Ronald M Lynch
Work Summary
Precise diagnosis and treatment of disease requires an ability to target agents to specific tissues and cell types within those tissues. We are developing agents that exhibit cell type specificity for these purposes.
Precise diagnosis and treatment of disease requires an ability to target agents to specific tissues and cell types within those tissues. We are developing agents that exhibit cell type specificity for these purposes.
The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion.
Complications in pregnancy elevate fetal norepinephrine (NE) concentrations. Previous studies in NE-infused sheep fetuses revealed that sustained exposure to high NE resulted in lower expression of α2-adrenergic receptors in islets and increased insulin secretion responsiveness after acutely terminating the NE infusion. In this study, we determined if the compensatory increase in insulin secretion after chronic elevation of NE is independent of hyperglycemia in sheep fetuses and whether it is persistent in conjunction with islet desensitization to NE. After an initial assessment of glucose-stimulated insulin secretion (GSIS) at 129 ± 1 days of gestation, fetuses were continuously infused for seven days with NE and maintained at euglycemia with a maternal insulin infusion. Fetal GSIS studies were performed again on days 8 and 12. Adrenergic sensitivity was determined in pancreatic islets collected at day 12. NE infusion increased (P 0.01) fetal plasma NE concentrations and lowered (P 0.01) basal insulin concentrations compared to vehicle-infused controls. GSIS was 1.8-fold greater (P 0.05) in NE-infused fetuses compared to controls at both one and five days after discontinuing the infusion. Glucose-potentiated arginine-induced insulin secretion was also enhanced (P 0.01) in NE-infused fetuses. Maximum GSIS in islets isolated from NE-infused fetuses was 1.6-fold greater (P 0.05) than controls, but islet insulin content and intracellular calcium signaling were not different between treatments. The half-maximal inhibitory concentration for NE was 2.6-fold greater (P 0.05) in NE-infused islets compared to controls. These findings show that chronic NE exposure and not hyperglycemia produce persistent adaptations in pancreatic islets that augment β-cell responsiveness in part through decreased adrenergic sensitivity.
Melanocortin receptors can be used as biomarkers to detect and possibly treat melanoma. To these ends, molecules bearing one, two, or three copies of the weakly binding ligand MSH(4) were attached to scaffolds based on phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane by means of the copper-assisted azide-alkyne cyclization. This synthetic design allows rapid assembly of multivalent molecules. The bioactivities of these compounds were evaluated using a competitive binding assay that employed human embryonic kidney cells engineered to overexpress the melanocortin 4 receptor. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (∼2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed.
Although changes in both pH(in) and [Ca(2+)](i) have been observed in response to a variety of agonists, it is not clear whether these ionic events work independently or are coordinated to lead to a specific physiological response. One of the fundamental problems in studying these ionic events is that changes in pH(in) modify Ca(2+) regulatory mechanisms and changes in Ca(2+) may modify pH regulation. It is desirable to use a technique that allows concomitant monitoring of these two ions in cell populations with high time resolution. Furthermore, like many Ca(2+) binding proteins, all Ca(2+)-sensitive fluoroprobes are inherently sensitive to pH owing to competition of H(+) for the Ca(2+)-binding sites. This chapter describes experimental paradigms that provide optimum conditions for simultaneous measurement of pH from the fluorescence emission of snarf-1, and Ca(2+) using fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca(2+) both in vitro and in vivo. Moreover, the ratio of the H(+)-sensitive wavelengths of snarf-1 is unaffected by Ca(2+), or the concomitant presence of fura-2 in cells. Although the fluorescence ratio of fura-2 is insensitive to the presence of snarf-1, it is affected by pH, as indicated above. We describe procedures to correct for this effect and to obtain calibration parameters for fura-2 and snarf-1 required to facilitate analysis of pH and Ca(2+) concentrations within cell populations.
Bortezomib, a first-generation proteasome inhibitor, induces an endoplasmic reticulum (ER) stress response, which ultimately leads to dysregulation of intracellular Ca(2+) and apoptotic cell death. This study investigated the role of the Ca(2+)-dependent enzyme, calpain, in bortezomib cytotoxicity. A novel therapeutic combination was evaluated in which HIV protease inhibitors were used to block calpain activity and enhance bortezomib cytotoxicity in myeloma cells in vitro and in vivo.