Rashmi Chandra

Early Fetal Hypoxia Leads to Growth Restriction and Myocardial Thinning

Hypoxia is necessary for fetal development; however, excess hypoxia is detrimental. Hypoxia has been extensively studied in the near-term fetus, but less is known about earlier fetal effects. The purpose of this study was to determine the window of vulnerability to severe hypoxia, what organ system(s) is most sensitive, and why hypoxic fetuses die. We induced hypoxia by reducing maternal-inspired O2 from 21% to 8%, which decreased fetal tissue oxygenation assessed by pimonidazole binding. The mouse fetus was most vulnerable in midgestation: 24 h of hypoxia killed 89% of embryonic day 13.5 (E13.5) fetuses, but only 5% of E11.5 and 51% of E17.5 fetuses. Sublethal hypoxia at E12.5 caused growth restriction, reducing fetal weight by 26% and protein by 45%. Hypoxia induced HIF-1 target genes, including vascular endothelial growth factor (Vegf), erythropoietin, glucose transporter-1 and insulin-like growth factor binding protein-1 (Igfbp-1), which has been implicated in human intrauterine growth restriction (IUGR). Hypoxia severely compromised the cardiovascular system. Signs of heart failure, including loss of yolk sac circulation, hemorrhage, and edema, were caused by 18-24 h of hypoxia. Hypoxia induced ventricular dilation and myocardial hypoplasia, decreasing ventricular tissue by 50% and proliferation by 21% in vivo and by 40% in isolated cultured hearts. Epicardial detachment was the first sign of hypoxic damage in the heart, although expression of epicardially derived mitogens, such as FGF2, FGF9, and Wnt9b was not reduced. We propose that hypoxia compromises the fetus through myocardial hypoplasia and reduced heart rate.

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.

Multiple effects of KPQ deletion mutation on gating of human cardiac Na+ channels expressed in mammalian cells

Several aspects of the effect of the KPQ deletion mutation on Na+ channel gating remain unresolved. We have analyzed the kinetics of the early and late currents by recording whole cell and single-channel currents in a human embryonic kidney (HEK) cell line (HEK293) expressing wild-type and KPQ deletion mutation in cardiac Na+ channels. The rate of inactivation increased three- to fivefold between -40 and -80 mV in the mutant channel. The rate of recovery from inactivation was increased twofold. Two modes of gating accounted for the late current: 1) isolated brief openings with open times that were weakly voltage dependent and the same as the initial transient and 2) bursts of opening with highly voltage-dependent prolonged open times. Latency to first opening was accelerated, suggesting an acceleration of the rate of activation. The ΔKPQ mutation has multiple effects on activation and inactivation. The aggregate effects may account for the increased susceptibility to arrhythmias.Several aspects of the effect of the KPQ deletion mutation on Na+ channel gating remain unresolved. We have analyzed the kinetics of the early and late currents by recording whole cell and single-channel currents in a human embryonic kidney (HEK) cell line (HEK293) expressing wild-type and KPQ deletion mutation in cardiac Na+ channels. The rate of inactivation increased three- to fivefold between -40 and -80 mV in the mutant channel. The rate of recovery from inactivation was increased twofold. Two modes of gating accounted for the late current: 1) isolated brief openings with open times that were weakly voltage dependent and the same as the initial transient and 2) bursts of opening with highly voltage-dependent prolonged open times. Latency to first opening was accelerated, suggesting an acceleration of the rate of activation. The delta KPQ mutation has multiple effects on activation and inactivation. The aggregate effects may account for the increased susceptibility to arrhythmias.

An Enteroendocrine Cell – Enteric Glia Connection Revealed by 3D Electron Microscopy

The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY – both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia – the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

CD36-dependent signaling mediates fatty acid-induced gut release of secretin and cholecystokinin

Genetic variants in the fatty acid (FA) translocase FAT/CD36 associate with abnormal postprandial lipids and influence risk for the metabolic syndrome. CD36 is abundant on apical enterocyte membranes in the proximal small intestine, where it facilitates FA uptake and FA‐initiated signaling. We explored whether CD36 signaling influences FA‐mediated secretion of cholecystokinin (CCK) and secretin, peptides released by enteroendocrine cells (EECs) in the duodenum/jejunum, which regulate events important for fat digestion and homeostasis. CD36 was immunodetected on apical membranes of secretin‐ and CCK‐positive EECs and colocalized with cytosolic granules. Intragastric lipid administration to CD36–/– mice released less secretin (–60%) and CCK (–50%) compared with wild‐type mice. Likewise, diminished secretin and CCK responses to FA were observed with CD36–/– intestinal segments in vitro, arguing against influence of alterations in fat absorption. Signaling mechanisms underlying peptide release were examined in STC‐1 cells stably expressing human CD36 or a signaling‐impaired mutant (CD36K/A). FA stimulation of cells expressing CD36 (vs. vector or CD36K/A) released more secretin (3.5‐ to 4‐fold) and CCK (2‐ to 3‐fold), generated more cAMP (2‐ to 2.5‐fold), and enhanced protein kinase A activation. Protein kinase A inhibition (H‐89) blunted secretin (80%) but not CCK release, which was reduced (50%) by blocking of calmodulin kinase II (KN‐62). Coculture of STC‐1 cells with Caco‐2 cells stably expressing CD36 did not alter secretin or CCK release, consistent with a minimal effect of adjacent enterocytes. In summary, CD36 is a major mediator of FA‐induced release of CCK and secretin. These peptides contribute to the role of CD36 in fat absorption and to its pleiotropic metabolic effects.—Sundaresan, S., Shahid, R., Riehl, T. E., Chandra, R., Nassir, F., Stenson, W. F., Liddle, R. A., Abumrad, N. A. CD36‐dependent signaling mediates fatty acid‐induced gut release of secretin and cholecystokinin. FASEB J. 27, 1191–1202 (2013). www.fasebj.org

Neural and hormonal regulation of pancreatic secretion.

Purpose of review The biology of the pancreas is exquisitely complex and involves both endocrine and exocrine functions that are regulated by an integrated array of neural and hormonal processes. This review discusses recent developments in the regulation of both endocrine and exocrine secretion from the pancreas. Recent findings New data suggest that cholecystokinin can stimulate neurons located in the dorsal motor nucleus of the vagus. Addressing a controversial topic, recent evidence suggests a direct secretory action of cholecystokinin on human acinar cells. An emerging concept is that some hormones and peptides such as melatonin, ghrelin, obestatin and leptin perform dual functions in the pancreas by regulating secretion and maintaining metabolic homeostasis. The regulation of pancreatic secretion by several appetite-controlling neuropeptides such as ghrelin, orexin A and neuropeptide Y is also discussed. Recent data highlight findings that mechanisms of hormone action may be different between species possibly due to a divergence in signaling pathways during evolution. Summary The regulation of the secretory function of the pancreas by numerous hormones suggests that there are multiple and perhaps redundant signals governing the control of this important organ. Understanding these diverse pathways is essential to the treatment of pancreatitis, diabetes and obesity.

Block of Wild-Type and Inactivation-Deficient Cardiac Sodium Channels IFM/QQQ Stably Expressed in Mammalian Cells

The role of inactivation as a central mechanism in blockade of the cardiac Na(+) channel by antiarrhythmic drugs remains uncertain. We have used whole-cell and single channel recordings to examine the block of wild-type and inactivation-deficient mutant cardiac Na(+) channels, IFM/QQQ, stably expressed in HEK-293 cells. We studied the open-channel blockers disopyramide and flecainide, and the lidocaine derivative RAD-243. All three drugs blocked the wild-type Na(+) channel in a use-dependent manner. There was no use-dependent block of IFM/QQQ mutant channels with trains of 20 40-ms pulses at 150-ms interpulse intervals during disopyramide exposure. Flecainide and RAD-243 retained their use-dependent blocking action and accelerated macroscopic current relaxation. All three drugs reduced the mean open time of single channels and increased the probability of their failure to open. From the abbreviation of the mean open times, we estimated association rates of approximately 10(6)/M/s for the three drugs. Reducing the burst duration contributed to the acceleration of macroscopic current relaxation during exposure to flecainide and RAD-243. The qualitative differences in use-dependent block appear to be the result of differences in drug dissociation rate. The inactivation gate may play a trapping role during exposure to some sodium channel blocking drugs.

Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.

The peptide tyrosine tyrosine (PYY) is produced and secreted from L cells of the gastrointestinal mucosa. To study the anatomy and function of PYY-secreting L cells, we developed a transgenic PYY-green fluorescent protein mouse model. PYY-containing cells exhibited green fluorescence under UV light and were immunoreactive to antibodies against PYY and GLP-1 (glucagon-like peptide-1, an incretin hormone also secreted by L cells). PYY-GFP cells from 15 μm thick sections were imaged using confocal laser scanning microscopy and three-dimensionally (3D) reconstructed. Results revealed unique details of the anatomical differences between ileal and colonic PYY-GFP cells. In ileal villi, the apical portion of PYY cells makes minimal contact with the lumen of the gut. Long pseudopod-like basal processes extend from these cells and form an interface between the mucosal epithelium and the lamina propria. Some basal processes are up to 50 μm in length. Multiple processes can be seen protruding from one cell and these often have a terminus resembling a synapse that appears to interact with neighboring cells. In colonic crypts, PYY-GFP cells adopt a spindle-like shape and weave in between epithelial cells, while maintaining contact with the lumen and lamina propria. In both tissues, cytoplasmic granules containing the hormones PYY and GLP-1 are confined to the base of the cell, often filling the basal process. The anatomical arrangement of these structures suggests a dual function as a dock for receptors to survey absorbed nutrients and as a launching platform for hormone secretion in a paracrine fashion.

β-adrenergic action on wild-type and KPQ mutant human cardiac Na+ channels: shift in gating but no change in Ca2+: Na+ selectivity

OBJECTIVE Prior studies of the modulation of the Na+ current by sympathetic stimulation have yielded controversial results. Separation of the Na+ and Ca2+ currents poses a problem in myocyte preparations. The gating of cloned Na+ channels is different in oocytes compared with mammalian expression systems. We have examined the sympathetic modulation of the alpha-subunit of the wild-type human cardiac Na+ channel (hH1) and the long QT-associated mutant, delta KPQ, expressed in human embryonic kidney cells. METHODS Stable cell lines of hH1 and delta KPQ were established in human embryonic kidney cells. Whole-cell and single-channel currents were measured with the patch-clamp technique. Sympathetic stimulation was effected by exposure to isoproterenol or 8-bromo-cAMP. Na+ channel activation and inactivation were determined using standard voltage clamp protocols. Ca2+:Na+ permeability ratio was determined under bi-ionic conditions. RESULTS We observed a qualitatively different effect of sympathetic stimulation on the cardiac Na+ current from that reported in frog oocytes: activation and inactivation kinetics were shifted to more negative potentials. This shift was similar for both hH1 and delta KPQ. [delta V0.5 for inactivation: 8.3 +/- 1.7 mV, p < 0.001 (hH1); 6.8 +/- 0.9 mV, p < 0.001 (delta KPQ)]. Increased rate of closed-state inactivation contributed to the shifting of the inactivation-voltage relationship. Open-state inactivation was not affected as mean open times were unchanged. Reversal potential measurement in hH1 suggested a low Ca2+:Na+ permeability ratio of 0.017, uninfluenced by sympathetic stimulation. In delta KPQ, the size of the persistent relative to the peak current was increased with 8-bromo-cAMP from 3.0 +/- 0.7% to 4.3 +/- 0.6% (p = 0.056). CONCLUSIONS Sympathetic stimulation exerts multiple effects on the gating of hH1. Similar effects are also seen in delta KPQ which may increase arrhythmia susceptibility in long QT syndrome by modifying the Na+ channel contribution to the action potential.

Pseudopod-like basal cell processes in intestinal cholecystokinin cells

Cholecystokinin (CCK) is secreted by neuroendocrine cells comprising 0.1%-0.5% of the mucosal cells in the upper small intestine. Using CCK promoter-driven green fluorescent protein (GFP) expression in transgenic mice, we have applied immunofluorescence techniques to analyze the morphology of CCK cells. GFP and CCK colocalize in neuroendocrine cells with little aberrant GFP expression. CCK-containing cells are either flask- or spindle-shaped, and in some cells, we have found dendritic processes similar to pseudopods demonstrated for gut somatostatin-containing D cells. Most pseudopods are short, the longest process visualized extending across three cells. Pseudopods usually extend to adjacent cells but some weave between neighboring cells. Dual processes have also been observed. Three-dimensional reconstructions suggest that processes are not unidirectional and thus are unlikely to be involved in migration of CCK cells from the crypt up the villus. Abundant CCK immunostaining is present in the pseudopods, suggesting that they release CCK onto the target cell. In order to identify the type of cells being targeted, we have co-stained sections with antibodies to chromogranin A, trefoil factor-3, and sucrase-isomaltase. CCK cell processes almost exclusively extend to sucrase-isomaltase-positive enterocytes. Thus, CCK cells have cellular processes possibly involved in paracrine secretion.