Amy L. Clark

Characterization of CA XV, a new GPI-anchored form of carbonic anhydrase

The main function of CAs (carbonic anhydrases) is to participate in the regulation of acid-base balance. Although 12 active isoenzymes of this family had already been described, analyses of genomic databases suggested that there still exists another isoenzyme, CA XV. Sequence analyses were performed to identify those species that are likely to have an active form of this enzyme. Eight species had genomic sequences encoding CA XV, in which all the amino acid residues critical for CA activity are present. However, based on the sequence data, it was apparent that CA XV has become a non-processed pseudogene in humans and chimpanzees. RT-PCR (reverse transcriptase PCR) confirmed that humans do not express CA XV. In contrast, RT-PCR and in situ hybridization performed in mice showed positive expression in the kidney, brain and testis. A prediction of the mouse CA XV structure was performed. Phylogenetic analysis showed that mouse CA XV is related to CA IV. Therefore both of these enzymes were expressed in COS-7 cells and studied in parallel experiments. The results showed that CA XV shares several properties with CA IV, i.e. it is a glycosylated glycosylphosphatidylinositol-anchored membrane protein, and it binds CA inhibitor. The catalytic activity of CA XV is low, and the correct formation of disulphide bridges is important for the activity. Both specific and non-specific chaperones increase the production of active enzyme. The results suggest that CA XV is the first member of the alpha-CA gene family that is expressed in several species, but not in humans and chimpanzees.

μ and κ Opioid Receptors Activate ERK/MAPK via Different Protein Kinase C Isoforms and Secondary Messengers in Astrocytes

Acute μ and κ opioids activate the ERK/MAPK phosphorylation cascade that represents an integral part of the signaling pathway of growth factors in astrocytes. By this cross-talk, opioids may impact neural development and plasticity among other basic neurobiological processes in vivo. The μ agonist, [d-ala2,mephe4,glyol5]enkephalin (DAMGO), induces a transient stimulation of ERK phosphorylation, whereas κ agonist, U69,593, engenders sustained ERK activation. Here we demonstrate that acute U69,593 and DAMGO stimulate ERK phosphorylation by utilization of different secondary messengers and protein kinase C (PKC) isoforms upstream of the growth factor pathway. Immortalized astrocytes transfected with either antisense calmodulin (CaM), a mutant μ opioid receptor that binds CaM poorly or a dominant negative mutant of PKCϵ were used as a model system to study μ signaling. Evidence was gained to implicate CaM and PKCϵ in DAMGO stimulation of ERK. DAMGO activation of PKCϵ and/or ERK was insensitive to selective inhibitors of Ca2+ mobilization, but it was blocked upon phospholipase C inhibition. These results suggest a novel mechanism wherein, upon DAMGO binding, CaM is released from the μ receptor and activates phospholipase C. Subsequently, phospholipase C generates diacylglycerides that activate PKCϵ. In contrast, U69,593 appears to act via phosphoinositide 3-kinase, PKCζ, and Ca2+ mobilization. These signaling components were implicated based on studies with specific inhibitors and a dominant negative mutant of PKCζ. Collectively, our findings on acute opioid effects suggest that differences in their mechanism of signaling may contribute to the distinct outcomes on ERK modulation induced by chronic μ and κ opioids.

μ- and κ-Opioids Induce the Differentiation of Embryonic Stem Cells to Neural Progenitors

Growth factors, hormones, and neurotransmitters have been implicated in the regulation of stem cell fate. Since various neural precursors express functional neurotransmitter receptors, which include G protein-coupled receptors, it is anticipated that they are involved in cell fate decisions. We detected μ-opioid receptor (MOR-1) and κ-opioid receptor (KOR-1) expression and immunoreactivity in embryonic stem (ES) cells and in retinoic acid-induced ES cell-derived, nestin-positive, neural progenitors. Moreover, these G protein-coupled receptors are functional, since [d-Ala2,MePhe4,Gly-ol5]enkephalin, a MOR-selective agonist, and U69,593, a KOR-selective agonist, induce a sustained activation of extracellular signal-regulated kinase (ERK) signaling throughout a 24-h treatment period in undifferentiated, self-renewing ES cells. Both opioids promote limited proliferation of undifferentiated ES cells via the ERK/MAP kinase signaling pathway. Importantly, biochemical and immunofluorescence data suggest that [d-Ala2,MePhe4,Gly-ol5]enkephalin and U69,593 divert ES cells from self-renewal and coax the cells to differentiate. In retinoic acid-differentiated ES cells, opioid-induced signaling features a biphasic ERK activation profile and an opioid-induced, ERK-independent inhibition of proliferation in these neural progenitors. Collectively, the data suggest that opioids may have opposite effects on ES cell self-renewal and ES cell differentiation and that ERK activation is only required by the latter. Finally, opioid modulation of ERK activity may play an important role in ES cell fate decisions by directing the cells to specific lineages.

Defective counterregulation and hypoglycemia unawareness in diabetes: mechanisms and emerging treatments.

For people with diabetes, hypoglycemia remains the limiting factor in achieving glycemic control. This article reviews recent advances in how the brain senses and responds to hypoglycemia. Novel mechanisms by which individuals with insulin-treated diabetes develop hypoglycemia unawareness and impaired counterregulatory responses are outlined. Prevention strategies for reducing the incidence of hypoglycemia are discussed.

Even silent hypoglycemia induces cardiac arrhythmias

While many studies have shown that intensive glycemic control can prevent the microvascular complications of diabetes, the benefits of intensive glycemic control in preventing macrovascular complications, including heart attacks, strokes, and overall mortality, have been less clear. Intensive glycemic control almost always increases the frequency and severity of hypoglycemic episodes. What remains unclear is whether hypoglycemia directly contributes to, or is merely associated with, the increased mortality noted in recent large trials (e.g., Normoglycaemia in Intensive Care Evaluation and Survival Using Glucose Algorithm Regulation [NICE-SUGAR], Control of Hyperglycaemia in Paediatric intensive care [CHiP], Action to Control Cardiovascular Risk in Diabetes [ACCORD]) (1–3). In the intensive care setting, noniatrogenic hypoglycemia serves as a harbinger of mortality, but it is unlikely to be a direct cause of mortality (4). By contrast, in the outpatient setting, insulin-induced hypoglycemia can be lethal. Among people with diabetes, the mortality rate due to hypoglycemia has been reported to be as high as 10% (5). Indeed, insulin-induced hypoglycemia has been considered responsible for nocturnal deaths in diabetic patients (6), and has been documented to be associated with the “dead-in-bed” syndrome (7). Therefore in the outpatient setting, the microvascular benefits of intensive glycemic control in people with diabetes have to be weighed against the apparent increased mortality associated with iatrogenic hypoglycemia. The mechanism(s) by which hypoglycemia may increase mortality remains unknown. In patients with cardiac disease, hypoglycemia has been associated with ischemic chest pain (8). Hypoglycemia also increases markers of thrombosis and inflammation, potentially increasing the risk of acute thrombotic events or accelerating development of atherosclerosis (9). Although hypoglycemia-associated …

Endoplasmic reticulum stress in beta cells and autoimmune diabetes

Type 1 diabetes results from the autoimmune destruction of pancreatic β cells, leading to insulin deficiency and hyperglycemia. Although multiple attempts have been made to slow the autoimmune process using immunosuppressive or immunomodulatory agents, there are still no effective treatments that can delay or reverse the progression of type 1 diabetes in humans. Recent studies support endoplasmic reticulum (ER) as a novel target for preventing the initiation of the autoimmune reaction, propagation of inflammation, and β cell death in type 1 diabetes. This review highlights recent findings on ER stress in β cells and development of type 1 diabetes and introduces potential new treatments targeting the ER to combat this disorder.

Hypercalcemia in Patients with Williams-Beuren Syndrome

OBJECTIVE To evaluate the timing, trajectory, and implications of hypercalcemia in Williams-Beuren syndrome (WBS) through a multicenter retrospective study. STUDY DESIGN Data on plasma calcium levels from 232 subjects with WBS aged 0-67.1 years were compared with that in controls and also with available normative data. Association testing was used to identify relevant comorbidities. RESULTS On average, individuals with WBS had higher plasma calcium levels than controls, but 86.7% of values were normal. Nonpediatric laboratories overreport hypercalcemia in small children. When pediatric reference intervals were applied, the occurrence of hypercalcemia dropped by 51% in infants and by 38% in toddlers. Across all ages, 6.1% of the subjects had actionable hypercalcemia. In children, actionable hypercalcemia was seen in those aged 5-25 months. In older individuals, actionable hypercalcemia was often secondary to another disease process. Evidence of dehydration, hypercalciuria, and nephrocalcinosis were common in both groups. Future hypercalcemia could not be reliably predicted by screening calcium levels. A subgroup analysis of 91 subjects found no associations between hypercalcemia and cardiovascular disease, gastrointestinal complaints, or renal anomalies. Analyses of electrogradiography data showed an inverse correlation of calcium concentration with corrected QT interval, but no acute life-threatening events were reported. CONCLUSIONS Actionable hypercalcemia in patients with WBS occurs infrequently. Although irritability and lethargy were commonly reported, no mortality or acute life-threatening events were associated with hypercalcemia and the only statistically associated morbidities were dehydration, hypercalciuria, and nephrocalcinosis.

Recurrent hypoglycemia: boosting the brain's metabolic flexibility.

For people with diabetes, recurrent episodes of hypoglycemia limit the brains ability to sense dangerously low blood sugar levels. In this issue of the JCI, the mechanisms behind this clinical problem of hypoglycemia unawareness are addressed by Herzog et al. The authors provide compelling evidence that recurrent hypoglycemia enhances transport of lactate into the brain and, although not itself a major alternative fuel source, lactate may preserve neuronal function during hypoglycemia by maintaining neuronal glucose metabolism. These findings redefine our understanding of the brains metabolic adaptations that result from recurrent hypoglycemia.

Increased opioid receptor binding and G protein coupling in the accumbens and ventral tegmental area of postnatal day 2 rats

In some regions of the developing rat brain such as the nucleus accumbens (Acb), mu opioid (MOP) receptor specific binding in the perinatal period exceeds that in the adult. To investigate the significance of these developmental changes, MOP and nociceptin/orphanin FQ (NOP) receptor binding and G protein coupling as determined by GTPgammaS binding experiments were examined in mesolimbic regions of postnatal day 2 (P2) pups and compared to those of their dams. Acb of the P2 pup exhibited 2-fold greater MOP receptor specific binding than that of the dam. In the ventral tegmental area (VTA), NOP specific binding was about 2-fold higher in the P2 pup. A correlation was found between MOP and NOP binding and their coupling to G protein on dam and P2 pup brain sections. However, the magnitude of increases in MOP and NOP receptor G protein coupling to G protein in P2 pups exceeded the 2-fold differences in binding between pups and dams. Furthermore, the amplitude of the MOP receptor G protein coupling in female P2 Acb was greater than increases in male P2 pup Acb. Differences in MOP and NOP binding and G protein coupling in other mesolimbic regions between P2 pups and dams were rarely observed. The data indicate that greater binding and G protein coupling of MOP and NOP receptors occur in discrete, mesolimbic regions of P2 pups when compared to their dams. It may be of significance that these brain regions, Acb and VTA, are undergoing maturation on P2.

Targeting cellular calcium homeostasis to prevent cytokine-mediated beta cell death

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.