Jeffrey D. Roizen

Block of rat brain recombinant SK channels by tricyclic antidepressants and related compounds

SK channels are small conductance, Ca(2+)-activated K(+) channels that underlie neuronal slow afterhyperpolarization and mediate spike frequency adaptation. Using the patch clamp technique, we tested the effects of eight clinically relevant psychoactive compounds structurally related to the tricyclic antidepressants, on SK2 subtype channels cloned from rat brain and functionally expressed in the human embryonic kidney cell line, HEK293. Amitriptyline, carbamazepine, chlorpromazine, cyproheptadine, imipramine, tacrine and trifluperazine blocked SK2 channel currents with micromolar affinity. The block was reversible and concentration-dependent. The potency differed according to chemical structure. In contrast, the cognitive enhancer linopirdine was ineffective at blocking these channels. Our results point to a distinct pharmacological profile for SK channels.

Patch-clamp analysis of anesthetic interactions with recombinant SK2 subtype neuronal calcium-activated potassium channels

Small conductance calcium-activated potassium channels (SK) mediate spike frequency adaptation and underlie the slow afterhyperpolarization in central neurons. We tested the actions of several anesthetics on the SK2 subtype of recombinant SK channels, cloned from rat brain and functionally expressed in a mammalian cell line. Butanol, ethanol, ketamine, lidocaine, and methohexital blocked recombinant SK2 channel currents, measured in the whole-cell patch clamp recording mode. The block was reversible, dose-dependent, and of variable efficacy. The inhaled anesthetics chloroform, desflurane, enflurane, halothane, isoflurane, and sevoflurane produced little or no block when applied at 1 minimum alveolar anesthetic concentration; varying degrees of modulation were observed at very large concentrations (10 minimum alveolar concentration). The extent of block by inhaled anesthetics did not appear to depend on concentration or membrane voltage. Implications: We describe differential effects of anesthetics on cloned small conductance calcium-activated potassium channels from brain that may play a role in generating the effects or side effects of anesthetics.

Oxytocin in the Circadian Timing of Birth

Background The molecular components determining the timing for birth remain an incompletely characterized aspect of reproduction, with important conceptual and therapeutic ramifications for management of preterm, post-term and arrested labor. Methodology/Principal Findings To test the hypothesis that oxytocin mediates circadian regulation of birth, we evaluated parturition timing following shifts in light cycles in oxytocin (OT)-deficient mice. We find that, in contrast to wild type mice that do not shift the timing of birth following a 6-h advance or delay in the light cycle, OT-deficient mice delivered at random times of day. Moreover, shifts in the light-dark cycle of gravid wild type mice have little impact on the pattern of circadian oxytocin release. Conclusions/Significance Our results demonstrate oxytocin plays a critical role in minimizing labor disruption due to circadian clock resetting.

CYP2R1 Mutations Impair Generation of 25-hydroxyvitamin D and Cause an Atypical Form of Vitamin D Deficiency

CONTEXT Production of the active vitamin D hormone 1,25-dihydroxyvitamin D requires hepatic 25-hydroxylation of vitamin D. The CYP2R1 gene encodes the principal vitamin D 25-hydroxylase in humans. OBJECTIVE This study aimed to determine the prevalence of CYP2R1 mutations in Nigerian children with familial rickets and vitamin D deficiency and assess the functional effect on 25-hydroxylase activity. DESIGN AND PARTICIPANTS We sequenced the CYP2R1 gene in subjects with sporadic rickets and affected subjects from families in which more than one member had rickets. MAIN OUTCOME MEASURES Function of mutant CYP2R1 genes as assessed in vivo by serum 25-hydroxyvitamin D values after administration of vitamin D and in vitro by analysis of mutant forms of the CYP2R1. RESULTS CYP2R1 sequences were normal in 27 children with sporadic rickets, but missense mutations were identified in affected members of 2 of 12 families, a previously identified L99P, and a novel K242N. In silico analyses predicted that both substitutions would have deleterious effects on the variant proteins, and in vitro studies showed that K242N and L99P had markedly reduced or complete loss of 25-hydroxylase activity, respectively. Heterozygous subjects were less affected than homozygous subjects, and oral administration of vitamin D led to significantly lower increases in serum 25-hydroxyvitamin D in heterozygous than in control subjects, whereas homozygous subjects showed negligible increases. CONCLUSION These studies confirm that CYP2R1 is the principal 25-hydroxylase in humans and demonstrate that CYP2R1 alleles have dosage-dependent effects on vitamin D homeostasis. CYP2R1 mutations cause a novel form of genetic vitamin D deficiency with semidominant inheritance.

Primary hyperparathyroidism in children and adolescents

&NA; Primary hyperparathyroidism (PHPT) is a common endocrine disorder in adults in whom the typical presentation is incidentally discovered as asymptomatic hypercalcemia. PHPT is much less common in children and adolescents, but has greater morbidity in this age group, as most young patients with PHPT will have symptomatic hypercalcemia or complications such as kidney stones, abdominal pain, and skeletal fragility. An important feature of PHPT in younger patients is the relatively high prevalence of germline inactivating mutations of the CASR gene, which encodes the calcium‐sensing receptor. Biallelic CASR mutations cause neonatal severe hyperparathyroidism, a life‐threatening condition that presents within days of life with marked hypercalcemia, respiratory distress, failure to thrive, and skeletal demineralization. By contrast, more common heterozygous CASR mutations are generally associated with a benign variant of PHPT termed familial hypocalciuric hypercalcemia. Appropriate management of PHPT in children and adolescents requires distinction between familial hypocalciuric hypercalcemia, which generally requires no specific treatment, and other forms of PHPT that are best treated by parathyroidectomy.

Resting Energy Expenditure Is Decreased in Pseudohypoparathyroidism Type 1A.

CONTEXT Pseudohypoparathyroidism type 1A (PHP1A) is caused by loss-of-function mutations on the maternally inherited GNAS allele and is associated with early-onset obesity, neurocognitive defects, and resistance to multiple hormones. The role of energy intake vs central regulation of energy expenditure in the pathophysiology of obesity remains unclear. OBJECTIVE The aim of this study was to evaluate resting energy expenditure (REE) in participants with PHP1A. DESIGN We assessed REE, biochemical, endocrine, and auxological status of 12 participants with PHP1A who had normal or elevated body mass index; controls were a cohort of 156 obese participants. SETTING This study took place at Childrens Hospital in Philadelphia and Sick Childrens Hospital in Toronto. MAIN OUTCOME MEASURES REE as a percent of predicted REE was the outcome measure. RESULTS PHP1A participants had normal endocrine status while receiving appropriate hormone replacement therapy, but had significantly decreased REE as a percent of predicted REE (using the modified Schofield equation). CONCLUSION Our results are consistent with REE being the principal cause of obesity in PHP1A rather than it being caused by excessive energy intake or endocrine dysfunction.

A meta-analysis comparing the biochemistry of primary hyperparathyroidism in youths to the biochemistry of primary hyperparathyroidism in adults.

CONTEXT The distinctive presentation of primary hyperparathyroidism (PHPT) in adults and youths suggest that PHPT is a fundamentally different disease in these two groups. OBJECTIVE To understand the difference in PHPT between adults and youths we compared the biochemistry of PHPT in these two groups. DESIGN This study is a systematic review and meta-analysis of retrospective studies published 1966-2014 on PHPT. DATA SOURCES All studies were obtained through Medline (1966-2014). STUDY SELECTION AND DATA EXTRACTION Only studies that included post-surgical subjects and that explicitly described biochemical results from more than one decade were included. Data were extracted from each article to generate the mean and SE for multiple biochemical parameters. DATA SYNTHESIS We analyzed 16 studies describing 268 unique youths and 2405 adults with PHPT. Youths with PHPT had significantly (P < .05) greater serum and urinary calcium than adults with PHPT (3.2 ± 0.1 mmol/L vs 2.8 ± 0.0 mmol/L for serum calcium, and 9.95 ± 1.26 mmol/d vs 7.15 ± 0.56 mmol/d for urine calcium, [mean ± SEM]). There were no significant differences in serum intact PTH, phosphorus, or alkaline phosphatase. CONCLUSIONS Juvenile PHPT has greater hypercalcemia and hypercalciuria than adult PHPT at similar concentrations of serum intact PTH. These observations suggest that there are differences in the pathophysiology of PHPT between juvenile and adult patients who reflect an apparent decrease in the sensitivity of the parathyroid adenoma to negative feedback by calcium and increased sensitivity of target tissues to the effects of PTH.

CYP3A4 mutation causes vitamin D–dependent rickets type 3

Genetic forms of vitamin D–dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR; however, using whole exome sequencing analysis, we identified a recurrent de novo missense mutation, c.902T>C (p.I301T), in CYP3A4 in both subjects that alters the conformation of substrate recognition site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than WT CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide insight into vitamin D metabolism and demonstrate that accelerated inactivation of vitamin D metabolites represents a mechanism for vitamin D deficiency.

Paraventricular Nucleus Sim1 Neuron Ablation Mediated Obesity Is Resistant to High Fat Diet

Single minded 1 (SIM1) is a transcription factor involved in brain patterning and control of energy balance. In humans, haploinsufficiency of SIM1 causes early-onset obesity. Mice deficient in the homologous gene, SIM1, also exhibit early onset obesity and increased sensitivity to a high fat diet. SIM1 is expressed in several areas of the brain implicated in control of energy balance including the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the medial amygdala and nucleus of the lateral olfactory tract. We have previously shown that mice with global Sim1 neuron ablation exhibit obesity with hyperphagia as the primary defect. The PVN has a critical role in feeding and in high-fat appetite, thus, we sought to determine the effect of Sim1 neuron ablation limited to the PVN. We achieved PVN-SIM1 limited ablation through stereotactic injection of diphtheria toxin into the PVN of Sim1Cre-iDTR mice. The specificity of this ablation was confirmed by immunohistochemistry and quantitative real time PCR of the PVN, supraoptic nucleus and the amygdala. Mice with PVN Sim1 neuron ablation, similar to mice with global Sim1 neuron ablation, exhibit early onset obesity with hyperphagia as the primary defect. However, PVN-Sim1 neuron ablated mice have a decreased response to fasting-induced hyperphagia. Consistent with this decrement, PVN-Sim1 neuron ablated mice have a decreased hyperphagic response to PVN injection of agouti-related peptide (AgRP). When PVN-Sim1 neuron ablated mice are placed on a high fat diet, surprisingly, their intake decreases and they actually lose weight. When allowed ad lib access to high fat diet and normal chow simultaneously, PVN-Sim1 neuron ablated mice exhibit overall decreased intake. That is, in PVN-Sim1 neuron ablated mice, access to fat suppresses overall appetite.

Mendelian randomization analysis demonstrates that low vitamin D is unlikely causative for pediatric asthma

To the Editor: Asthma is the most common chronic childhood disease, affecting approximately 8% of children in the United States. Multiple studies show strong associations between low serum 25-hydroxyvitamin D (25(OH)D) concentration and childhood asthma incidence and severity. Vitamin D deficiency is highly prevalent in atopic pediatric patients, and vitamin D has been hypothesized to contribute to childhood asthma through regulation of airway reactivity, sensitivity to corticosteroids, or modulation of immune function. Vitamin D supplementation studies, however, have failed to reveal a benefit for vitamin D in preventing or treating asthma. Recently,Mendelian randomization has been used to help dissect direction and extent of causality identified in association studies between modifiable risk factors and disease. Our overarching goal was to use Mendelian randomization to examine the hypothesis that low 25(OH)D concentration contributes significantly to asthma prevalence or exacerbations. First, we performed cross-sectional analyses to confirm the inverse association between serum 25(OH)D concentration and asthma in our cohort. We then performed a genome-wide association study (GWAS) to confirm that previously identified genetic determinants of vitamin D status were similarly determinative of 25(OH)D concentration in our cohort. Finally, we used the genetic determinants confirmed in our GWAS to perform our Mendelian randomization. The Children’s Hospital of Philadelphia Center for Applied Genomics biorepository consisted of 56,835 subjects; 12,842 were used in the Asthma Study Cohort. Asthma severity was inferred from the electronic medical record (see Figs E1 and E2 in this article’s Online Repository at www.jacionline.org) per the National Asthma Education and Prevention Program guidelines. Our cross-sectional analysis was done using Stata 13.1. Our