Scott A. Rivkees

Molecular cloning of the rat A2 adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum

A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this cDNA in COS cells revealed high affinity (Kd = 38.6 nM) and saturable binding of the A2 adenosine receptor-selective ligand [3H]CGS 21680. Agonist displacement profile of [3H]CGS 21680 binding was consistent with an adenosine receptor of the A2 subtype (NECA greater than (R)-PIA greater than CPA greater than (S)-PIA). In situ hybridization demonstrated that rat A2 adenosine receptor mRNA was co-expressed in the same striatal neurons as D2 dopamine receptor mRNA, and never co-expressed with striatal D1 dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A2 subtype of adenosine receptor in the forebrain. The co-expression of D2 dopamine and A2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior.

Localization and characterization of melatonin receptors in rodent brain by in vitro autoradiography

Little is known of the neural sites of action for the pineal hormone, melatonin. Thus, we developed an in vitro autoradiographic method using 125I-labeled melatonin (I-MEL) to study putative melatonin receptors in rodent brain. We first determined optimal in vitro labeling conditions for autoradiographic detection of I-MEL binding sites in rat median eminence, the most intensely labeled area in the rat brain. We then assessed the pharmacologic and kinetic properties of I-MEL binding sites in rat median eminence by quantitative autoradiography. These sites have high affinity for I-MEL (equilibrium dissociation constant = 43 pM). I-MEL binding was inhibited by nanomolar concentrations of melatonin or 6-chloromelatonin, but was not inhibited by serotonin, dopamine, or norepinephrine (100 microM). These results suggest that I- MEL binding sites identified by in vitro autoradiography represent specific, high-affinity melatonin receptors. Studies of the distribution of I-MEL binding in rat, Syrian hamster, and Djungarian hamster brain confirm that the median eminence and suprachiasmatic nucleus are major sites of I-MEL binding in rodent brain; other brain areas labeled in one or more of these species were the thalamus (paraventricular, anteroventral, and reuniens nuclei, nucleus of the stria medullaris, and medial part of the lateral habenular nucleus), hypothalamus (dorsomedial nucleus), subiculum, and area postrema. The presence of putative melatonin receptors in the suprachiasmatic nuclei and median eminence of these rodent species suggests that these brain regions are important loci for melatonin effects on circadian rhythms and reproduction.

Neuregulin-1 promotes formation of the murine cardiac conduction system

The cardiac conduction system is a network of cells responsible for the rhythmic and coordinated excitation of the heart. Components of the murine conduction system, including the peripheral Purkinje fibers, are morphologically indistinguishable from surrounding cardiomyocytes, and a paucity of molecular markers exists to identify these cells. The murine conduction system develops in close association with the endocardium. Using the recently identified CCS-lacZ line of reporter mice, in which lacZ expression delineates the embryonic and fully mature conduction system, we tested the ability of several endocardial-derived paracrine factors to convert contractile cardiomyocytes into conduction-system cells as measured by ectopic reporter gene expression in the heart. In this report we show that neuregulin-1, a growth and differentiation factor essential for ventricular trabeculation, is sufficient to induce ectopic expression of the lacZ conduction marker. This inductive effect of neuregulin-1 was restricted to a window of sensitivity between 8.5 and 10.5 days postcoitum. Using the whole mouse embryo culture system, neuregulin-1 was shown to regulate lacZ expression within the embryonic heart, whereas its expression in other tissues remained unaffected. We describe the electrical activation pattern of the 9.5-days postcoitum embryonic mouse heart and show that treatment with neuregulin-1 results in electrophysiological changes in the activation pattern consistent with a recruitment of cells to the conduction system. This study supports the hypothesis that endocardial-derived neuregulins may be the major endogenous ligands responsible for inducing murine embryonic cardiomyocytes to differentiate into cells of the conduction system.

Immunohistochemical detection of A1 adenosine receptors in rat brain with emphasis on localization in the hippocampal formation, cerebral cortex, cerebellum, and basal ganglia

Polyclonal antisera were generated against two identical regions of rat and human A1 adenosine receptors using synthetic multiple-antigenic-peptides as immunogens. Western blotting showed that the antisera recognized a single protein in brain of the expected size for A1 receptors. Immunohistochemistry of CHO cells transfected with the rat or human A1 adenosine receptor cDNAs showed robust labeling of the cell surface. In contrast, labeling was not apparent over non-transfected CHO cells, nor over CHO cells expressing A2a receptors. The pattern of immunoreactivity in rat brain was similar to that expected for A1 adenosine receptors. In contrast to receptor autoradiography or in situ hybridization methods, immunohistochemistry allowed identification of individually labeled cells and processes. Heavy labeling was apparent in many brain regions. In the hippocampal formation, strong labeling was present on granule cell bodies and dendrites, mossy fibers, and pyramidal neurons. In cerebellum, basket cells were the most heavily labeled cell type. Less intense staining was present over granule cells. In cerebral cortex, pyramidal cells were the most heavily labeled cell type, and some interneurons were also labeled. In the basal ganglia, 43% of neurons in the globus pallidus were labeled. In the caudate-putamen region, 38% of neurons were labeled. Heavy labeling was present in most thalamic nuclei, and moderate to heavy labeling was seen in many brainstem nuclei. These data identify specific cellular sites of A1 receptor expression and support the concept of cellular specificity of A1 adenosine receptor action.

Long-term growth in juvenile acquired hypothyroidism: the failure to achieve normal adult stature.

It has been suggested that complete catch-up growth is achieved with treatment in patients with juvenile acquired hypothyroidism. We tested this assumption by examining long-term growth in 18 girls (mean [+/- SD] age, 11.4 +/- 2.7 years; bone age, 6.2 +/- 3.1 years) and 6 boys (age, 10.6 +/- 4.7 years; bone age, 6.4 +/- 2.7 years) with severe primary hypothyroidism (serum thyroxine level 1.1 +/- 0.3 micrograms per deciliter [13 +/- 4 nmol per liter]). At diagnosis, heights were 4.04 +/- 0.5 and 3.15 +/- 0.4 SD below the mean heights for age of normal girls and boys, respectively. The patients were treated with levothyroxine (3.4 +/- 0.3 micrograms per kilogram of body weight per day) to maintain normal thyroid function. During the first 18 months of therapy, the childrens skeletal maturation exceeded the maturation expected for their statural growth, regardless of whether or not they were undergoing pubertal development. Predictions of decreased adult height were based on these observations. At maturity, girls and boys stood approximately 2 SD below normal adult stature, at 149 +/- 5.0 cm and 168 +/- 5.1 cm, respectively. Heights at maturity were also lower than midparental heights (P less than 0.01) and lower than pre-illness standard-deviation scores for height (P less than 0.01). The deficit in adult stature was significantly related to the duration of hypothyroidism before treatment (P less than 0.01). We conclude that despite treatment, prolonged juvenile acquired hypothyroidism results in a permanent height deficit related to the duration of thyroxine deficiency before treatment.

Clinical and Economic Outcomes of Thyroid and Parathyroid Surgery in Children

CONTEXT Clinical and economic outcomes after thyroidectomy/parathyroidectomy in adults have demonstrated disparities based on patient age and race/ethnicity; there is a paucity of literature on pediatric endocrine outcomes. OBJECTIVE The objective was to examine the clinical and demographic predictors of outcomes after pediatric thyroidectomy/parathyroidectomy. DESIGN This study is a cross-sectional analysis of Healthcare Cost and Utilization Project-National Inpatient Sample hospital discharge information from 1999-2005. All patients who underwent thyroidectomy/parathyroidectomy were included. Bivariate and multivariate analyses were performed to identify independent predictors of patient outcomes. SUBJECTS Subjects included 1199 patients 17 yr old or younger undergoing thyroidectomy/parathyroidectomy. MAIN OUTCOME MEASURES Outcome measures included in-hospital patient complications, length of stay (LOS), and inpatient hospital costs. RESULTS The majority of patients were female (76%), aged 13-17 yr (71%), and White (69%). Whites were more often in the highest income group (80% vs. 8% for Hispanic and 6% for Black; P < 0.01) and had private/HMO insurance (76% vs. 10% for Hispanic and 5% for Black; P < 0.001) rather than Medicaid (13% vs. 32% for Hispanic and 41% for Black; P < 0.001). Ninety-one percent of procedures were thyroidectomies and 9% parathyroidectomies. Children aged 0-6 yr had higher complication rates (22% vs. 15% for 7-12 yr and 11% for 13-17 yr; P < 0.01), LOS (3.3 d vs. 2.3 for 7-12 yr and 1.8 for 13-17 yr; P < 0.01), and higher costs. Compared with children from higher-income families, those from lower-income families had higher complication rates (11.5 vs. 7.7%; P < 0.05), longer LOS (2.7 vs. 1.7 d; P < 0.01), and higher costs. Children had higher endocrine-specific complication rates than adults after parathyroidectomy (15.2 vs. 6.2%; P < 0.01) and thyroidectomy (9.1 vs. 6.3%; P < 0.01). CONCLUSIONS Children undergoing thyroidectomy/parathyroidectomy have higher complication rates than adult patients. Outcomes were optimized when surgeries were performed by high-volume surgeons. There appears to be disparity in access to high-volume surgeons for children from low-income families, Blacks, and Hispanics.

Putting Propylthiouracil in Perspective

The antithyroid drugs propylthiouracil (PTU) and methimazole (MMI) have played central roles in the management of hyperthyroidism for more than 50 yr. Although both drugs effectively control hyperthyroidism, observations over several decades have shown that MMI and its prodrug carbimazole are better than PTU in controlling more severe hyperthyroidism, having higher adherence rates, and causing less toxicity, especially when prescribed in lower doses (1).Thishas led to therecommendationthatMMIshouldbe the first-line drug when antithyroid drug therapy is initiated, either for primary treatment or to prepare a patient for radioiodine or surgery. An exception to this rule has been pregnancy, during which PTU has been preferred because of rare reports of birth defects associated with MMI(2).PTUhasalsobeenused inpatientswhohadminor reactions to MMI but, nonetheless, preferred to continue antithyroid drug therapy. PTU may also be preferable in patients with life-threatening thyrotoxicosis because of its additional inhibition of T4 to T3 conversion. It is in thiscontext thatcontinuedPTUuseasasecond-lineagent, a first-line agent in pregnancy, and routinely by some practitioners has been reevaluated at two meetings in the last 6 months. The first meeting, which was sponsored by the Eunice Kennedy Shriver National Institute of Child Health and Development on October 28, 2008, examined PTU safety in children because of accumulating reports of PTU-related liver failure and death in children (3). The second meeting, sponsored by the American Thyroid Association and the Food and Drug Administration (FDA) on April 18, 2009, reevaluated the role of PTU during pregnancy, given what is known about PTU-related hepatotoxicity and MMI-related birth defects (4). At both meetings, the world’s literature on PTU hepatotoxicity was reviewed. Representatives from the FDA provided data on currentPTUandMMIprescribingpractices. Informationpertaining to PTUhepatotoxicity fromtheFDAAdverseEventReportingSystem (AERS) MedWatch Program was examined. Data on hepatic transplantations for PTU-related hepatotoxicity, provided by The United Network for Organ Sharing (UNOS), were also reviewed. At the second meeting, MMI-related aplasia cutis and more severe teratogenesis were discussed; however, no new information about the frequency or the cause of this problem was presented. At both meetings, mechanisms of drug-related hepatic injury were reviewed, and the role of biochemical monitoring of liver integrity in patients taking drugs known to cause hepatic damage was discussed. A complex and incomplete but, nonetheless, worrisome picture emerged from these meetings. There are 33 published reports of severe PTU-related liver failure in adults and 14 in children (see Supplemental Table 1 and Supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). UNOS reported 16 liver transplants in adults and seven in children between 1990 and 2007 due to PTU-induced liver failure (5, 6) (supplemental data). Although MMI can cause liver injury, too, it is typically characterized by serious cholestatic dysfunction rather than hepatocellular inflammation (1). Indeed, over the same 17-yr period when one to three PTU-related liver transplants occurred per year, there were no liver transplants in the United States attributed to MMI toxicity. The FDA AERS databases, which overlap published reports and are subject to underreporting, detail instances of severe liver injury in 22 adults over the past 20 yr, nine of whom died and five who received liver transplants. Over the same period, 12 pediatric patients sustained severe liver injury resulting in three deaths and six liver transplants. The average daily dose of PTU associated with liver failure was approximately 300 mg in both children and adults. Liver failure occurred after 6 to 450 d (median, 120 d) of treatment. In the AERS data, there were also two reports of serious maternal liver injury due to PTU during pregnancy and two reports of liver injury in fetuses whose mothers took PTU. Because the true incidence of severe liver injury among patients taking PTU is unknown, efforts were made at the two meetings to define this figure more precisely. Based on published age-specific annual incidence data for hyperthyroidism (7) and the age distribution of the U.S. population from the 2000 census, it can be calculated thatapproximately60,000adultsdevelophyperthyroidism each year. Based on data reported at the two meetings, PTU is prescribed to one fourth of patients treated with antithyroid drugs forhyperthyroidismintheUnitedStates (8).Approximately15,000 adults are, therefore, estimated tobeginPTUtherapyperyear. If the frequency of PTU-related severe liver damage is approximately

Protective effects of caffeine on chronic hypoxia‐induced perinatal white matter injury

Periventricular white matter injury (PWMI) is the major cause of cerebral palsy and cognitive impairment in prematurely born infants. PWMI is characterized by reductions in cerebral myelination and cerebrocortical volumes and is associated with secondary ventriculomegaly. In neonatal rodents, these features of PWMI can be induced by rearing in chronic hypoxia or by activation of A1 adenosine receptors. We determined: (1) whether altered maturation or development of one or more oligodendrocyte (OL) lineage stages plays a role in the pathogenesis of the myelination disturbances associated with exposure to chronic hypoxia, and (2) whether blockade of A1 adenosine receptor action with the adenosine antagonist caffeine can prevent hypoxia‐induced white matter injury.

A1 adenosine receptors mediate hypoxia-induced ventriculomegaly

Periventricular leukomalacia is characterized by a reduction in brain matter and secondary ventriculomegaly and is a major cause of developmental delay and cerebral palsy in prematurely born infants. Currently, our understanding of the pathogenesis of this condition is limited. In animal models, features of periventricular leukomalacia can be induced by hypoxia and activation of A1 adenosine receptors (A1ARs). Using mice that are deficient in the A1AR gene (A1AR–/–), we show that A1ARs play a prominent role in the development of hypoxia-induced ventriculomegaly in neonates. Supporting a role for adenosine in the pathogenesis of developmental brain injury, ventriculomegaly was also observed in mice lacking the enzyme adenosine deaminase, which degrades adenosine. Thus, adenosine acting on A1ARs appears to mediate hypoxia-induced brain injury ventriculomegaly during early postnatal development.

The Treatment of Differentiated Thyroid Cancer in Children: Emphasis on Surgical Approach and Radioactive Iodine Therapy

Pediatric thyroid cancer is a rare disease with an excellent prognosis. Compared with adults, epithelial-derived differentiated thyroid cancer (DTC), which includes papillary and follicular thyroid cancer, presents at more advanced stages in children and is associated with higher rates of recurrence. Because of its uncommon occurrence, randomized trials have not been applied to test best-care options in children. Even in adults that have a 10-fold or higher incidence of thyroid cancer than children, few prospective trials have been executed to compare treatment approaches. We recognize that treatment recommendations have changed over the past few decades and will continue to do so. Respecting the aggressiveness of pediatric thyroid cancer, high recurrence rates, and the problems associated with decades of long-term follow-up, a premium should be placed on treatments that minimize risk of recurrence and the adverse effects of treatments and facilitate follow-up. We recommend that total thyroidectomy and central compartment lymph node dissection is the surgical procedure of choice for children with DTC if it can be performed by a high-volume thyroid surgeon. We recommend radioactive iodine therapy for remnant ablation or residual disease for most children with DTC. We recommend long-term follow-up because disease can recur decades after initial diagnosis and therapy. Considering the complexity of DTC management and the potential complications associated with therapy, it is essential that pediatric DTC be managed by physicians with expertise in this area.