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Biomedical subjects

J M Beach

Publications and source records attributed to J M Beach.

14 recordsLinked to original sources
PubMed2001

Fluorescence emission spectral shift measurements of membrane potential in single cells.

Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between -80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.

Cell Line↗
PubMed2001

Role of calcium in acute hyperthermic myocardial injury.

INTRODUCTION: We hypothesized that intracellular calcium overload may play an important role in heat-induced myocardial injury. This postulate was investigated using a model of isolated guinea pig papillary muscle in which resting tension was measured as an indirect indicator of cytosolic free-calcium concentration and the fluorescence changes of Fluo-3 AM dye was measured as a direct indicator of cytosolic free-calcium concentration. METHODS AND RESULTS: Excised guinea pig right ventricular papillary muscles were attached to a force transducer in a high-flow tissue bath and superfused with Tyrode's solution at 37 degrees +/- 0.5 degrees C. The temperature was rapidly changed to between 38.0 degrees and 56.0 degrees C for 60 seconds and then returned to 37.0 degrees C. Hyperthermia caused a reversible increase in resting tension at temperatures between 45 degrees and 50 degrees C and irreversible contracture at > or =50 degrees C. Rapid cooling contracture experiments and experiments measuring fluorescence of myocytes loaded with 5 microM Fluo-3 AM dye demonstrated that the hyperthermia-induced rise in resting tension was likely due to an increase in intracellular calcium content. Inhibition of the sarcoplasmic reticulum calcium pump with 20 microM thapsigargin resulted in irreversible contracture of the papillary muscles at temperatures between 45 degrees and 50 degrees C and significant increases in Fluo-3 fluorescence at 48 degrees C. Blockade of sarcolemmal calcium channels with 0.5 mM cadmium or 40 microM verapamil did not attenuate the heat-induced increase in resting tension and Fluo-3 fluorescence. CONCLUSION: Hyperthermia causes an increase in resting tension of cardiac muscle that most likely is mediated by a calcium channel-independent increase in calcium permeability of the sarcolemmal membrane and/or release of stored intracellular calcium.

Acute Disease↗
PubMed1999

Oximetry of retinal vessels by dual-wavelength imaging: calibration and influence of pigmentation.

A method for noninvasive measurement of Hb O2 saturation (SO2) in retinal blood vessels by digital imaging was developed and tested. Images of vessels were recorded at O2-sensitive and O2-insensitive wavelengths (600 and 569 nm, respectively) by using a modified fundus camera with an image splitter coupled to an 18-bit digital camera. Retinal arterial SO2 was varied experimentally by having subjects breathe mixtures of O2 and N2 while systemic arterial SO2 was monitored with a pulse oximeter. Optical densities (ODs) of vascular segments were determined using a computer algorithm to track the path of reflected light intensity along vessels. During graded hypoxia the OD ratio (ODR = OD600/OD569) bore an inverse linear relationship to systemic SO2. Compensation for the influence of choroidal pigmentation significantly reduced variation in the arterial SO2 measurements among subjects. An O2 sensitivity of 0.00504 +/- 0.00029 (SE) ODR units/%SO2 was determined. Retinal venous SO2 at normoxia was 55 +/- 3.38% (SE). Breathing 100% O2 increased venous SO2 by 19.2 +/- 2.9%. This technique, when combined with blood flow studies in human subjects, will enable the study of retinal O2 utilization under experimental and various disease conditions.

Adolescent↗
PubMed1998

Retinal oxygen consumption during hyperglycemia in patients with diabetes without retinopathy.

OBJECTIVE: This study aimed to determine whether retinal oxygen consumption in patients with diabetes without retinopathy is affected by acute changes in blood glucose. DESIGN: The study design was a nonrandomized, interventional clinical study. PARTICIPANTS: The study consisted of 12 patients with diabetes without ophthalmoscopically visible diabetic retinopathy whose ages were 20 to 59 years (mean, 33.7 years). INTERVENTION: Retinal vessel oximetry was performed on subjects during normoglycemia and during acutely induced hyperglycemia. Hyperglycemia was achieved with the ingestion of an oral glucose load. MAIN OUTCOME MEASURES: Changes in retinal arterial and venous blood oxygen saturation resulting from systemic blood glucose changes were measured. Measurements were made using a noninvasive, two-wavelength digital imaging retinal vessel oximeter. RESULTS: In each subject, the measured retinal venous blood oxygen saturation decreased significantly during hyperglycemia. The amount of decrease in venous oxygen saturation associated with hyperglycemia was positively correlated with the duration of diabetes. The measured retinal arterial oxygen saturation did not change significantly during hyperglycemia. CONCLUSIONS: These results, taken with the observations of numerous other investigators that show increased blood flow during acute hyperglycemia, provide evidence of substantially increased retinal oxygen consumption during hyperglycemia. The results also complement previous observations of the effect of diabetes on retinal autoregulation.

Adult↗
PubMed1998

Capillaries demonstrate changes in membrane potential in response to pharmacological stimuli.

It has been proposed that capillaries can detect changes in tissue metabolites and generate signals that are communicated upstream to resistance vessels. The mechanism for this communication may involve changes in capillary endothelial cell membrane potentials which are then conducted to upstream arterioles. We have tested the capacity of capillary endothelial cells in vivo to respond to pharmacological stimuli. In a hamster cheek pouch preparation, capillary endothelial cells were labeled with the voltage-sensitive dye di-8-ANEPPS. Fluorescence from capillary segments (75-150 microns long) was excited at 475 nm and recorded at 560 and 620 nm with a dual-wavelength photomultiplier system. KCl was applied using pressure injection, and acetylcholine (ACh) and phenylephrine (PE) were applied iontophoretically to these capillaries. Changes in the ratio of the fluorescence emission at two emission wavelengths were used to estimate changes in the capillary endothelial membrane potential. Application of KCl resulted in depolarization, whereas application of the vehicle did not. Application of ACh and PE resulted in hyperpolarization and depolarization, respectively. The capillary responses could be blocked by including a receptor antagonist (atropine or prazosin, respectively) in the superfusate. We conclude that the capillary membrane potential is capable of responding to pharmacological stimuli. We hypothesize that capillaries can respond to changes in the milieu of surrounding tissue via changes in endothelial membrane potential.

Acetylcholine↗
PubMed1998

Capillaries and arterioles are electrically coupled in hamster cheek pouch.

In this report we demonstrate electrical communication in the microcirculation between arterioles and capillary networks in situ. Microvessel networks in the hamster cheek pouch, which included capillaries and their feeding arterioles, were labeled with the voltage-sensitive dye di-8-ANEPPS by intraluminal perfusion through a micropipette. Pulses of 140 mM potassium solution were applied by pressure ejection from micropipettes positioned on arterioles several hundred micrometers upstream from capillaries. Potassium caused membrane potential changes of 3-11 mV in capillary segments up to 1,200 micrometers distal to the stimulation site, with time delays of <1 s. Capillary membrane potential changes were biphasic, with initial depolarizations followed by hyperpolarizations. The size of the response decreased exponentially with the distance between the arteriole and capillary, with a 1/e distance of 590 micrometers. The time to peak depolarization of both arteriolar and capillary segments was similar. The time to peak response was significantly faster than that for responses from direct stimulation of capillaries. Capillary responses were also obtained when blood flow was either blocked or directed toward sites of stimulation. Acetylcholine (10(-4) M) and phenylephrine (10(-5) M) applied to the arterioles by iontophoresis produced monophasic hyperpolarizing and depolarizing responses, respectively, in capillaries with <1-s delay between stimulus and onset of the membrane potential change. These results provide evidence in situ of a pathway for electrical communication between arteriolar and capillary levels of the microcirculation.

Animals↗
PubMed1997

A LED light calibration source for dual-wavelength microscopy.

An LED light source for dual-wavelength calibration of light microscope recording systems is described. A 1 mm2 circular field of bi-chromatic illumination is formed by passing the crossed beams of two light emitting diodes (LEDS) through an optical diffuser and circular mask. The source is constructed using LEDs which emit light at wavelengths overlapping those of many of the dual-emission molecular probes used for measurement of calcium, pH and membrane potential. The light output can be independently varied to set the ratio of light intensities recorded at two wavelengths. Internally sampled voltages which are proportional to LED light output intensity provide an internal reference for comparison with optical recordings. The LED source is useful for obtaining the optical response of dual emission recording equipment for specific recording conditions defined by the filtering and light detection components of the system.

Calibration↗
PubMed1996

Ratiometric measurement of endothelial depolarization in arterioles with a potential-sensitive dye.

A fluorescence ratio technique based on the voltage-sensitive dye 1-(3-sulfonatopropyl)-8-[beta-[2-di-n-butylamino)-6-naphythyl++ +]vinyl] pyridinium betaine (di-8-ANEPPS)has been developed for recording membrane potential changes during vascular responses of arterioles. Perfusion of hamster cheek pouch arterioles with the dye labeled the endothelial cell layer. voltage responses from the endothelium of intact arterioles were determined by analysis of voltage-induced shifts in fluorescence emission wavelengths from dye spectra imaged from the vessel wall. Membrane depolarization caused the dye spectrum to shift toward blue wavelengths, with maximal fluorescence changes near 560 and 620 nm. In isolated nonperfused arterioles, comparison of continuous dual-wavelength recordings with simultaneous microelectrode recordings showed that the ratio of fluorescence intensities (fluorescence at 620 nm to fluorescence at 560 nm) accurately followed changes in membrane potential (6-21 mV) during vasoconstriction. The dye response was linear with respect to potential changes from -56 to -6 mV, with a voltage sensitivity of 9.7% change in the ratio per 100 mV. Membrane potential responses from in vitro and in vivo arterioles after potassium stimulation consisted of rapid ( < 0.5 -s) depolarization followed by slow repolarization over several seconds. Potassium-induced depolarizations were conducted along arterioles, and the values of the electrical length constant for conducted depolarization determined by optical and microelectrode methods were in agreement. We conclude that ratio analysis of di-8-ANEPPS fluorescence emission can be used to accurately record membrane potential changes on the time scale of seconds during vasomotor activity from arterioles.

Animals↗
PubMed1993

A light-emitting diode light standard for photo- and videomicroscopy.

A light calibration system consisting of a compact light-emitting diode (LED) source with feedback control of intensity is described. The source is positioned in the focal plane of the microscope objective and produces flat-field illumination of up to 31 microW. The source can be easily used to determine the performance of microscope optics and camera response. It can also be used as a standard light source for calibration of experimental systems. Selectable light intensities are produced by controlling the LED input power via a feedback circuit consisting of a photodiode that detects output light intensity. Spectral coverage extends between 550 and 670 nm using green, yellow and red LEDs mounted side by side, which are selected individually. The LED chips are encapsulated in plastic diffusers which homogenize the light, and a flat field of illumination is obtained through a thin 1-mm-diameter aperture positioned directly over each chip. Provision is made for insertion of Ronchi rulings over the aperture to enable measurements of contrast modulation in a uniform field. The light may be pulse-modulated to assess camera response times and the device can be synchronized with video frames. Narrow bandpass interference filters can be placed between the objective lens and the LED source to produce monochromatic light without affecting the spacing of controlled light intensities since emission spectra do not shift appreciably over the range of LED powers chosen in this design. Results of tests using controlled light intensity and uniform illumination are presented.

Calibration↗
PubMed1993

Modification of vascular reactivity by alteration of intimal permeability: effect of TNF-alpha.

We investigated the effects of alterations in intimal permeability on microvascular reactivity to small hydrophilic agents in isolated, cannulated, perfused arterioles (65 +/- 6 microns ID) from hamster cheek pouches. Arterioles are 300-fold less responsive to the hydrophilic alpha 1-agonist, phenylephrine, applied to the lumen than when applied to the adventitia. Luminal treatment with tumor necrosis factor-alpha (TNF-alpha, 0.625 micrograms/ml, 1-2 h) potentiated reactivity to luminally applied phenylephrine, but the treatment did not change reactivity to adventitially applied phenylephrine. Similar results were obtained with a brief treatment with the detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS; 0.3%, < 30 s). To confirm that a change in permeability had occurred, we measured the movement across the arteriolar wall of a low-molecular-weight hydrophilic fluorescent molecule, fluorescein, before and after luminal treatment with TNF-alpha or CHAPS. Either TNF-alpha or CHAPS significantly increased the rate of movement of fluorescein across the arteriolar wall. These data suggest that one element in the pathophysiology of TNF-alpha is an increase in arteriolar permeability to small, water-soluble agents, which may modify reactivity to circulating vasoactive substances.

Animals↗
PubMed1991

Functional equivalence of metarhodopsin II and the Gt-activating form of photolyzed bovine rhodopsin.

Absorption of a photon by the visual pigment rhodopsin leads to the formation of an activated conformational state, denoted rho*, which is capable of activating the visual G-protein, Gt. The bleaching of rhodopsin can be resolved into a series of spectrally distinct photointermediates. Previous studies suggest that the photointermediate metarhodopsin II (meta II, lambda max of 380 nm) corresponds to the physiologically active form rho*. In the studies reported herein, spectral and enzymological data were analyzed and compared so as to evaluate the temporal correspondence between meta II and rho*. This information was obtained by direct observation of the meta II and rho* decay times in parallel experiments utilizing identical preparations of urea-stripped, bovine retinal rod outer segment disk membranes at pH 8.0, 20 degrees C. Postflash spectra were deconvolved to resolve the meta II absorbance at 380 nm, and a decay time for the loss of meta II of 8.2 min (SD = 0.5 min) was obtained from fitting these data to a single-exponential decay process. The diminishing ability of bleached rhodopsin to activate Gt was measured by monitoring the level of catalyzed exchange of Gt-bound GDP for a nonhydrolyzable GTP analogue. Analysis of the decrease in the initial velocity of nucleotide exchange, measured at various postflash incubation times, yielded a rho* decay time of 7.7 min (SD = 0.5 min) when analyzed as a single-exponential process. The similarity of these decay times provides direct evidence that meta II and rho* are present over the same time regime, and further supports the equivalence of these two forms of photoactivated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Lipid-protein interactions mediate the photochemical function of rhodopsin.

We have investigated the molecular features of recombinant membranes that are necessary for the photochemical function of rhodopsin. The magnitude of the metarhodopsin I to metarhodopsin II phototransient following a 25% +/- 3% bleaching flash was used as a criterion of photochemical activity at 28 degrees C and pH 7.0. Nativelike activity of rhodopsin can be reconstituted with an extract of total lipids from rod outer segment membranes, demonstrating that the protein is minimally perturbed by the reconstitution protocol. Rhodopsin photochemical activity is enhanced by phosphatidylethanolamine head groups and docosahexaenoyl (22:6 omega 3) acyl chains. An equimolar mixture of phosphatidylethanolamine and phosphatidylcholine containing 50 mol% docosahexaenoyl chains results in optimal photochemical function. These results suggest the importance of both the head-group and acyl chain composition of the rod outer segment lipids in the visual process. The extracted rod lipids and those lipid mixtures favoring the conformational change from metarhodopsin I to II can undergo lamellar (L alpha) to inverted hexagonal (HII) phase transitions near physiological temperature. Interaction of rhodopsin with membrane lipids close to a L alpha to HII (or cubic) phase boundary may thus lead to properties which influence the energetics of conformational states of the protein linked to visual function.

Animals↗