was esterified, while most of the retinoids in bone marrow were unesterified.

We also studied the post-prandial increase in chylomicron associated retinyl esters in rabbit leukocytes in vivo. After administering an oral load of 26μmoles retinyl palmitate, retinoids increased four-fold in blood leukocytes after 5h. There was almost no difference in retinoid uptake in leukocytes in Watanabe Heritable Hyperlipidaemic rabbits compared to normal rabbits, suggesting that chylomicron remnant retinyl esters are taken up in peripheral blood leukocytes independently of the low density lipoprotein receptor.

In conclusion, bone marrow does not store high amounts of retinoids, and retinyl ester transport and storage appear normal in absence of functional low density lipoprotein receptors.     

Castration blocks chronic sulpiride-induced desensitization of striatal D1 receptor-stimulated adenylate cyclase activity in male rats

Gonadal hormones have been shown to modulate adaptive responses of the mesostriatal dopaminergic system to antipsychotic challenge. We examined the role of endogenous gonadal steroids in the regulation of D1 receptor function after chronic treatment with sulpiride, a D2 specific antagonist. Chronic sulpiride treatment induced a desensitization of striatal D1 receptor-simulated adenylate cyclase activity in intact male rats with no change in the number of D1 or D2 receptors. This desensitization of D1-stimulated adenylate cyclase activity was expressed as a decrease in Vmax with no change in the activation constant. Castration of male rats blocked the chronic sulpiride-induced desensitization of D1 receptor function. Castration of male rats also resulted in a decrease in the number of D1 receptors as measured by [3H]SCH23390 binding. Ovariectomy of female rats had no effect on striatal D1 receptor-stimulated adenylate cyclase activity. Preliminary studies showed no effect of chronic sulpiride treatment on D1 receptor function in intact or ovariectomized female rats. We conclude that testicular hormones have a permissive effect on the expression of the chronic sulpiride-induced desensitization of D1 receptor function.     

Effects of acute ischemia in the dog on myocardial blood flow, beta receptors, and adenylate cyclase activity with and without chronic beta blockade.

We ligated the left anterior descending coronary artery for 1 or 2 h in 31 purebred beagles. We did not detect any changes in beta-adrenergic receptor density or affinity when normal and ischemic zones were compared, either in the subendocardium or in the subepicardium. In the ischemic zones, there was a significant decline in all measures of adenylate cyclase activity, including activity mediated by the beta-adrenergic receptor. By contrast, after chronic beta-adrenergic blockade (1.5 mg/kg propranolol i.v. twice daily for 7 d), there was an increase in adenylate cyclase activity stimulated by (-)-isoproterenol relative to adenylate cyclase activity stimulated by guanyl-5'imidodiphosphate (GppNHp) in both normal and ischemic tissue, suggesting that one effect of chronic beta blockade may be to enhance coupling between the stimulatory guanine nucleotide regulatory protein (Gs) and the beta-adrenergic receptor, despite a reduction in the number or function of Gs units. Chronic beta blockade also led to up regulation of beta-adrenergic receptor density in subepicardial regions. After 20 min of reperfusion following 2 h of ischemia, adenylate cyclase activity tended to return to control levels, particularly in the subepicardium, where (-)-isoproterenol-stimulated adenylate cyclase activity was not different from normal myocardium. We conclude that chronic beta-adrenergic blockade may have beneficial effects during prolonged episodes of myocardial ischemia by preserving signal transduction mediated by the beta-adrenergic receptor.     

Inhibition of tissue factor limits the growth of venous thrombus in the rabbit

Summary.  Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring ¹²⁵I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.     

Uptake of chylomicron remnant retinyl esters in human leukocytes in vivo

Retinoids have been successfully used in the treatment of some forms of leukaemia, suggesting that such cells have an efficient uptake mechanism for circulating retinoids. Therefore, we have studied the uptake of lipoprotein-associated retinyl esters in human leukocytes in vivo. After an oral load of 100 mumol retinyl palmitate (30,000 retinol equivalents) per square meter given to healthy adults, the concentration of retinoids in circulating leukocytes was determined. A peak was measured after 5 h, which coincided with a peak of retinyl esters in plasma. To test whether low-density lipoprotein receptors are necessary for the postprandial uptake of retinoids, we studied retinoid uptake in leukocytes from two patients homozygous for familial hypercholesterolaemia. After an oral load of retinoids we found that leukocytes from these patients took up at least as much retinoid as leukocytes in normal individuals, suggesting that uptake of chylomicron remnant retinyl esters may proceed independent of the low-density lipoprotein receptor. The expression of mRNA for the low density lipoprotein receptor-related protein, which is a putative chylomicron remnant receptor, was similar in leukocytes from a patient homozygous for familial hypercholesterolaemia and normal individuals. Six hours after vitamin A administration, recovery of unesterified retinol was 71% in normal leukocytes, however, only 9% unesterified retinol was recovered in leukocytes from the two patients with familial hypercholesterolaemia. Thus, the apparent rate of retinyl ester hydrolysis was markedly reduced in leukocytes from these patients, indicating different intracellular traffic of chylomicron remnants in normal individuals and patients homozygous for familial hypercholesterolaemia.     

Leukocyte Glucocerebrosidase Deficiency Diagnostic in Adult Gaucher's Disease with Negative Bone Marrow Biopsy. Some Properties of the Enzyme in Leukocytes and Spleen

Abstract. A sixty-one year-old man with Gaucher's disease is described. The disease was manifested by splenomegaly, hypersplenism and elevations of serum acid phosphatase and immunoglobulin but no bone lesions were roentgenologically demonstrable and bone marrow biopsy failed to reveal Gaucher cells. The diagnosis was established prior to splenectomy by the finding of decreased β-D-gIucoside glucohydrolase (β-D-GGH) activity in his peripheral leukocytes. Examination of the surgically removed spleen revealed Gaucher cells, a large excess of glucocerebroside and decreased β-D-GGH activity. Splenectomy was followed by normalization of haemoglobin, white cell and platelet counts and of the serum acid phosphatase and immunoglobulin levels. The patient's leukocyte β-D-GGH activity showed an optimum pH similar to that of normal leukocyte enzyme, about 5.4, using both glucocerebroside (Glc-Cer) and 4-methylumbelliferyl-β-D-glu-copyranoside (MU-Glc) as substrate. The patient's spleen β-D-GGH activity showed a pH optimum of about 4.5, which differed slightly from that of normal spleen enzyme. The Km values of β-D-GGH in the patient's leukocytes for Glc-Cer and MU-Glc were 1.35 × 10^-4 M and 3.6 × 10^-3 M, respectively, i.e. in the normal range. The patient's splenic tissue Km values were for Glc-Cer and MU-Glc 0.52×10^-4M and 5.8×10^-3 M, respectively, as compared to 0.30×10^-4 M and 5.0 × 10^-3 M in a normal control.     

Chronic norepinephrine elicits desensitization by uncoupling the beta-receptor.

The goal of this study was to determine the mechanism of beta-adrenergic receptor desensitization after chronic elevation of circulating NE levels. Osmotic minipumps containing either NE or saline were implanted subcutaneously in dogs for 3-4 wk. Physiologic desensitization to isoproterenol was confirmed in conscious dogs, i.e., left ventricular dP/dt increased in response to isoproterenol (0.4 micrograms/kg per min) by 5,625 +/- 731 mmHg/s in control dogs with saline pumps, and significantly less, P less than 0.01, by 2,093 +/- 263 mmHg/s in dogs with NE pumps. Myocardial beta-adrenergic receptor density as determined with 125I-cyanopindolol binding was 49% higher (p less than 0.05) in the NE pump group. However, beta-adrenergic receptor agonist binding with isoproterenol demonstrated a significant shift into the low affinity state for the animals with NE pumps. Basal, GTP plus isoproterenol, 5'-guanylylimidodiphosphate, sodium fluoride, and forskolin-stimulated adenylate cyclase activity in the NE pump group were significantly depressed (P less than 0.05) by amounts ranging from 20 to 40%. The functional activity of the guanine nucleotide binding protein Gs was also reduced (P less than 0.05) in animals with NE pumps. Thus, the process of desensitization in response to chronic elevation of NE levels in intact, normal dogs does not involve a decrease in beta-adrenergic receptor density. Rather, it is characterized by reduced adenylate cyclase activation and uncoupling of the beta-adrenergic receptor in association with decreased activity of the GTP-coupling protein Gs.     

Reciprocal regulation of platelet responses to P2Y and thromboxane receptor activation

Summary.  Background:  Thromboxane A₂ and ADP are two major platelet agonists that stimulate two sets of G protein-coupled receptors to activate platelets. Although aggregation responses to ADP and thromboxane desensitize, there are no reports currently addressing whether activation by one agonist may heterologously desensitize responses to the other. Objectives:  To demonstrate whether responses to ADP or U46619 may be modulated by prior treatment of platelets with the alternate agonist, revealing a level of cross-desensitization between receptor systems. Results:  Here we show that pretreatment of platelets with either agonist substantially desensitizes aggregation responses to the other agonist. Calcium responses to thromboxane receptor activation are desensitized by preactivation of P2Y₁ but not P2Y₁₂ receptors. This heterologous desensitization is mediated by a protein kinase C (PKC)-independent mechanism. Reciprocally, calcium responses to ADP are desensitized by pretreatment of platelets with the thromboxane analogue, U46619, and P2Y₁₂-mediated inhibition of adenylate cyclase is also desensitized by pretreatment with U46619. In this direction, desensitization is comprised of two components, a true heterologous component that is PKC-independent, and a homologous component that is mediated through stimulated release of dense granule ADP. Conclusions:  This study reveals cross-desensitization between ADP and thromboxane receptor signaling in human platelets. Cross-desensitization is mediated by protein kinases, involving PKC-dependent and independent pathways, and indicates that alterations in the activation state of one receptor may have effects upon the sensitivity of the other receptor system.     

Stress-induced 5-HT1A receptor desensitization: protective effects of Ginkgo biloba extract (EGb 761)

Summary— The effects of sub-chronic cold stress on the functioning of hippocampal 5-HT_1A receptors in old isolated rats and the possible protective effects of Ginkgo biloba extract (EGb 761) were investigated. Cold exposure during five days, produced a significant reduction of the inhibitory effect of 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity. In contrast, neither the affinity nor the density of hippocampal [³H]8-OH-DPAT binding sites were affected indicating that the reduced sensitivity of 5-HT_1A receptors induced by stress is probably due to a modification of their coupling mechanisms to adenylyl cyclase. The stress-induced desensitization of 5-HT_1A receptors was prevented by the administration of EGb 761 (50 mg/kg per os /14 days). These results clearly indicate that 5-HT_1A receptors are desensitized by stress and point out the reduced capacity of old rats to cope with the adverse effects of a chronic stressor. EGb 761 appears to restore the age-related decreased capacity to adapt to a chronic stressor.     

Abnormality of adenylate cyclase regulation in human platelet membranes in renal insufficiency

Adenylate cyclase in human platelets is under dual control of prostaglandins (PGI2 and PGE1) and catecholamines. The adenylate cyclase complex in membranes of platelets from ten patients with uraemia was investigated. The activation of the platelet cyclase by PGE1 is increased in the uraemic state, Vmax 4436 +/- 607 pmol cAMP mg-1 15 min-1. In the normal state Vmax is 2098 +/- 309 pmol cAMP mg-1 15 min-1. The alpha 2-adrenergic receptor was assayed with 3H-yohimbine binding. The density of receptors was equal in the uraemic (175 fmol mg-1 membrane protein) and the normal (170 fmol mg-1 membrane protein) states. Norepinephrine/3H-yohimbine competition binding revealed that catecholamines were bound with normal affinity in platelets in uraemia. Yet the inhibition of adenylate cyclase through the alpha 2-adrenergic receptor was diminished since Vmax values of adenylate cyclase with PGE1 and PGE2 + norepinephrine did not significantly differ. In the normal state, norepinephrine significantly (P less than 0.05) inhibited the PGE1 stimulated cyclase. It is concluded that platelet adenylate cyclase in the uraemia has an increased capacity for activation which is the result of both a sensitized stimulatory mechanism (prostaglandin mediated) and a deficient inhibitory mechanism (catecholamine mediated). It is suggested that a defect exists in the inhibitory nucleotide binding protein (NI) which is the coupling unit between the adenylate cyclase catalytic subunit (C).     

Adenylate cyclase activity and cAMP content of human platelets in uraemia

Abstract. We investigated the effects of uraemia and haemodialysis on the basal activity of adenylate cyclase and the cyclic-AMP content of human platelets in patients with end-stage renal insufficiency, patients receiving maintenance haemodialysis, and as controls healthy voluntary subjects.
Basal adenylate cyclase activity in terminal renal disease (creatinine clearance less than 15 ml/min/1·73 m²) was 824 ± SEM 57, in comparison to the healthy subjects with 453 ± SEM 28 ( P < 0·001). We also found significant elevation ( P < 0·001) of platelet cAMP levels as compared to the controls. Basal adenylate cyclase activity and platelet cAMP levels were approximately normal in the dialysed patients.
These results show that uraemic toxins adversely affect the platelet AC-cAMP system, possibly causing impaired platelet aggregation and the bleeding diathesis of uraemia.     

Development of functional dependence on ethanol in dopaminergic systems

Withdrawal of mice from chronic ethanol treatment results in a decreased responsiveness of striatal (but not mesolimbic) dopamine-sensitive adenylate cyclase activity to stimulation by dopamine. This subsensitivity is not apparent at the time of withdrawal from chronic feeding of ethanol, when animals are still intoxicated, but becomes evident as ethanol is eliminated from the animals. Addition of ethanol in vitro to tissue homogenates from ethanol-withdrawn animals, at concentrations similar to those found in brain at the time of withdrawal, normalizes the response of the adenylate cyclase to dopamine. No difference is evident between control and ethanol-withdrawn animals in stimulation of adenylate cyclase by sodium fluoride. The specificity of the response of striatal adenylate cyclase to stimulation by dopamine, as compared to other transmitters, is unaltered by chronic ethanol feeding. Chronic treatment with ethanol and withdrawal also does not affect the specific binding of spiroperidol in either striatal or mesolimbic regions. It is suggested that the decreased response of adenylate cyclase to dopamine in ethanol-withdrawn animals results from decreased efficiency of coupling between dopamine "receptor" sites and catalytic units of adenylate cyclase.     

Beta Adrenergic Receptors of Polymorphonuclear Particulates in Bronchial Asthma

We have tested the beta adrenergic receptor theory of bronchial asthma by determining the number and affinity of binding sites of the beta adrenergic radioligand [(3)H]dihydroalprenolol (DHA) and the activity of adenylate cyclase in broken cell preparations of polymorphonuclear leukocytes (PMN). We studied 31 control subjects (group 1), 30 asthmatics receiving no systemic adrenergic medication (group 2), and 17 asthmatics receiving adrenergic agonists systemically (group 3). Control subjects and asthmatics taking no adrenergic drugs bound similar amounts of DHA at 0.5 nM and 30 nM DHA and had about 900 binding sites per PMN. In contrast, asthmatics receiving adrenergic agonists had a >70% decrease in their number of DHA binding sites per PMN (254+/-57). In a subset of our three groups of subjects (eight from group 1, six from group 2, and five from group 3) we measured DHA binding at several DHA concentrations and found similar values (0.4-0.7 nM) for the dissociation constant of DHA among these subjects.In further studies we examined the interaction of the agonist (-)-isoproterenol with beta adrenergic receptors in 8 normal subjects and 10 asthmatics not receiving adrenergic medication. We tested the ability of isoproterenol to compete for DHA binding sites and to stimulate adenylate cyclase in sonicates prepared from PMN and examined under identical conditions. The dissociation constants for the competition of isoproterenol for DHA binding sites in normal and asthmatic subjects were virtually identical ( approximately 1.0 muM). In addition, the (activation constant) values for stimulation of adenylate cyclase were similar (0.16-0.19 muM) in the two groups of subjects.Thus, these data suggest that asthma per se is not associated with alteration in either the number or affinity of beta adrenergic receptors in PMN. Our findings indicate that previous reports of abnormal beta adrenergic receptor function in asthmatic patients may in part be explained by prior treatment of such patients with adrenergic agonists. Because the asthmatics who received adrenergic agonists in our study tended to be more ill and to receive additional medication compared to subjects in group 2, we cannot rule out unequivocally that severe asthma may be associated with decreased binding to beta adrenergic receptors. Nevertheless, we conclude that beta adrenergic receptors on PMN from asthmatics are relatively normal unless such patients are treated with adrenergic agonists.     

Thyroid-stimulating immunoglobulins in insulin-dependent diabetes mellitus

Abstract. Increased frequencies of thyroid diseases and thyroid microsomal antibodies have been observed in insulin-dependent diabetes mellitus. However, the exact prevalence of thyroid-stimulating immunoglobulins has not been established. In the present study these antibodies were measured by both a radioreceptor and an adenylate-cyclase stimulation assay.
In forty-six patients with insulin-dependent diabetes mellitus without endogeneous insulin production (C-peptide concentration ≤ 0·06 nmol l^-1) the receptor assay was positive in ten and the stimulation assay in fifteen patients. The immunoglobulins of four patients inhibited the adenylate cyclase, and one of these was positive in the receptor assay. In nine patients with post-prandial C-peptide 0·07–0·19 nmol l^-1, five had adenylate-cyclase-stimulating antibodies, while none were positive in the receptor assay. Thyroid hormones and thyrotropin concentrations were not different in the forty-six patients without endogenous insulin production with thyroid-stimulating immunoglobulins compared with patients without these antibodies. Patients with thyroid-stimulating immunoglobulins required a daily median amount of 0·71 IE of insulin kg^-1 compared to median of 0·57 IE kg^-1 in patients without these antibodies ( P < 0·03), despite a similar degree of diabetic regulation.
The level of tri-iodothyronine was correlated to the antibody level in patients with adenylate-cyclase-stimulating antibodies. While the prognostic and possibly pathogenetic importance of these antibodies in Graves' disease have been established, their significance in insulin-dependent diabetes mellitus remains to be demonstrated.     

Impaired cardiac muscarinic receptor function in dogs with heart failure.

Prior physiological studies have suggested that parasympathetic control is altered in heart failure. The goal of our studies was to investigate the influence of heart failure on the muscarinic receptor, and its coupling to adenylate cyclase. Ligand binding studies using [3H]quinuclidinyl benzilate and enriched left ventricular (LV) sarcolemma, demonstrated that muscarinic receptor density in heart failure declined 36% from a control of 5.6 +/- 0.6 pmol/mg, with no change in antagonist affinity. However, agonist competition studies with both carbachol and oxotremorine showed that it was a loss of high affinity agonist binding sites in the sarcolemma from failing LV that accounted for this difference. The functional efficacy of the muscarinic receptor was also examined. When 1 microM methacholine was added to 0.1 mM GTP and 0.1 mM isoproterenol, adenylate cyclase stimulated activity was inhibited by 15% in normal LV but only 5% in LV sarcolemma from animals with heart failure even when the reduced adenylate cyclase in these heart failure animals was taken into account. Even at 100-fold greater concentrations of methacholine, significantly less inhibition of adenylate cyclase activity was observed in LV failure as compared with normal LV sarcolemma. Levels of the GTP-inhibitory protein known to couple the muscarinic receptor to adenylate cyclase, as measured with pertussis toxin labeling, were not depressed in LV failure. Thus, the inhibitory pathway regulating LV adenylate cyclase activity is defective in heart failure. The decrease in muscarinic receptor density, and in particular the specific loss of the high affinity agonist binding component of this receptor population, appears to be the major factor underlying this abnormality.     

Canine renal receptors for parathyroid hormone. Down-regulation in vivo by exogenous parathyroid hormone.

Chronic elevation of circulating parathyroid hormone (PTH) is associated with decreased target cell responsiveness to PTH. To study the subcellular mechanism of this phenomenon we evaluated PTH receptors and adenylate cyclase activity in renal cortical membranes prepared before and after infusion of bovine parathyroid gland extract (PTE) into thyroparathyroidectomized dogs. PTE infusion resulted in a 53% decrease in the number of high-affinity receptors (P less than 0.01) associated with a 66% decrease in PTH-stimulated adenylate cyclase (P less than 0.01) relative to paired base-line values. Both the equilibrium constant of dissociation (KD) for PTH binding and the concentration of PTH that caused half-maximal stimulation of adenylate cyclase were in the range of 1 to 4 nM, and were unaffected by the PTE infusion. Responsiveness of the renal adenylate cyclase to sodium fluoride was 88% of base-line values. Infusion of the PTE vehicle alone did not affect PTH receptor number or blunt the adenylate cyclase response to PTH. Pretreatment of the membranes made after PTE infusion with guanosine triphosphate (GTP), which is known to produce dissociation of receptor-bound PTH, failed to restore either receptor number or PTH-stimulated adenylate cyclase. This finding was not due to a lack of efficacy of the GTP pretreatment, because identical GTP pretreatment restored PTH binding to base-line values in membranes partially occupied by incubation with PTH in vitro. Thus, simple residual occupancy of PTH receptors by the infused hormone did not appear to account for the observed receptor loss. The results of this study suggest that target cell resistance to PTH in patients with hyperparathyroidism might occur, at least in part, due to down-regulation of PTH receptors by circulating hormone.