We examined the effects of hypoxia on the release of dopamine (DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cells and assessed the involvement of Ca2+ and protein kinases in stimulus-secretion coupling. Catecholamine release was monitored by microvoltammetry using a carbon fiber electrode as well as by HPLC coupled with electrochemical detection (ECD). Microvoltammetric analysis showed that hypoxia-induced catecholamine secretion (Po 2 of medium ∼40 mmHg) occurred within 1 min after the onset of the stimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysis revealed that, at any level of Po 2, the release of NE was greater than the release of DA. In contrast, in response to K+ (80 mM), DA release was ∼11-fold greater than NE release. The magnitude of hypoxia-induced NE and DA releases depended on the passage, source, and culture conditions of the PC-12 cells. Omission of extracellular Ca2+ or addition of voltage-gated Ca2+ channel blockers attenuated hypoxia-induced release of both DA and NE to a similar extent. Protein kinase inhibitors, staurosporine (200 nM) and bisindolylmaleimide I (2 μM), on the other hand, attenuated hypoxia-induced NE release more than DA release. However, protein kinase inhibitors had no significant effect on K+-induced NE and DA releases. These results demonstrate that hypoxia releases catecholamines from PC-12 cells and that, for a given change in Po 2, NE release is greater than DA release. It is suggested that protein kinases are involved in the enhanced release of NE during hypoxia.

No abstract

Diffusional microtitration can be used to precisely titrate extremely small samples from tens of microliters to a few hundred femtoliters. A cylindrical diffusion membrane of macroscopic dimensions is used for reagent delivery into macroscopic samples (>100 nL). Microscopic samples (<100 nL) are titrated with a microscopic device called a diffusional microburet, DMB. This is a pulled glass capillary filled with the reagent solution and a miniature agar gel plug in its conical tip. Apart from a negligibly short initial transient period, the reagent diffuses through this plug into the sample at a constant rate. This results in a linear DMB calibration with a negligible offset term. This experimental observation is quantitatively predicted by a mathematical model derived in this work for diffusive mass transport through a conical membrane. The delivery rate is limited by the taper angle and inner diameter

Microscopic aqueous sample droplets of nano- and picoliter volumes were formed on the bottom of a polystyrene dish under water-saturated heptane. The electrochemical cell consisted of a beveled carbon fiber microdisk working electrode and a reference electrode with a miniature junction, both inserted into the studied droplet. Both electrodes had a nominal tip diameter of 7.5 microns. Cyclic voltammetry and chronoamperometry in droplets of 3.3 mM ruthenium hexaammine trichloride in 0.1 M KCl solution were performed with this system. The experiments revealed for the first time major deviations in both voltammetric and amperometric microelectrode behavior in picoliter domains as compared to nanoliter volume and bulk solution. The concept and criteria of electrochemical microscopicity of volume is discussed. This work also provides a simple and robust experimental model system to verify electrochemical experiments in restricted domains such as in or near single biological cells or in microscopic tissue cavities. The methodology developed has, however, more general analytical and physicochemical applications as well.

Acid/base titrations of pico- and femtoliter microsamples have been performed previously using a diffusional microburet (DMB) for reagent delivery in a simple droplet-heptane system (Gratzl, M.; Yi, C. Anal. Chem. 1993, 65, 2085-2088). The lowest delivery rate achieved with a DMB was about 6 fmol/s, which would correspond to about a 1 microL/year volumetric flow rate with a hypothetical equivalent mechanical delivery scheme (Yi, C.; Gratzl, M. Anal. Chem. 1994, 66, 1976-1982). In this work, the feasibility of complexometric titrations in microscopic samples is explored. Stability of pH in the microdroplets required for different determinations and the effects of DMB shank geometry on titration characteristics are also studied. Diffusional microtitrations of Fe(III), Zn(II), and Cu(II) have been performed with EDTA. Xylenol orange and Eriochrome Black T provide clear color changes at the end point of the respective titrations, despite the microscopic size of the samples (between 16 and 1570 pL, corresponding to diameters between 30 and 144 microns). Random errors of the determinations relative to full scale were 6.6% for Fe(III), 5.8% for Cu(II), and 7.9% for Zn(II). The pH required for EDTA titrations of the individual metal ions stays stable in the acidic range. This makes the microscopic titration of a number of metal ions, such as Fe(III), Fe(II), Cu(II), and Pb(II), feasible in a simple droplet-heptane system without any modification. With a higher density of strongly alkaline buffer droplets (about 100 droplets/mm2) sprayed on the bottom of the Petri dish, or by flushing N2 above the heptane, the microscopic samples can also be kept alkaline despite ambient CO2 present. In this way, Zn(II) can also be titrated in microdroplets, requiring a pH around 10. This work renders it possible to perform a variety of complexometric titrations and other chemical manipulations in microdroplets even if they need to be kept alkaline. Similar titrations in single biological cells to assess intracellular buffer capacities of different metal ions, such as Ca(II) and Mg(II), are underway.