Peggy Sue Bowman

Effects of Hypoxia on Isometric Force, Intracellular Ca2+, pH, and Energetics in Porcine Coronary Artery

When exposed to hypoxic conditions, coronary arteries dilate, which is an important protective response. Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we tested the major classes of hypotheses by measuring the effects of hypoxia on energetics, [Ca(2+)](i), K(+) channel function, and pH(i). Hypoxia relaxes porcine coronary arteries stimulated with either KCl or U46619. The extent of relaxation is dependent on both the degree and kind of stimulation. [Ca(2+)](i) was measured in endothelium-denuded arteries using fura 2-AM and ratiometric fluorescent techniques. At lower stimulus levels, hypoxia decreased both force and [Ca(2+)](i). Inhibitor studies suggest that K(Ca) and K(ATP) channels are not involved in the hypoxic relaxation, whereas K(V) channels may play a minor role, if any. Despite the hypoxia-mediated decrease in force, [Ca(2+)](i) was unchanged or increased at high levels of stimulation. Despite a marked increase in lactate content, pH(i) (measured with the ratiometric fluorescent dye BCECF) was also little affected by hypoxia. Measurement of the phosphagen and metabolite profile of freeze-clamped arteries with analytical isotachophoresis indicated that hypoxia increased lactate content by 4-fold and decreased phosphocreatine to 60% of control. However, neither ATP nor P(i) was affected by hypoxia. Interestingly, additional stimulation under hypoxia increased force but not ATP utilization, as estimated from measurements of anaerobic lactate production. Thus, surprisingly, the economy of force maintenance is increased under hypoxia. In porcine coronary artery, both Ca(2+)-dependent and, importantly, Ca(2+)-independent mechanisms are involved in hypoxic vasodilatation. For the latter, mechanisms involving either ATP, [Ca(2+)](i), pH(i), or P(i) cannot be invoked. This novel oxygen sensing mechanism involves a decreased Ca(2+) sensitivity.

Effects of hypoxia on [Ca2+]i, pHi and myosin light chain phosphorylation in guinea-pig taenia caeci.

1 Hypoxia (achieved by bubbling with N2 instead of O2) reduces the force of a KCl (40 mm)‐induced contracture to ?10 % of the control value in guinea‐pig taenia caeci. The underlying mechanism of this relaxation in response to hypoxia was investigated by measuring the major cell signalling parameters, intracellular Ca2+ concentration ([Ca2+]i) and myosin regulatory light chain (LC20) phosphorylation (MLC‐Pi), as well as intracellular pH (pHi), a factor often suggested to mediate hypoxic relaxation of muscle. 2 [Ca2+]i, measured using the ratiometric fluorescent dye fura‐2, increased when 40 mm KCl was added to physiological saline solution (PSS) (peak value assigned 100%), and the steady state after 15 min was 92.8 %. There were no detectable decreases in [Ca2+]i during hypoxia. 3 MLC‐Pi, measured using isoelectric focusing‐polyacrylamide gel electrophoresis and identified using Western blotting, increased from 9 % of the total LC20 in Ca2+‐free PSS to a peak value of 51 % in 40 mM KCl‐PSS. The steady‐state value in hypoxia of 43 % was not significiantly different from that in control oxygenated conditions at the same point in time. 4 pHi, measured using the ratiometric fluorescent dye 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF), under quiescent conditions (Ca2+‐free PSS) was 7.23 and increased to 7.36 with 40 mM KCl. After imposition of hypoxia pHi remained unchanged despite the known increase in both lactate content and production. 5 As [Ca2+]i and MLC‐Pi, key factors in activation, were not decreased by hypoxia and changes in pHi were minor, hypoxic relaxation in guinea‐pig taenia caeci appears to be directly related to energy limitation rather than any oxygen‐sensing mechanism.

Na+-K+-ATPase and Ca2+ clearance proteins in smooth muscle: a functional unit

The Na(+)-K(+)-ATPase (NKA) can affect intracellular Ca(2+) concentration regulation via coupling to the Na(+)-Ca(2+) exchanger and may be important in myogenic tone. We previously reported that in mice carrying a transgene for the NKA alpha(2)-isoform in smooth muscle (alpha(2sm+)), the alpha(2)-isoform protein as well as the alpha(1)-isoform (not contained in the transgene) increased to similar degrees (2-7-fold). Aortas from alpha(2sm+) mice relaxed faster from a KCl-induced contraction, hypothesized to be related to more rapid Ca(2+) clearance. To elucidate the mechanisms underlying this faster relaxation, we therefore measured the expression and distribution of proteins involved in Ca(2+) clearance. Na(+)-Ca(2+) exchanger, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and plasma membrane Ca(2+)-ATPase (PMCA) proteins were all elevated up to approximately fivefold, whereas actin, myosin light chain, and calponin proteins were not changed in smooth muscle from alpha(2sm+) mice. Interestingly, the corresponding Ca(2+) clearance mRNA levels were unchanged. Immunocytochemical data indicate that the Ca(2+) clearance proteins are distributed similarly in wild-type and alpha(2sm+) aorta cells. In studies measuring relaxation half-times from a KCl-induced contraction in the presence of pharmacological inhibitors of SERCA and PMCA, we estimated that together these proteins were responsible for approximately 60-70% of relaxation in aorta. Moreover, the percent contribution of SERCA and PMCA to relaxation rates in alpha(2sm+) aorta was not significantly different from that in wild-type aorta. The coordinate expressions of NKA and Ca(2+) clearance proteins without change in the relative contributions of each individual protein to smooth muscle function suggest that NKA may be but one component of a larger functional Ca(2+) clearance system.

Point-of-Care Device for Quantification of Bilirubin in Skin Tissue

Steady state diffuse reflectance spectroscopy is a nondestructive method for obtaining biochemical and physiological information from skin tissue. In medical conditions such as neonatal jaundice excess bilirubin in the blood stream diffuses into the surrounding tissue leading to a yellowing of the skin. Diffuse reflectance measurement of the skin tissue can provide real time assessment of the progression of a disease or a medical condition. Here we present a noninvasive point-of-care system that utilizes diffuse reflectance spectroscopy to quantifying bilirubin from skin reflectance spectra. The device consists of an optical system integrated with a signal processing algorithm. The device is then used as a platform to study two different spectral databases. The first spectral database is a jaundice animal model in which the jaundice reflectance spectra are synthesized from normal skin. The second spectral database is the spectral measurements collected on human volunteers to quantify the different chromophores and other physical properties of the tissue such as Hematocrit, Hemoglobin, etc. The initial trials from each of these spectral databases have laid the foundation to verify the performance of this bilirubin quantification device.

Device for Quantification of Bilirubin in Cerebral Spinal Fluid

In North America, an estimated 30 000 patients annually experience an aneurysmal subarachnoid hemorrhage (SAH). In approximately five percent of these patients, the hemorrhage is not visible on computerized tomography scans due to the inability to image blood at time intervals greater than 12 h post symptom onset. For these patients (many of which have experienced a sentinel hemorrhage that is a precursor to a more significant rupture), a method is needed for accurately analyzing cerebral spinal fluid (CSF) for evidence of SAH. Further, it is necessary to differentiate blood associated with the SAH from blood associated with the spinal tap procedure. This letter presents a point-of-care device that is capable of performing such an analysis. The stand-alone prototype device uses commercially available embedded system components to implement a point-of-care device that is capable of collecting and analyzing optical absorbance spectra. A mathematical model for the hemorrhagic CSF sample is then developed by using a partial-least-squares-regression-based regression methodology that is able to differentiate between SAH and blood associated with the spinal tap. This differentiation is achieved by quantifying bilirubin (associated with the breakdown of old blood) in the CSF. Initial testing on the prototype device suggests that the device is able to quantify bilirubin in the presence of hemoglobin over concentrations ranges that are clinically relevant to the patient population of interest.

Effects of Endothelium on Metabolic and Mechanical Parameters in Porcine Coronary Artery

This study examined the influence of the endothelium on metabolic and mechanical properties of porcine coronary arteries. Oxygen consumption rates (JO2) and lactate release (Jlac) under basal conditions were similar for intact (+E) and denuded (-E) rings. While passive tension was not different in2+E and -E arteries, the -E rings did develop a significant level of basal tone. With 50 mM KC1 stimulation, JO2 and Jlac in both +E and -E arteries increased to similar levels, and developed force also was not different. While calculated total rates of ATP utilization (JATP) showed an increase in both +E and -E rings with 50 mM KCI stimulation, there were no differences attributable to the presence of the endothelium. In summary, the endothelium does not significantly alter the metabolic or mechanical properties of porcine coronary arteries, although it does appear to play a role in the suppression of basal tone.

Improved quantification of CSF bilirubin in the presence of hemoglobin using least squares curve-fitting

Subarachnoid hemorrhage (SAH) is a dangerous neurological event with a very short time window for early diagnosis. Clinical diagnoses performed in a lab seek to quantify bilirubin in cerebrospinal fluid (CSF) as a biomarker for SAHs; however laboratory assays suffer from lengthy protocols, interference from hemoglobin, and the availability of expertise. Substantial improvements in the determination of bilirubin concentration in the presence of he moglobin in CSF are demonstrated in this work. Concentration estimates within 15% for bilirubin in the range of 0.2 to 1.6 mg /dl were determined for CSF samples containing fresh hemoglobin concentrations ranging from 0.05 to 0.25 g/dl. To demonstrate extensibility of the system with respect to more complete mock SAH samples, sample sets with one additional species of both hemoglobin and bilirubin, methemoglobin and alpha-bilirubin, respectively, were tested and yielded results within 25% of actual values, as measured by standard chemical assays of preparations prior to mixing.

Development of a point-of-care device for the quantification of bilirubin in cerebral spinal fluid

In North America, an estimated 30,000 patients annually experience an aneurysmal subarachnoid hemorrhage (SAH). In approximately five percent of these patients, the hemorrhage is not visible on computerized tomography scans due to the inability to image blood at time intervals greater than 12 hours post symptom onset. For these patients (many of which have experience a sentinel hemorrhage that is a precursor to a more significant rupture) a method is needed for accurately analyzing cerebral spinal fluid (CSF) for evidence of SAH. Further, it is necessary to differentiate blood associated with the SAH from blood associated with the spinal tap procedure. This paper presents the development of a point-of-care device that is capable of performing such an analysis. The stand alone prototype device uses commercially available embedded system components to implement a hardware platform that is capable of collecting and analyzing optical absorbance spectra. A mathematical model for the hemorrhagic CSF sample is then developed using a PLSR based regression methodology that is able to differentiate between SAH and blood associated with the spinal tap. This differentiations in achieved by quantifying bilirubin (associated with the breakdown of old blood) in the CSF. Initial testing on the prototype device suggests that the device is able to quantify bilirubin in the presence of hemoglobin over concentrations ranges that are clinically relevant to the patient population of interest.