Two approaches for addressing electrochemical electrode arrays with reduced external connections.

Although patterning hundreds or thousands of electrochemical electrodes on lab-on-a-chip devices is straightforward and cost-effective using photolithography, easily making connections between hundreds of electrodes and external amplifiers remains a bottleneck. Here we describe two electrode addressing approaches using multiple fluid compartments that can potentially reduce the number of external connections by ~100-fold. The first approach enables all compartments on the device to be filled with solution at the same time, and then each fluid compartment is sequentially electrically activated to make the measurements. The second approach achieves lower measurement noise by sequentially filling recording chambers with solution. We propose an equivalent circuit to explain measurement noise in these recording configurations and demonstrate application of the approaches to measure quantal exocytosis from individual cells. A principle advantage of using these approaches is that they reduce the fraction of the microchip area that needs to be dedicated to making external connections and therefore reduces the cost per working electrode.

Measuring secretion in chromaffin cells using electrophysiological and electrochemical methods.

Our present understanding of exocytosis of catecholamines has benefited tremendously from the arrival of single-cell electrochemical methods (amperometry and voltammetry), electrophysiological techniques (whole-cell and patch capacitance) and from the combination of both techniques (patch amperometry). In this brief review, we will outline the strengths and limitations of amperometric and electrophysiological methods and highlight the major contribution obtained with the use of these techniques in chromaffin cells.

Naturally occurring Sarcocystis infection in domestic cats (Felis catus).

Equine protozoal myeloencephalitis is an important neurological disease of horses in the United States. Consequently, there is an active research effort to identify hosts associated with the primary causative agent, Sarcocystis neurona. The purpose of this study was to determine whether the domestic cat (Felis catus) is a natural host for S. neurona. Muscle sections from 50 primarily free-roaming domestic cats were examined for the presence of sarcocysts. Serum from cats in this group and another group of 50 free-roaming cats were evaluated for the presence of S. neurona antibody. Sarcocysts were found in five of 50 (10%) cats, and S. neurona antibody in five of 100 (5%) cats. Morphological, molecular (including ribosomal RNA genes), and biological characterisation of these sarcocysts showed that they were not S. neurona or S. neurona-like. Sarcocysts found in the cats were identified morphologically as Sarcocystis felis, a common parasite of wild felids. The life cycle of S. felis is not known, and prior to this study, no molecular marker for S. felis existed. Although cats were found to be infected with S. felis sarcocysts, serological data provided evidence of possible infection with S. neurona as well. Further work is needed to determine the role of the domestic cat in the life cycle of S. neurona.

The relationship between cAMP, Ca(2)+, and transport of CFTR to the plasma membrane.

The mechanism whereby cAMP stimulates Cl(-) flux through CFTR ion channels in secretory epithelia remains controversial. It is generally accepted that phosphorylation by cAMP-dependent protein kinase increases the open probability of the CFTR channel. A more controversial hypothesis is that cAMP triggers the translocation of CFTR from an intracellular pool to the cell surface. We have monitored membrane turnover in Calu-3 cells, a cell line derived from human airway submucosal glands that expresses high levels of CFTR using membrane capacitance and FM1-43 fluorescence measurements. Using a conventional capacitance measurement technique, we observe an apparent increase in membrane capacitance in most cells that exhibit an increase in Cl(-) current. However, after we carefully correct our recordings for changes in membrane conductance, the apparent changes in capacitance are eliminated. Measurements using the fluorescent membrane marker FM1-43 also indicate that no changes in membrane turnover accompany the activation of CFTR. Robust membrane insertion can be triggered with photorelease of caged Ca(2)+ in Calu-3 cells. However, no increase in Cl(-) current accompanies Ca(2)+-evoked membrane fusion. We conclude that neither increases in cAMP or Ca(2)+ lead to transport of CFTR to the plasma membrane in Calu-3 cells. In addition, we conclude that membrane capacitance measurements must be interpreted with caution when large changes in membrane conductance occur.

The noise of membrane capacitance measurements in the whole-cell recording configuration.

High-resolution measurement of membrane capacitance in the whole-cell-recording configuration can be used to detect small changes in membrane surface area that accompany exocytosis and endocytosis. We have investigated the noise of membrane capacitance measurements to determine the fundamental limits of resolution in actual cells in the whole-cell mode. Two previously overlooked sources of noise are particularly evident at low frequencies. The first noise source is accompanied by a correlation between capacitance estimates, whereas the second noise source is due to "1/f-like" current noise. An analytic expression that summarizes the noise from thermal and 1/f sources is derived, which agrees with experimental measurements from actual cells over a large frequency range. Our results demonstrate that the optimal frequencies for capacitance measurements are higher than previously believed. Finally, we demonstrate that the capacitance noise at high frequencies can be reduced by compensating for the voltage drop of the sine wave across the series resistance.

Admittance-based measurement of membrane capacitance using the EPC-9 patch-clamp amplifier.

A software lock-in amplifier (SLIA) was developed to allow high-time-resolution measurement of membrane capacitance as a single-cell assay of exocytosis. The unique feature of this "virtual instrument" is that it is thoroughly integrated with a computer-controlled patch-clamp amplifier (EPC-9) to allow estimation of equivalent circuit parameters based upon calibrated admittance measurements rather than just relative changes. Since the same software package ("PULSE") controls both the EPC-9 and the SLIA, instrument settings which affect admittance calculations (gain, filtering, etc.) are always "known" by the SLIA. Attenuation and phase shifts introduced within the EPC-9 by low-pass filters and other circuitry are modelled and automatically corrected by the software. In addition, changes in the measured signal introduced by whole-cell capacitance and series resistance compensation are accounted for. The noise of capacitance measurements is nearly optimal and resistive parameters can vary over a large range without inducing artifactual changes in capacitance estimates.

Effect of eltenac in horses with induced endotoxaemia.

Ten horses were used in a crossover study to evaluate the effectiveness of eltenac against endotoxaemia. Eltenac (0.5 mg/kg bwt) or saline control was given i.v. then 15 min later, intravenous infusion of endotoxin was begun and continued for 120 min (total dose 100 ng/kg bwt). Horses were monitored for heart and respiratory rates, pulmonary and carotid arterial pressure and core body temperature. Blood was sampled at intervals for measurement of haematological variables and plasma concentrations of lactate, prostanoid metabolites, tumour necrosis factor (TNF) and stress hormones. In comparison with saline-treatment, use of eltenac significantly protected against endotoxin-induced changes in respiratory rate, core temperature, systemic arterial blood pressure (SAP), pulmonary arterial pressure, PCV, and plasma protein, 6-keto prostaglandin F1 alpha, thromboxane B2, epinephrine, and cortisol concentrations. Despite statistical effect of eltenac on SAP, values in both treatment groups remained well above baseline throughout the evaluation period. Significant protective effect of eltenac was not found for heart rate, white blood cell count, plasma lactate concentration or TNF activity. On the basis of these results, it is expected that use of eltenac will provide clinical benefit in horses with naturally occurring endotoxaemia.

Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme.

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant (tauc) decreased with increasing [ATP] to a minimum value of approximately 0.43 s at [ATP] >1.00 mM. The open time constant (tauo) increased with increasing [ATP] with a minimal tauo of approximately 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of approximately 250 ms is dominant at low ATP concentrations (10 microM) and a much longer open state with a time constant of approximately 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent.

Mechanisms of transmission of Aujeszky's disease virus originating from feral swine in the USA.

To understand the possible mechanisms of transmission of Aujeszky's disease virus (pseudorabies or PRV) from a feral pig reservoir, intranasal infections were initiated in domestic pigs and in pigs from a herd derived from captured feral pigs. Virus strains originating from feral pigs and from domestic pigs were compared. Similar shedding patterns were obtained in both feral-derived and domestic pigs, however, virus strains from feral pigs were markedly attenuated. Virus could be isolated after acute infection from nasal secretions, tonsils and occasionally from genital organs. In studies of transmission of PRV by cannibalism, either latently infected or acutely infected tissue was fed to both domestic and feral-derived pigs. In two similar experiments, latently infected tissue did not transmit virus, but tissues from acutely infected pigs did transmit infection. Cannibalism was observed typically in both types of pigs older than 6 weeks of age. It was concluded that transmission of PRV originating from feral pigs can occur by several mechanisms including the respiratory route and by cannibalism of pigs that die of acute infection. Transmission of PRV from feral swine may, however, result in sub-clinical infection.

Kinetics of exocytosis in adrenal chromaffin cells.

Upon repetitive or maintained stimulation, chromaffin cells secrete catecholamines initially at a very high rate which then relaxes with multiple kinetic components. The complex kinetics are often modeled as resulting from the successive depletion of several functional pools of secretory granules which may reflect specific protein-mediated steps in granule maturation. The fastest component represents granules fully primed for exocytosis. This 'readily releasable pool' may, under some circumstances, consist of only about a dozen granules which can be released within tens of milliseconds. Modulating the size of this pool may be an important way for cells to regulate secretion.

Current trends in physicians' practice arrangements. From owners to employees.

OBJECTIVE: To examine current trends in practice organization among postresident patient care physicians in the United States. DESIGN AND SETTING: The American Medical Association's Socioeconomic Monitoring System (SMS), a series of periodic surveys of nationally representative samples of nonfederal postresident patient care physicians. Physicians were divided into 3 categories based on the organization of their main practice. They were classified as employee physicians if they had no ownership interest in their practice, as self-employed solo physicians if they were in 1-physician practices in which they had an ownership interest, and as self-employed group physicians if they were in multiple-physician practices in which they had an ownership interest. PARTICIPANTS: Nonfederal, postresident patient care physicians who provided more than 47 000 responses to SMS surveys between 1983 and 1994. MAIN OUTCOME MEASURE: The proportion of nonfederal postresident patient care physicians who were employees between 1983 and 1994. RESULTS: Between 1983 and 1994, the proportion of patient care physicians practicing as employees rose from 24.2% to 42.3% (P<.001), the proportion self-employed in solo practices fell from 40.5% to 29.3% (P<.001), and the proportion self-employed in group practices fell from 35.3% to 28.4% (P<.001). Most of these changes occurred in the latter half of the 12-year period. These trends, which are evident in virtually every segment of the patient care physician population, are especially prominent among young physicians. The growing proportion of employee physicians is associated with increases in the earnings of employee physicians relative to those of self-employed solo physicians. CONCLUSIONS: Current trends in the US health care system are rapidly changing the career opportunities of patient care physicians and, hence, physicians' choice of practice arrangement.

Protein kinase C enhances exocytosis from chromaffin cells by increasing the size of the readily releasable pool of secretory granules.

We have used membrane capacitance measurements to assay Ca2+-triggered exocytosis in single bovine adrenal chromatin cells. Brief application of phorbol ester (PMA) enhances depolarization-evoked exocytosis severalfold while actually decreasing the Ca2+ current. Ca2+ metabolism is unchanged. Three different protocols were used to show that PMA increases the size of the readily releasable pool of secretory granules. PMA treatment leads to a large increase in amplitude, but little change in the time course of the exocytic burst that results from rapid elevation of [Ca2+]i upon photolysis of DMI-Nitrophen. Thus, PKC appears to affect a late step in secretion but not the Ca2+ sensitivity of the final step.

Use of enzyme-linked immunosorbent assay and radioimmunoassay to determine serum and urine dexamethasone concentrations in thoroughbreds after intravenous administration of the steroid.

OBJECTIVE: To develop a simple and sensitive ELISA for detection of dexamethasone in horse serum and urine. SAMPLE POPULATION: Blood and urine samples from 3 thoroughbred mares. PROCEDURE: A dexamethasone oxime was prepared and conjugated to hemocyanin, bovine serum albumin and to horseradish peroxidase. One- and two-step double-antibody ELISA methods, as well as a radioimmunoassay method, were performed. The one-step ELISA was used to test urine from 3 Thoroughbred mares injected with 5 mg of dexamethasone, IV. RESULTS: The ELISA could detect dexamethasone in the range of 0.01 to 50 ng/ml, with intra- and interassay variations of 8.92 and 9.42%, respectively. Serum dexamethasone concentration reached a peak of 20 to 35 ng/ml 15 minutes after steroid administration and decreased to 1 ng/ml in 2.5 hours. Urine dexamethasone concentration 18 to 50 ng/ml 1 to 2 hours after drug administration and decreased to 1 ng/ml at 10 hours. CONCLUSION: The developed assay is sensitive as well as simple for detecting dexamethasone in horse serum and urine, and is comparable to radioimmunoassay. CLINICAL RELEVANCE: This method can be useful for screening samples from racehorses, because it is sensitive and does not require sample preparation or sophisticated equipment.

Characterization of a prolactin-synthesizing rat pituitary cell line immortalized by the temperature-sensitive mutant SV40.

A prolactin (PRL) synthesizing rat anterior pituitary (AP) cell line named tAP-CLC-2 was established by transformation with mutant temperature-sensitive A (tsA) simian virus 40 (SV40). The transformed cells were temperature-sensitive for morphology, cell propagation, and expression of phenotypic genes. Monolayer primary rat AP cells were transfected with a mutant SV40 (tsA 205) at 10 times the multiplicity of infection (MOI) at 34 degrees C. After cell clones formed, each clone was isolated and transferred to separate flask until sufficient cells were grown for freezing and characterization. At the permissive temperature (34 degrees C), these cells were spindle-shaped and grew rapidly like tumor cells. However, at the nonpermissive temperature (40 degrees C), the cells exhibited a rounded shape and ceased to propagate because the gene for maintenance of transformation was not expressed. Cell extracts from the cell line tAP-CLC-2 showed an inhibition curve parallel to that of normal rat pituitary cell extracts in a PRL radioimmunoassay (RIA). The gel filtration profile of immunoassayable PRL obtained from fast performance liquid chromatography (FPLC) showed that tAP-CLC-2 cell extract exhibited a PRL peak coincidental with primary rat pituitary cell extracts. Western blot showed that cell extracts from the tAP-CLC-2 cell line and from normal rat pituitary glands shared a similar, major immunoreactive PRL band. The tAP-CLC-2 cell line was responsive to estradiol (E 2; 10(-7) M), progesterone (10(-8) M), and gonadotropin-releasing hormone (GnRH; 10(-9) M) treatments. These hormonal treatments increased (p < 0.05) cell PRL content. Interestingly, treatment with a high dose (10(-7) M) of ovine luteinizing hormone (oLH) also increased (p < 0.05) PRL content in the cell line; a low dose (10(-9) M) of oLH did not. The cell line appeared to synthesize growth hormone (GH) as well. These results indicate this cell line has properties shared by primary AP cells and can provide a unique model for the study of the synthesis and gene regulation of PRL in vitro.

Physician responses to Medicare payment reform: an update on access to care.

This paper updates previous research on physician-based measures of access to care for Medicare beneficiaries following the implementation of Medicare payment reform in 1992. Using data collected in spring 1993, the results show that the majority of physician-based access indicators did not change significantly from 1992 levels. Those indicators that did change generally offset reductions in access identified immediately after the implementation of Medicare physician payment reform.

Enhancers of cytosolic cAMP augment depolarization-induced exocytosis from pancreatic B-cells: evidence for effects distal to Ca2+ entry.

We have investigated the effects of cAMP-enhancing agents on depolarization-induced membrane capacitance increases (delta Cm) in single rat pancreatic B-cells. Concentrations of IBMX, 8-CPT cAMP and forskolin, which enhance cAMP and insulin release, all enhance depolarization-induced delta Cm's seen in response to single voltage-clamp pulses and reduce the depression of delta Cm responses often seen with trains of pulses. These effects often occur in the absence of changes in peak Ca2+ current or the total Ca2+ charge entry during the depolarizing pulse. These data suggest that cAMP-modulating maneuvers may directly affect the mechanism of insulin granule mobilization into a readily releasible store or fusion at a discharge site.

Physician responses to Medicare physician payment reform: preliminary results on access to care.

This paper examines the impact of Medicare physician payment reform on access to care by comparing several physician-based access measures in the pre- and post-reform periods. The results suggest that the broad goals of payment reform may have been at least partially achieved: the proportion of physician revenues derived from Medicare increased for primary care physicians and decreased for nonprimary care MDs; there was little change in the absolute or relative number of visits provided to Medicare patients; and an increasing number of physicians charged no more than the Medicare payment amount. Some signs of deteriorating access were found, however. Fewer physicians were willing to treat all new Medicare patients and more physicians accepted no new Medicare patients. Furthermore, there was an increase in the proportion of physicians who reduced or stopped providing to Medicare patients certain types of services that they continued to provide to other patients.

Assessing the validity of the geographic practice cost indexes.

Data on physician practice inputs were used to test the degree to which the geographic practice cost indexes (GPCIs) of the Medicare physician payment schedule reflect geographic variation in input prices. For purposes of this study, input quantity information was collected through the American Medical Association's Socioeconomic Monitoring System survey in 1990 and 1991. These data, along with practice expense information, were used to construct unit input prices. The GPCIs were correlated with input prices; however, "real" or GPCI-adjusted prices varied significantly across locations. We conclude that the GPCIs are useful, but imperfect measures of geographic differences in physician practice input prices.

Electrophysiology of stimulus-secretion coupling in human beta-cells.

Herein, we review the applicability to human beta-cells of an electrophysiologically based hypothesis of the coupling of glucose metabolism to insulin secretion. According to this hypothesis, glucose metabolism leads to the generation of intracellular intermediates (including ATP), which leads to closure of ATP-sensitive K+ channels. Channel closure results in membrane depolarization, the onset of electrical activity, and voltage-dependent Ca2+ entry. The resultant rise in cytosolic Ca2+ leads to Ca(2+)-dependent exocytosis of insulin granules. We found that most of the published experimental evidence for human beta-cells supports this hypothesis. In addition, we present three other emerging lines of evidence in support of this hypothesis for human islet beta-cells: 1) the effects of pHi-altering maneuvers on insulin secretion and electrical activity; 2) preliminary identification of LVA and HVA single Ca2+ channel currents; and 3) validation of the feasibility of Cm measurements to track insulin granule exocytosis. On the basis of this last new line of evidence, we suggest that combinations of Cm measurements and electrical activity/membrane current measurements may help define the roles of diverse electrical activity patterns, displayed by human beta-cells, in stimulus-induced insulin secretion.

Stimulus-secretion coupling in beta-cells of transplantable human islets of Langerhans. Evidence for a critical role for Ca2+ entry.

With human islets isolated for transplantation, we examined the applicability to humans of a metabolic fuel hypothesis of glucose transduction and a Ca2+ hypothesis of depolarization-secretion coupling, both previously proposed for rodent islet beta-cells. We report that several features of human beta-cell physiology are well accounted for by these hypotheses. With whole-islet perifusion, we demonstrated that insulin secretion induced by glucose, tolbutamide, or elevated K+ is dependent on extracellular Ca2+. Insulin release induced by these secretagogues is enhanced by the dihydropyridine Ca2+ channel agonist BAYk8644 and depressed by the dihydropyridine Ca(2+)-channel antagonist nifedipine. All of the aforementioned secretagogues provoke increases in cytosolic free Ca2+, which are dependent on extracellular Ca2+ and are altered by the dihydropyridine drugs. Individual beta-cells in the islet display diminished resting membrane conductance, graded depolarization, and complex electrical patterns, including bursts of action potentials in response to stimulatory concentrations of glucose or tolbutamide. Individual islet beta-cells display voltage-dependent Ca2+ currents that are activated at membrane potentials traversed during the excursion of the action potential. In most cells, the Ca2+ currents are enhanced by BAYk8644 and depressed by nifedipine at concentrations that have parallel effects on secretagogue-induced increases in cytosolic Ca2+ and insulin secretion. These survey studies should provide the basis for more detailed investigations of the relationship of voltage-dependent ionic currents to electrical activity patterns and of electrical activity patterns to granule exocytosis in single human beta-cells.