Basal ganglia calcification in hypoparathyroidism and pseudohypoparathyroidism: local and systemic metabolic mechanisms.

BACKGROUND: Hypoparathyroidism and pseudohypoparathyroidism are rare disorders of mineral metabolism which may be associated with soft tissue calcification in the basal ganglia in the brain, and occasionally the skin and other tissues. The basal ganglia are the most common sites of calcification in the central nervous system in these disorders, and were first associated with this manifestation in a report from the Mayo Clinic in 1939. The reasons why the basal ganglia are a common site of soft tissue calcification in these rare disorders has been a matter of investigation for many years. FINDINGS: Due to recent increased understanding of phosphate transport and new insights gained from mRNA expression in the basal ganglia, the pathophysiology of basal ganglia calcification (BGC) is now clearer. There is evidence that the absence of parathyroid hormone in hypoparathyroidism may play a direct role, but this is clearly not the case in pseudohypoparathyroidism, which is associated with increased parathyroid hormone levels. Maintaining the calcium/phosphorus ratio as close to normal as possible, and maintaining normal serum phosphate levels, may help mitigate the progression of BGC. There is no evidence of regression of BGC with conventional treatment, and long-term data with adjunctive or replacement therapy with parathyroid hormone or its analogues are not yet available. PURPOSE OF THE REVIEW: This review will focus on the pathophysiology of BGC in hypoparathyroidism and pseudohypoparathyroidism, and review the proposed pathophysiologic mechanisms, as well as the clinical implications of BGC on patient quality of life.

Primary hyperparathyroidism: review and recommendations on evaluation, diagnosis, and management. A Canadian and international consensus.

The purpose of this review is to assess the most recent evidence in the management of primary hyperparathyroidism (PHPT) and provide updated recommendations for its evaluation, diagnosis and treatment. A Medline search of "Hyperparathyroidism. Primary" was conducted and the literature with the highest levels of evidence were reviewed and used to formulate recommendations. PHPT is a common endocrine disorder usually discovered by routine biochemical screening. PHPT is defined as hypercalcemia with increased or inappropriately normal plasma parathyroid hormone (PTH). It is most commonly seen after the age of 50 years, with women predominating by three to fourfold. In countries with routine multichannel screening, PHPT is identified earlier and may be asymptomatic. Where biochemical testing is not routine, PHPT is more likely to present with skeletal complications, or nephrolithiasis. Parathyroidectomy (PTx) is indicated for those with symptomatic disease. For asymptomatic patients, recent guidelines have recommended criteria for surgery, however PTx can also be considered in those who do not meet criteria, and prefer surgery. Non-surgical therapies are available when surgery is not appropriate. This review presents the current state of the art in the diagnosis and management of PHPT and updates the Canadian Position paper on PHPT. An overview of the impact of PHPT on the skeleton and other target organs is presented with international consensus. Differences in the international presentation of this condition are also summarized.

Long-term outcomes in patients with multiple endocrine neoplasia type 1 and pancreaticoduodenal neuroendocrine tumours.

BACKGROUND: In patients with multiple endocrine neoplasia type 1 (MEN-1), pancreaticoduodenal (PD) neuroendocrine tumours (NETs) are associated with early mortality, yet the best treatment strategy remains uncertain. AIM: To assess patient important outcomes (mortality and metastasis) of PD-NETs and predictors of outcomes in patients with MEN-1. METHODS: Retrospective cohort of patients with MEN-1 who attended the Mayo Clinic, Rochester, MN from 1997 to 2014. RESULTS: We identified 287 patients with MEN-1; 199 (69%) patients had 217 PD-NETs. Among those with a PD-NETs, 129 (65%) had surgery of which 90 (70%) had their primary surgery performed at Mayo Clinic. The median postoperative follow-up was 8 years during which 13 (14%) patients died. The mean (±standard deviation) age of death was 51 (±9) years. Tumour size, metastasis at surgery or tumour type were not predictive of mortality, but for every year older at surgery, the odds of metastasis increased by 6%. Surgery was not performed in 70 (35%) patients. Among those who were observed/medically managed without known metastatic disease, mean tumour growth was 0·02 cm/year (range, -0·13-0·4 cm/year). Four patients (7%) died at a median age of 77 (range, 51-89) years. CONCLUSION: PD-NETs are common in patients with MEN-1 and are associated with early mortality even after surgical intervention. Active surveillance is a viable option in nonaggressive PD-NETs, although definitive factors identifying such patients are lacking. Therefore, counselling regarding risks and benefits of current treatment options remains integral to the care of patients with MEN-1.

Ossification of the posterior longitudinal ligament in vitamin D-resistant rickets: case report and review of the literature.

STUDY DESIGN: A case report of cervical myelopathy caused by ossification of the posterior longitudinal ligament in a patient with vitamin D-resistant rickets is presented together with a review of literature. OBJECTIVE: To report the diagnosis of ossification of the posterior longitudinal ligament in a white woman with vitamin D-resistant rickets. SUMMARY OF BACKGROUND DATA: The association between ossification of the posterior longitudinal ligament and untreated vitamin D-resistant rickets has been reported in Japan, but infrequently in white populations. In whites, ossification of the posterior longitudinal ligament is closely associated with diffuse idiopathic skeletal hyperostosis. A clear association between ossification of the posterior longitudinal ligament and vitamin D-resistant rickets in white populations has not yet been established. METHODS: The medical record and imaging studies of a patient treated at the authors' institution for cervical myelopathy caused by ossification of the posterior longitudinal ligament in the setting of treated vitamin D-resistant rickets were reviewed. A Medline search of the medical literature between 1966-1999 was performed to identify pertinent studies and similar case reports. RESULTS: The occurrence of spinal stenosis in untreated adults with vitamin D-resistant rickets has been reported in all regions of the spine in Japanese patients. The association between ossification of the posterior longitudinal ligament and untreated vitamin D-resistant rickets was first reported in Japan, where ossification of the posterior longitudinal ligament is endemic. This association may be incidental, because reports on ossification of the posterior longitudinal ligament in whites are not as frequent as in Japanese, reflecting the higher prevalence of this condition in Japan. CONCLUSION: Ossification of the posterior longitudinal ligament and ossification of the posterior longitudinal ligament associated with deranged calcium or phosphate metabolism may be different pathologic entities sharing a common outcome. Adequate treatment of vitamin D-resistant rickets may not always prevent or reverse ossification of the posterior longitudinal ligament.

Diabetes insipidus and anterior pituitary insufficiency as presenting features of Wegener's granulomatosis.

Central diabetes insipidus (DI) is a rare complication of Wegener's granulomatosis (WG), which usually presents after pulmonary or kidney involvement. Anterior pituitary dysfunction secondary to WG has been extremely rare, documented in only three cases. We report a case of a 47-year-old postmenopausal woman who was diagnosed with hypopituitarism in November 1999 and started on vasopressin, thyroxine, and hydrocortisone. She sought treatment at the Mayo Clinic in February 2000 with a purpuric rash, fever, cough, shortness of breath, and blood in the sputum. Computed tomography of the chest showed a 6-cm irregular mass in the right lower lobe, and a biopsy of the mass showed marked reactive atypia and necrosis. Positive C-antineutrophil cytoplasmic antibodies (ANCA) and skin biopsy of a purpuric lesion showing leukocytoclastic vasculitis confirmed the diagnosis of WG. Hormonal studies showed low gonadotropins, thyroid-stimulating hormone (TSH), and prolactin. Magnetic resonance imaging (MRI) of the head showed cystic enlargement of the pituitary gland that did not enhance with gadolinium. Two months into the treatment with cyclophosphamide and prednisone, she had persistent pituitary dysfunction, despite the normal appearance of the pituitary gland on repeat MRI. We conclude that WG should be included in the differential diagnosis of DI and anterior pituitary dysfunction in the proper clinical setting. Early diagnosis and treatment may be crucial in preventing pituitary gland destruction and long-term endocrine sequelae. We suggest screening for anterior pituitary failure in the presence of the WG-associated DI.

Adrenocorticotropic hormone controls Concanavalin A activation of rat lymphocytes by modulating IL-2 production.

The initiation of DNA synthesis and secretion of Interleukin 2 (IL-2) was measured in isolated rat splenic lymphocytes following activation with Concanavalin A (ConA). The extent of 3H-thymidine incorporation into activated cells was tested when cultured with various concentrations of Adrenocorticotropic hormone (ACTH). A paradoxical dose-response curve resulted when ACTH caused a biphasic response of augmenting and inhibiting 3H-thymidine uptake in lymphocytes depending on the hormone concentration. Low levels of ACTH (0.001-1-nM) augmented 3H-thymidine uptake and high levels (10-1000 nM) reversed the effect. The optimal ACTH concentration was 10 pM ACTH in the presence of 5 ug/ml ConA and there was no ACTH effect on quiescent cells (no ConA). Conditioned media from splenic lymphocytes treated with various concentrations of ConA or ACTH was tested for increased uptake of 3H-thymidine by the IL-2 growth dependent Cytotoxic T Lymphocyte Leukemia (CTLL-2) cells. ConA conditioned medium could sustain the CTLL-2 cells indicating the presence of IL-2. Conditioned medium from splenic lymphocytes treated with both ConA and 100 pM ACTH further increased CTLL-2 cell proliferation indicating an additional increase of IL-2 secretion. The identity of IL-2 was confirmed by using an anti-rat IL-2 antibody to neutralize the growth potential of the conditioned medium. ACTH alone had no effect on the CTLL-2 cell proliferation indicating the effect is due solely to induced IL-2 found in the conditioned medium. IL-2 levels in the conditioned media were quantitated by ELISA assay; splenic lymphocytes produced 4.2 ng/ml to ConA only, 19.2 ng/ml in ConA plus 10 nM ACTH, and no detectable IL-2 at ConA plus 10 uM ACTH. These results demonstrated that ACTH modulates IL-2 secretion from activated lymphocytes, which is both biphasic and concentration dependent.

Osteomalacia associated with adult Fanconi's syndrome: clinical and diagnostic features.

OBJECTIVE: Osteomalacia associated with adult acquired Fanconi's syndrome is thought to result from hypophosphataemia and relative 1,25-dihydroxyvitamin D deficiency. We have followed the clinical and diagnostic features of patients with osteomalacia associated with adult Fanconi's syndrome, with particular emphasis on their responses to treatment with calcium, phosphate and vitamin D. DESIGN: Retrospective Mayo Clinic case-note review from 1975 to 1994 and prospective follow-up study, combined with literature review. PATIENTS: Eleven patients (7 male, 4 female) were identified who satisfied criteria for diagnosis of osteomalacia and adult Fanconi's syndrome. Twenty-five additional patients were identified in a literature review from 1954 to the present. METHODS: Clinical history and physical examination, serum and urine bone and mineral parameter, X-ray radiography and iliac crest bone histomorphometry. RESULTS: All patients presented with typical symptoms of osteomalacia, including lower extremity or low back bone pain, and all had fractures, pseudofractures, and/or bone demineralization on X-ray radiography. Osteomalacia and Fanconi's syndrome were diagnosed concurrently in 10 patients, whereas osteomalacia preceded diagnosis of Fanconi's syndrome by 5 years in one patient. Pre-treatment bone biopsies in 9 of the 11 patients demonstrated increased osteoid surface, volume and width. In the one patient labelled with tetracycline prior to biopsy, mineralization lag time was prolonged at 111 days (normal 19.2 +/- 1.0 days). Hypophosphataemia, inappropriately low 1,25-dihydroxyvitamin D levels, renal insufficiency, and chronic acidosis due to bicarbonate leak and uraemia, contributed to the osteomalacia in these patients. Secondary hyperparathyroidism was present in two patients. Eight of the 11 patients with osteomalacia associated with Fanconi's syndrome had monoclonal disorders, including multiple myeloma or lymphoma, many of them manifest by light-chain proteinuria. Over a mean patient follow-up period of 46 months (range 1-239 months), patients responded symptomatically to calcium, phosphate, and vitamin D replacement typically within 1-6 months. In 8 patients in whom follow-up data were available, post-treatment serum phosphate and 1,25-dihydroxyvitamin D levels improved in the setting of stable mild renal insufficiency; only one patient developed end-stage renal failure after 20 years, suggesting that these patients do not invariably progress rapidly to renal failure. CONCLUSIONS: Regardless of the underlying cause, osteomalacia associated with adult acquired Fanconi's syndrome appears to respond well to calcium, phosphate, and vitamin D replacement. These patients do not appear to necessarily require 1,25-dihydroxyvitamin D replacement.

Calcium uptake by ACTH-stimulated lymphocytes: what is the physiological significance?

Adrenocorticotropic hormone (ACTH) increases cAMP and cGMP concentrations in both adrenal and lymphoid cells, and requires extracellular Ca to have biological activity. The requirement for Ca has been difficult to characterize in terms of the channel identity and whether the committing step for steroidogenesis in the adrenal cells requires Ca. In lymphocytes, ACTH has a biphasic effect on functions such as proliferation and immunoglobin secretion. Current information is consistent with suppressive effects of high ACTH concentrations being mediated by cAMP. Stimulatory effects of ACTH concentrations are hypothesized to be mediated by Ca uptake. This review will discuss the localization of Ca signals to discrete domains within cells and the receptor- and tissue-specificity of their subcellular distribution. Considering the diversity of possible mechanisms, a hypothesis for the role of ACTH-stimulated Ca uptake during mitogen activation of T-cell lymphocytes will be presented.

Adrenocorticotropic hormone stimulates a transient calcium uptake in rat lymphocytes.

Freshly isolated rat lymphocytes were tested for corticotropin (ACTH)-dependent calcium uptake. Physiological levels of corticotropin (0.01-1 nM) were found to stimulate both an uptake of 45Ca2+ and a rise in cAMP. The calcium uptake was delayed by 2 min after ACTH addition, but was rapid and transient after the onset of uptake. The extent of calcium uptake was dose dependent on the corticotropin concentration and reached a maximum by 1 nM. Several fragments of corticotropin were tested for activity; both full-length 1-39 and a functional truncated form, 1-25, had equivalent effects on 45Ca influx at 1 nM; however, alpha MSH-(1-13), ACTH-(11-24), or a mixture of alpha MSH and ACTH-(11-24) had no effect on 45Ca influx. Extracellular calcium uptake was blocked by the calcium channel blockers lanthanum, diltiazem, nifedipine, and omega-conotoxin. Splenic lymphocytes that express ACTH receptors had ligand-dependent calcium uptake, but thymocytes that lack ACTH receptors had no ligand-dependent calcium uptake. A mouse adrenal cell line, Y-1, showed the same 45Ca uptake kinetics. These findings demonstrate that both lymphocytes and adrenal cells have a functional ACTH-dependent calcium uptake mechanism.

Distribution of noncollagenous proteins in the matrix of adult human bone: evidence of anatomic and functional heterogeneity.

The microanatomic distribution of several noncollagenous proteins (NCPs) in bone matrix was examined by immunohistochemical analysis of glycol-methyl methacrylate-embedded normal adult human bone biopsies. Osteopontin and bone sialoprotein stained throughout the lamellae of both trabecular and cortical bone. Cement lines (cortical and trabecular) and the mineralized matrix immediately adjacent to each Haversian canal were intensely stained. Osteocalcin was detected in cement lines; however, lamellar staining varied depending on the location within the individual unit of bone. In cortical bone, the inner concentric lamellae of osteons were often unstained but the outer lamellae were heavily stained for osteocalcin. Osteonectin was not detected in cement lines and in most specimens revealed a pattern similar to that of osteocalcin with respect to the absence of immunostaining within the inner concentric lamellae. Decorin was prominent in the perilacunar matrix, the canaliculi of osteocytes, and the matrix immediately adjacent to quiescent Haversian canals. Biglycan appeared evenly distributed throughout cortical and trabecular bone matrix. These results suggest that the incorporation of NCPs into matrix may vary depending on the stage of formation of individual bone units. The specific distribution and spatial relationship of these NCPs may be related to the function of each protein during bone resorption and formation. The distinct patterns of NCP localization in bone support the hypothesis that in addition to their structural and mineral-inducing properties, these proteins may influence the events associated with bone remodeling, such as recruitment, attachment, differentiation, and activity of bone cells.

A monoclonal anti-peptide antibody mimics adrenocorticotropic hormone activity.

A monoclonal antibody that specifically recognizes the adrenocorticotropic receptor (ACTH) on rat adrenal cells was tested for hormonal activity. The antibody behaved as an agonist based on three different biological activities associated with ACTH. An antibody concentration of 16 micrograms/ml stimulated isolated rat adrenal cells to secrete 800 ng/10(4) cells of corticosterone with a concomitant 10-fold increase of cAMP to 30 pmol/10(5) cells. Antibody concentrations above 16 micrograms/ml inhibited mitotic activity in mouse Y-1 adrenal cells. A radio-immunoassay using an anti-ACTH antibody showed that the monoclonal anti-adrenocorticotropic receptor antibody and ACTH are antigenically related. These findings suggest that the anti-receptor antibody recognizes the ligand binding domain of the ACTH receptor.

Modulation of IgM secretion and H chain mRNA expression in CH12.LX.C4.5F5 B cells by adrenocorticotropic hormone.

The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP.

Steroidogenic activity of a peptide specified by the reversed sequence of corticotropin mRNA.

The molecular recognition theory predicts that a reversed (3'----5') reading of an mRNA should yield a peptide that is structurally and functionally similar to that specified in the 5'----3' direction. We tested this idea by synthesizing a corticotropin (ACTH) analogue using a reverse reading of bovine mRNA for ACTH-(1-24). This peptide, designated ACTH-3'----5', had a similar hydropathic profile to native ACTH-5'----3' but had only 30% sequence homology and eight different charge substitutions. ACTH-3'----5' specifically bound to the surface of mouse Y-1 adrenal cells and to polyclonal anti-ACTH antibody. Additionally, ACTH-3'----5' stimulated cAMP synthesis and steroidogenesis in adrenal cells. These findings show that ACTH-3'----5' mimics the corticotropic properties of native ACTH, thereby further validating the molecular recognition theory.

The surface activity of the same subpopulation of galactosyl receptors on isolated rat hepatocytes is modulated by colchicine, monensin, ATP depletion, and chloroquine.

In this study, we characterized and compared the ligand-independent loss of surface galactosyl (Gal) receptor activity on isolated rat hepatocytes treated with monensin, chloroquine, microtubule depolymerizing agents, or NaN3 and NaF at 37 degrees C. Freshly isolated hepatocytes exhibit predominately one subset of surface Gal receptors, termed State 1 receptors (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). During equilibration at 37 degrees C, these cells also express a second subset of Gal receptors at the surface, termed State 2 receptors, and routinely double their total surface Gal receptor activity. Following equilibration at 37 degrees C and then inhibitor treatment, hepatocytes bound 40-60% less 125I-asialoorosomucoid (ASOR) at 4 degrees C than did untreated cells. Treated cells maintained a basal nonmodulated level of surface receptor activity regardless of temperature, perturbant concentration, or incubation time. Loss of surface Gal receptor activity on cells treated with multiple inhibitors simultaneously or sequentially was not additive. Thus, all treatments affected the same subpopulation of surface Gal receptors. None of these inhibitors decreased surface State 1 Gal receptor activity, but all prevented the normal appearance of State 2 Gal receptors on freshly isolated cells during incubation at 37 degrees C. The endocytic capability of residual surface State 1 Gal receptors on inhibitor-treated cells varied depending on the inhibitor. Hepatocytes treated first at 24 degrees C or with colchicine at 37 degrees C internalized greater than 85% of surface-bound 125I-ASOR. In contrast, monensin- or chloroquine-treated cells internalized approximately 50% of surface-bound 125I-ASOR. Azide-treated cells internalized less than 20% of surface-bound 125I-ASOR. We conclude that only surface State 2 Gal receptor activity is sensitive to these various perturbants. State 1 Gal receptor activity is not modulated. These data are consistent with the conclusion that only State 2 Gal receptors constitutively recycle.

Differential expression of functional adrenocorticotropic hormone receptors by subpopulations of lymphocytes.

In an effort to investigate the presence of adrenocorticotropic hormone (ACTH) receptors on rat lymphocytes, cells were separated by a panning procedure into T and B cell populations. By using the radiolabeled ACTH agonist, (125I-Tyr23) phenylalanine2-norleucine4-ACTH1-24, substantial numbers of ACTH binding sites were detected on T and B lymphocytes, but not on thymocytes. Scatchard analysis revealed two types of binding sites on each cell population, one with Kd1 = 0.088 +/- 0.025 nM and one with Kd2 = 4.2 +/- 0.6 nM; however, the absolute number of binding sites per cell was different. B lymphocytes expressed approximately three times the number of Kd1 binding sites per cell when compared with T lymphocytes. However, ACTH receptor expression by these cell populations was not static as suggested by the ability to induce receptor expression via mitogens. B or T cells and thymocytes stimulated with the mitogens LPS or Con A, respectively, substantially increased their number of Kd1 binding sites per cell (approximately three-fold). Even more dramatic increases in Kd1 receptor expression (approximately 100-fold) were observed when comparing "normal" and stimulated thymocytes. To demonstrate that these ACTH binding sites were in fact functional, cAMP levels were measured in lymphocytes 10 min after exposure to varying concentrations of ACTH. Dose-dependent increases in cAMP levels were observed, with significant stimulation occurring with as little as 0.1 nM ACTH added. Taken together, these studies demonstrate the presence of functional ACTH receptors on normal, rat T and B lymphocytes.

Recycling of the asialoglycoprotein receptor in isolated rat hepatocytes. ATP depletion blocks receptor recycling but not a single round of endocytosis.

Continuous endocytosis of 125I-asialo-orosomucoid (ASOR) mediated by the galactosyl receptor in rat hepatocytes is a cyclic process. 125I-ASOR-receptor complexes are internalized, processed, and the ligand is degraded while the receptor is returned to the cell surface for reutilization. Since a true cycle has a thermodynamic requirement for the input of external energy, we examined the effects of changes in intracellular ATP levels on the function of the receptor cycle. Hepatocytes were depleted of ATP to various extents prior to endocytosis by incubating cells at 15 degrees C in the presence of 2 mM NaF and 0-20 mM NaN3. A luciferase-luciferin bioluminescence assay was used to quantitate the amount of cellular ATP. ATP-depleted cells were allowed to bind 125I-ASOR at 0 degrees C, washed through discontinuous Percoll gradients, and only viable cells were isolated and incubated at 37 degrees C to initiate a synchronous single round of endocytosis. The extent of internalization of this surface-bound 125I-ASOR was unaffected by an ATP depletion to less than 1% of the control level. The rate of internalization of surface-bound ligand was unaffected until the ATP levels decreased to 30% or less; at greater than 98% ATP depletion the initial rate decreased by a maximum of 55% and the kinetics became biphasic. In contrast, continuous endocytosis in the presence of excess ASOR was inhibited by only a 25% decline in cellular ATP content and demonstrated a very sharp threshold response to changing ATP levels. Continuous endocytosis, which requires receptor recycling, was completely inhibited when the total cellular ATP level decreased by only 40%. We conclude that the internalization phase of endocytosis is not dependent on ATP but that the processing and/or externalization phases of the complete receptor cycle are either directly or indirectly dependent on ATP and very sensitive to changes in cellular ATP content.