Temporal monitoring of intracellular Ca2+ signaling and origins of Ca2+ oscillations

Abstract

This thesis examined parameters influencing stimulated cytoplasmic free Ca2+ concentration ([Ca2+]i) oscillations in hepatocytes and pancreatic beta-cells. Hepatic glucose output is regulated in part by hormones such as vasopressin that act through [Ca 2+]i oscillations. Pulsatile [Ca2+]i in beta-cells parallels insulin secretion and this results in potently controlled blood glucose homeostasis. Employing temporal [Ca2+]i measurements and related biochemical assays, efforts were made to understand how [Ca2+]i transients are generated and maintained.

In studies of hepatocyte [Ca2+]i signaling, we have demonstrated the existence of a phospholipase C (PLC) linked receptor driven Ca2+ entry pathway that, unlike capacitative Ca2+ entry, is independent of Ca2+ release from intracellular stores. These findings were challenged on the grounds that we used the cell permeant fluorescent Ca2+ indicator Fura-2 AM, which in some cases can be compartimentalized. The microinjection technique was established to rapidly introduce cell impermeant fura-2 into hepatocytes and subsequently monitor [Ca 2+]i. Extracellular Mn2+, which quenches fura-2 fluorescence upon entering the cell, was used as a Ca 2+ surrogate. Following depletion of intracellular Ca2+ stores by thapsigargin or 2,5di-(tbutyl)hydroquinone, vasopressin accelerated Mn2+ influx. Microinjected or loaded indicator yielded similar results. The source of Ca 2+ was extracellular. Hepatocytes therefore possess at least one carrier for extracellular Ca2+ entry into the cytosol (i.e., non-capacitative) in addition to the capacitative Ca 2+ entry pathway.

The methodology used above was extended to pancreatic beta-cells to determine if the second messenger cyclic adenosine diphosphate ribose (cADPR) influences glucose induced [Ca2+]i changes. Glucose was fully able to increase [Ca2+]i whether fura-2 was microinjected alone, or together with cADPR or its competitive inhibitor 8NH2-cADPR. Similar results were obtained with carbamylcholine and KC1. cADPR did not alter insulin release at any [Ca2+] in permeabilized beta-cells. A direct and rapid role for cADPR in normal insulin secretion was rejected.

The possibility that oscillatory insulin secretion is caused by glycolytic oscillations generated by muscle phosphofructokinase (PFK-M) was examined. According to this model, islet PFK-M experiences periodic bursts in activity via autocatalytic activation by its product, fructose 1,6-bisphosphate (F16BP). Primary pancreatic beta-cells were treated with dihydroxyacetone (DHA), which enters glycolysis as the glycolytic intermediate dihydroxyacetone phosphate (DHAP). DHA was found to trigger [Ca2+]i oscillations, most likely by increasing [F16BP] into the autocatalytic range at which PFK-M can oscillate. Substimulatory glucose (4 mM) was required in addition to DHA, indicating that glycolytic flux through PFK-M is obligatory. Attempts to measure [F16BP] in response to both glucose and DHA revealed large variability in this parameter. [Ca2+]i oscillations tightly paralleled those of the ATP/ADP ratio.

Metabolic features of a transgenic mouse with a disruption in the promoter region of the plan gene were studied. This animal displayed tissue-specific loss of PFK-M functional expression with about 95% reduction in pancreatic islets. Glucose induced [Ca2+]i oscillations were unaltered, but average amount and amplitude of insulin secretion were impaired. This might be explained by observations that maximum glucose utilization is about 100 times below PFK Vmax. This animal displayed impaired glucose tolerance in vivo without insulin resistance. The incomplete loss of PFK-M combined with impaired insulin secretion without impairment in [Ca2+]i response suggested a minor impediment in glycolytic flux that is more detrimental to insulin exocytosis than [Ca2+]i transients. No difference in the ATP/ADP ratio was detected.

Because fructose 2,6-bisphosphate (F26BP) can serve as a potent PFK-M activator, this was examined in beta-cells. Microinjection of F26BP did not alter subsequent glucose induced [Ca 2+]i oscillations, consistent with published knowledge that the affinity of F26BP binding to PFK-M is reduced by the high intracellular citrate levels thought to exist in beta-cells. An enzymatic assay established to measure [F26BP] in small numbers of islets indicated that [F26BP] remains at or below 3 µM in islets cultured at 11 mM glucose. Thus, at physiological glucose levels, [F26BP] is relatively constant in islets and cannot in any event affect [Ca2+]i oscillations.

The hepatocyte study demonstrated that multiple Ca2+ entry pathways are activated by hormones known to trigger [Ca2+]i oscillations. This could offer different permutations for driving and maintaining oscillations. This is likely applicable to other cell types. The pancreatic beta-cell studies collectively show that glucose metabolism is a prerequisite for the slow [Ca2+]i oscillations that parallel pulsatility in insulin release. Moreover, insulin secretion is more sensitive to reduced islet PFK-M expression than are increases in ATP/ADP ratio and oscillations in [Ca2+]i.