Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: antiproliferative effects of peptide analogues.

Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.

The management of diabetes insipidus in adults.

The management of patients with diabetes insipidus can be confusing because of the disorder's variable pathophysiology, the numerous medications used, and the possible complications related to their use. Nevertheless, the primary care physician, rather than the subspecialist, will increasingly be called on to manage patients with such relatively uncommon conditions in the future. If a few basic facts and principles are kept in mind, the care of most patients with diabetes insipidus can be successful. A comprehensive, practical review of the short- and long-term therapy for patients with diabetes insipidus, including central diabetes insipidus, nephrogenic diabetes insipidus, and the "excessive vasopressinase syndrome," is presented. The use of single and multidrug regimens, and of the newly marketed oral formulation of desmopressin acetate, is described for common clinical settings.

Silent thyrotoxic thyroiditis in association with chronic adrenocortical insufficiency.

A 23-year-old woman had silent thyrotoxic thyroiditis and chronic adrenocortical insufficiency (Addison's disease). Recent evidence suggests that silent thyroiditis is an autoimmune disease characterized by lymphocytic infiltration of the thyroid and by transient hyperthyroidism, followed occasionally by transient hypothyroidism and eventual recovery. This case would seem to expand the spectrum of autoimmune thyroid diseases (Graves' disease, lymphocytic [Hashimoto's] thyroiditis, and hypothyroidism) associated with chronic adrenocortical insufficiency.

Hyporeninemia and hypoaldosteronism in diabetes mellitus.

The changes in plasma renin activity (PRA) and plasma aldosterone concentration (PA) in response to postural stimuli were evaluated in 12 patients with stable diabetes mellitus and in five volunteers. Seven diabetic patients had hyperkalemia, and several had renal insufficiency and neurological complications. Five diabetics and had normal serum potassium concentration, a mean creatinine clearance within the normal range, and few complications. PRA and PA were measured in these patients and in the control subjects, all of whom were receiving a diet containing 10 mEq of sodium and 50 mEq of potassium while they were in a supine position, after they were tilted to a 90 degrees position, and after upright posture for two hours. The results indicate that impaired responsiveness of PRA and PA may occur in patients with complicated and those with uncomplicated diabetes and may be responsible in part for a relatively high prevalence of hyperkalemia especially in those diabetic patients with reduced renal function.

Decreased serum cholesterol-binding reserve in diabetes mellitus.

Serum cholesterol-binding reserve (SCBR), the capacity of a serum sample to solubilize additional cholesterol in excess of its cholesterol content, was measured in 43 white male patients with maturity-onset diabetes in the age range of 35--59 years who were under treatment with insulin. The values were compared with those of 194 nondiabetic controls of the same race, sex, and age range. The mean +/- S.D. of SCBR of patients (71.9 +/- 29.3 mg./dl.) was lower than that of controls (88.9 +/- 30.9 mg./dl.) (p less than 0.001). Age in the range of 35 to 59 years had no correlation with SCBR in either patients or controls. SCBR was positively correlated with serum levels of cholesterol (SC) and triglycerides (TG) in both patients and controls. After adjustment for SC and TG, the difference in SCBR between patients and controls persisted (p less than 0.001). In 15 of 20 (75 per cent) patient-control pairs matched for SC and TG to within 5 per cent, the patient had lower SCBR (paired t-test, p less than 0.002). In 16 patients without elevation of serum lipid levels (SC below 250 and TG below 150 mg./dl.), the mean +/- S.D. of SCBR (59.1 +/- 17.7 mg./dl.) was lower than that of 49 controls having serum lipids in the same range (77.4 +/- 31.7 mg./dl.) (p less than 0.03). These results indicate an association of decreased SCBR with diabetes and are consistent with the hypothesis that low SCBR is associated with accelerated atherosclerosis and enhanced risk for coronary heart disease.

Steroid enzyme activities in extraadrenal pheochromocytomas.

The adrenal medulla and tumors derived from it have been shown to be capable of converting radioactive steroid intermediates into glucocorticoid end products in vitro. This capacity for partial steroid synthesis was explored in two large extraadrenal pheochromocytomas and compared to the results of parallel studies with intraadrenal pheochromocytomas and adrenal cortex; in one experiment, all three types of tissue were obtained from a single patient and studied simultaneously. Slice preparations of extraadrenal pheochromocytoma transformed 14C-labeled pregnenolone (3 beta-hydroxypregn-5-en-20-one) into corticosterone and cortisol. Incubations of subcellular fractions demonstrated that, as in the adrenal cortex and intraadrenal chromaffin tissue, delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase, 17 alpha-hydroxylase, and 21-hydroxylase activities in extraadrenal pheochromocytomas were associated with the microsomes and 11 beta-hydroxylase was associated with the mitochondria, where metyrapone was an effective inhibitor, these findings tend to dissociate steroid-metabolizing activity in the adjacent chromaffin tissue perfused by glucocorticoid intermediates in high concentrations.

Dual sites of inhibition by metyrapone of human adrenal steroidogenesis: correlation of in vivo and in vitro studies.

In a patient with pituitary ACTH-dependent adrenal hyperplasia (AH), the standard oral metyrapone test resulted in a decrease in "apparent 11beta-hydroxylase activity" (-48%) accompanied by an increase in "apparent cholesterol cleavage activity" (+318%). When incubated adrenal mitochondria from this patient were studied, metyrapone inhibited both 11beta-hydroxylation of labeled 11-deoxycorticosterone and cleavage of labeled cholesterol, although at 0.1 and 1.0 mM metyrapone concentrations, depression of cholesterol cleavage (23 and 54%, respectively) was less than that of 11beta-hydroxylation (62 and 84%, respectively). The inhibition of cholesterol cleavage by metyrapone (26 and 62%, at 0.1 and 1.0 mM concentrations, respectively) was also demonstrable in adrenal mitochondria from a patient with hypercorticism resulting from an ACTH-independent adrenal adenoman (AA). Metyrapone administration to AA resulted in a significant depression of both 11beta-hydroxylase (-62%) and cholesterol cleavage (-36%) "apparent activities"; when metyrapone and ACTH were given together to this patient, however, only 11beta-hydroxylase "apparent activity" diminished (-26%), while cholesterol cleavage "apparent activity" was greatly augmented (+231%), thereby simulating the results of the standard metyrapone test in AH. These data demonstrate that metyrapone inhibits both mitochondrial reactions involved in cortisol synthesis--initial cholesterol cleavage and final 11beta-hydroxylation; these effects probably result from interference by this agent with the interaction between substrate and related cytochrome P - 450. Since ACTH has a major stimulatory effect on cholesterol cleavage but not on 11beta-hydroxylation, the outcome of metyrapone administration is thus determined by whether a change in ACTH level ensues: while 11beta-hydroxylation is inhibited by metyrapone under any circumstances, total steroid output rises when a compensatory ACTH increase overcomes metyrapone inhibition of cholesterol conversion into pregnenolone and falls when metyrapone inhibition of this reaction is unopposed.

Alpha1 connexin 43 gap junctions are decreased in human adrenocortical tumors.

Gap junctional communication disorders have been implicated in the etiology of benign and malignant tumors. Understanding the type, distribution, and frequency of gap junctions in adrenal disorders should provide insight into the role of gap junctions in adrenal carcinogenesis as well as information that may be useful in developing improved diagnosis and treatment of adrenal diseases. Using immunocytochemical techniques, we have characterized and compared alpha1 connexins 43 gap junction protein levels in normal adrenal glands to those in benign and malignant adrenocortical human tumors. In addition, gap junction protein levels were studied in a human adrenal cancer cell line (H295). In both normal and neoplastic adrenal tissues, only alpha1 connexin 43 could be detected, whereas beta1 connexin 32 and beta2 connexin 26 were not found. In the normal adrenal gland, the zona fasciculata was demonstrated to have the highest number of gap junctions per cell (mean +/- SEM, 13.78 +/- 1.93). In contrast, in benign adrenocortical adenomas, the number of gap junctions per cell compared to that detected in normal adrenal glands was significantly reduced (mean +/- SEM, 4.6 +/- 1.17; P < or = 0.05), and the lowest number was found in malignant adrenocortical tumors (1.42 +/- 0.58; P < or = 0.05). Similarly, there were few or no alpha1 connexin 43 gap junctions in the H295 population. There was a progressive decrease in gap junction plaques in adrenocortical cancer cell populations compared to those in normal cell populations. Therefore, analysis of gap junction protein may be helpful for the differential diagnosis of benign and malignant adrenal tumors. The induction of gap junctions in malignant cells may provide a novel therapeutic strategy for adrenal cancer.

Heparin-induced aldosterone suppression and hyperkalemia.

PURPOSE: To review the effects of heparin and heparinoid compounds on aldosterone physiology and associated induction of hyperkalemia. MATERIALS AND METHODS: A comprehensive literature search (of human and animal data) was carried out by computer and by using reference citations from primary sources. RESULTS: Heparin and its congeners are predictable, potent inhibitors of aldosterone production. This inhibitory effect is specific for the zona glomerulosa; other corticosteroids are not affected. Aldosterone suppression occurs within a few days of initiation of therapy, is reversible, and is independent of either anticoagulant effect or route of administration. Decreases in aldosterone levels may occur with heparin dosages as low as 5,000 U BID. The most important, but probably not the only mechanism of aldosterone inhibition appears to involve reduction in both the number and affinity of the angiotensin-II receptors in the zona glomerulosa. Prolonged use of heparin causes marked reduction in the width of the adrenal zona glomerulosa. CONCLUSIONS: Aldosterone suppression results in natriuresis and less predictably in decreased excretion of potassium. Greater than normal serum potassium levels occur in about 7% of patients, but marked hyperkalemia generally requires the presence of additional factors perturbing potassium balance (in particular, renal insufficiency, diabetes mellitus, or the use of certain medications). Heparin-induced increases in serum potassium need to be better anticipated by clinicians. Serum potassium levels should be monitored periodically in patients being given heparin for 3 or more days, and in patients at relatively high risk for hyperkalemia, the monitoring interval should probably be no greater than 4 days.

Familial pheochromocytoma.

During a 15-year period, 24 patients were treated operatively for pheochromocytoma at this medical center. In this group a father and three daughters were recognized to have the familial variety of this disease. Malignant hypertension, which began at an early age, was the most prominent characteristic. Operative treatment of the three sisters occurred at the ages of 9, 15, and 17 years. All pheochromocytomas were bilateral and were confined to the adrenal glands except for hepatic extension in the father. In contrast to most recent reports of familial pheochromocytoma, there was no evidence of multiple endocrine adenomatosis in this family. Total adrenalectomy was performed in only the youngest patient and, although normotensive, she requires close observation and steroid replacement. Three patients underwent a controversial partial adrenalectomy after excision of bilateral tumors. The father has remained hypertensive for 8 years and has evidence of a residual tumor in the liver. The two daughters who have had subtotal adrenalectomies remain normotensive, require no medication, and have normal urine catecholamine values 7 and 3 years after operation. These results show that removal of bilateral pheochromocytomas without total adrenalectomy may allow normal adrenal function free of recurrence for several years in patients without multiple endocrine adenomatosis.

Ambulatory parathyroidectomy for primary hyperparathyroidism.

OBJECTIVE: To evaluate whether the combined application of preoperative localization and intraoperative monitoring of intact parathyroid hormone (iPTH) levels could facilitate safe outpatient parathyroidectomy. DESIGN: Consecutive patients, who had no antecedent social or medical conditions mandating hospitalization, were prospectively offered ambulatory parathyroidectomy with a mean follow-up of 7 months (range, 1-25 months). SETTING: Tertiary care referral center PATIENTS: From 85 patients who had primary hyperparathyroidism with hypercalcemia and elevated iPTH levels, 57 were offered outpatient parathyroidectomy. Nineteen patients were asymptomatic, 3 had hypercalcemic crisis, and the others gave a history of renal stones or had complaints consistent with bone disease. INTERVENTIONS: Technetium Tc 99m sestamibi scintiscans were used for preoperative localization. Monitoring iPTH levels during parathyroidectomy quantitatively assured the surgeon (G.L.I. only) when all hyperfunctioning glands were excised. MAIN OUTCOME MEASURE: The number of patients without complications and with short operative times who were discharged without hospital admission or overnight stay. RESULTS: The combination of preoperative localization of abnormal parathyroid glands and a decline in circulating iPTH levels predicting postoperative normocalcemia after excision of all hyperfunctioning glands resulted in successful parathyroidectomy in 84 of 85 patients. A decreased operative time (average, 52 minutes) with minimal neck dissection permitted outpatient parathyroidectomy in 42 of 57 eligible patients. CONCLUSIONS: The combination of preoperative parathyroid scintiscan localization and iPTH level monitoring during surgery permitted successful parathyroidectomy in an ambulatory setting in half of a consecutive series of patients with primary hyperparathyroidism. The safety, success, and likely cost savings of this approach suggest wider application.

Effects of marine sponge extracts on mitogen-activated protein kinase (MAPK/ERK(1,2)) activity in SW-13 human adrenal carcinoma cells.

Some species of marine sponge have been shown to produce metabolites with endocrine-altering and cell growth regulatory properties. Since cell division and differentiation are controlled, in part, by the mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK/ERK) cascade, we tested extracts (1.0mg/ml) from six shallow water marine species obtained in the Florida Keys for effects on MAPK/ERK(l,2) (sub-variant of EC 2.7.1.37) activity in incubations with SW-13 human adrenal carcinoma cells in culture. In these short-term incubations, extracts from two species, the purple bleeding sponge (Iotrochota birotulata) and the West Indian bath sponge (Spongia barbara), significantly inhibited MAPK/ERK(1,2) activity (to 51 and 44% of control levels, respectively) without altering cell survival. Western blots for phosphorylated and total ERK showed that ERK(2) predominated over ERK(1) by a factor of about 4:1 and that the phosphorylated forms of these isozymes were strongly suppressed by active extracts from both sponges. Another species, the green sponge (Haliclona veridis), whose extract has been shown previously to activate guanylate cyclase and to inhibit adenylate cyclase in a variety of mammalian tissues, was found not to affect MAPK/ERK(1,2) in human adrenal carcinoma cultures but did lyse and kill most of these cultured cells. Extracts from the sheepswool sponge (Hippospongia lachne) and the bleeding sponge (Oligoceras hemorrhages) did not significantly affect either MAPK/ERK(1,2) activity or the survival of attached cells. An extract from the fire sponge (Tedania ignis) did not alter MAPK/ERK(1,2) activity but did modestly decrease cell viability. These studies document for the first time species-specifc effects of marine sponge extracts on the MAPK/ERK(1,2) cascade and on the growth and survival of human adrenal carcinoma cells in culture.

Tyrosine hydroxylase gene expression in varying forms of human pheochromocytoma.

We performed a comparative study of catecholamine content, tyrosine hydroxylase (TH) activity, and TH mRNA levels in normal human adrenals and various clinical forms of human pheochromocytoma. We studied sporadic, benign intra-adrenal chromaffin tumors and other non-malignant intra-adrenal tumors associated with multiple endocrine neoplasia type 2B (MEN 2B) and von Hippel-Lindau disease along with one extra-adrenal malignant pheochromocytoma. Our findings suggest substantial differences in TH transcriptional rates or the stability of TH mRNA or both may contribute to altered TH expression in human chromaffin cells associated with "normal" adrenal tissues and various forms of pheochromocytoma and distinctive patterns of expression in the different settings in which these tumors arise.

Studies of the neuronal transdifferentiation process in cultured human pheochromocytoma cells: effects of steroids with differing functional groups on catecholamine content and cell morphology.

The neuronal differentiation of adrenal pheochromocytoma cells from human subjects was studied in vitro for periods of up to 65 days. Changes with time in culture were observed in both intracellular catecholamine content (progressive decreases in epinephrine, norepinephrine, and dopamine, except for a possible transient early increase in the latter) and in morphology (increases in neurite outgrowth) of cells cultured in control medium; supplementation of cultures with nerve growth factor resulted in a substantial increase in neurite formation. The effects on these changes of the presence in the culture medium of various steroids were examined. The addition of 11-oxygenated steroids (aldosterone, corticosterone, cortisol, or dexamethasone) at 10(-5) M concentrations caused at least 2.5-fold increases in mean intracellular dopamine and norepinephrine levels; with dexamethasone, 9-10-fold increases were observed. Intracellular epinephrine content was also enhanced by 11,17-oxygenated steroids (dexamethasone and cortisol), but not by the other 11-oxygenated compounds studied. These two 11,17-oxygenated glucocorticoids also inhibited the morphologic changes seen with extended periods in culture, decreasing the outgrowth of neurite projections and causing cells to attain a vacuolated and granular appearance; the presence of dexamethasone strongly inhibited the morphologic changes induced by nerve growth factor. 11-Deoxy steroid intermediates (pregnenolone, 11-deoxycorticosterone, and 11-deoxycortisol) had little or no effect on catecholamine content or on morphology. Preliminary observations suggest that C-18 and C-19 sex steroid hormones (17 beta-estradiol and testosterone) may have morphologic effects opposite to those of the 11-oxygenated compounds, showing a slight stimulatory influence on the formation of neurite projections, but no significant effect on catecholamine content.

Biosynthesis of androgens by pheochromocytomas.

Homogenates from four adrenal pheochromocytomas converted 4-14C-labeled pregnenolone, 17-hydroxyprogesterone, and dehydroepiandrosterone into androstenedione and testosterone. In addition to these androgens, labeled pregnane substrates were also transformed into corticosteroids, as previously reported, and this conversion occurred in even higher yield. The formation of labeled metabolites of either pathway was greater in homogenates from intraadrenal pheochromocytomas than in those derived from an extraadrenal tumor, but less than in preparations of hyperplastic adrenal cortex. Incubations of subcellular fractions isolated from an adrenal pheochromocytoma showed that the enzyme activities involved in androgen formation from the radioactive substrates studied were associated with the microsomes and required exogenous cofactors. In contrast to adrenocortical tissue, chromaffin cell preparations uniformly failed to convert substrate [4-14C] cholesterol into either androgens or corticosteroids. The data available demonstrate the presence in chromaffin tissue of all of the enzyme activities required for the biosynthesis of androgens and corticosteroids except for those involved in the side-chain scission of cholesterol.

Review of the literature: severe hyperphosphatemia.

A patient with a markedly elevated serum phosphorus level (23.9 mg/dL) is described, followed by a brief review of severe hyperphosphatemia. Elevated serum phosphorus levels may be artifactual or true. True hyperphosphatemia is usefully subdivided according to (a) whether phosphorus is added to the extracellular fluid from a variety of exogenous or endogenous sources, or (b) whether the urinary excretion of phosphorus is reduced from either decreased glomerular filtration or increased tubular reabsorption. Severe hyperphosphatemia, defined herein as levels of 14 mg/dL or higher, is almost invariably multifactorial--usually resulting from addition of phosphorus to the extracellular fluid together with decreased phosphorus excretion. The hyperphosphatemia of the patient described herein appeared to result from a combination of dietary phosphorus supplementation, acute renal failure, acute pancreatitis, and ischemic bowel disease, complicated by lactic acidosis.

Biosynthesis and metabolism of steroid hormones by human adrenal carcinomas.

Over a 15-year period, our university-based laboratory obtained 125 adrenal tumors, of which 15 (12%) were adrenal cortical carcinomas. Of these, 6 (40% of the carcinomas) occurred in patients with clear clinical manifestations of steroid hormone excess. Adrenal cortical carcinoma cells derived from the surgically resected tumors in 4 of these patients were isolated and established in primary culture. Radiotracer steroid interconversion studies were carried out with these cultures and also on mitochondria isolated from homogenized tissues. Large tumors had the lowest steroidogenic activities per weight, whereas small tumors had more moderately depressed enzyme activities relative to cells from normal glands. In incubations with pregnenolone as substrate, 1 mM metyrapone blocked the synthesis of corticosterone and cortisol and also the formation of aldosterone. Metyrapone inhibition was associated with a concomitant increase in the formation of androgens (androstenedione and testosterone) from pregnenolone. Administration of metyrapone in vivo before surgery in one patient resulted in a similar increase in plasma androstenedione, though plasma testosterone levels were not significantly affected. In cultures of two of four tumors examined, dibutyryl cAMP stimulated 11ss-hydroxylase activity modestly; ACTH also had a significant stimulatory effect in one of these tumors. Unlike results obtained with normal or adenomatous adrenal cortical tissues, mitochondria from carcinomatous cells showed a lack of support of either cholesterol side-chain cleavage enzyme complex or steroid 11ss-hydroxylase activity by Krebs cycle intermediates (10 mM isocitrate, succinate or malate). This finding is consistent with the concept that these carcinomas may tend to function predominantly in an anaerobic manner, rather than through the oxidation of Krebs cycle intermediates.

The piriformis syndrome.

Piriformis syndrome is an often misdiagnosed cause of sciatica, leg, or buttock pain, and disability. The sciatic nerve may be compressed within the buttock by the piriformis muscle, with pain increased by muscular contraction, palpation, or prolonged sitting. A thorough medical history and physical examination are essential to proper diagnosis. Diagnostic testing may be used to differentiate piriformis syndrome from other causes of sciatica, lower extremity weakness, and pain. This article reviews the pathophysiology and management of piriformis syndrome.