Biomedical subjects
Publications and source records attributed to M Fujimoto.
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Using the bullfrog retina, depth profiles of Burkhardt's PNR and of the ERG are compared under various recording conditions. When the retina is voltage-annulled so that there is no ERG potential outside the retina, the depth profile of the ERG changes to resemble that of the PNR. When there is current annullment so that there is no ERG current outside, the depth profile of the PNR changes to resemble that of the ERG. Our evidence suggests that Burkhardt's PNR can be further analyzed into a PNR proper, which consists of an initial fast negativity followed by a slow negativity, and a local ERG dominated by the b-wave.
Two kinds of pH-sensitive microelectrodes were constructed: 1) glass microelectrode with a pH-sensitive tip of 50--100 micrometers in length, and 2) antimony (Sb) microelectrode with a tip of 1--5 micrometers in outside diameter. Comparisons of the two were made on the change in the electromotive force (EMF) in response to various pHs and biological applicability. The pH sensitivity (slope constant) at 20 degrees C of the glass and Sb-microelectrodes averaged 51.7 and 51.9 (-mV/pH), respectively. Over the pH range of 2 to 9, both electrodes responded linearly to the change of pH. The pH-dependent EMF of the Sb-microelectrode was influenced by phosphate buffers, but not by Tris-HCl buffers. Through the glass microelectrode showed a linear response to all the buffers tested, its slope constant tended to be limited by the surface area of the pH-sensitive tip. The stability of the EMFs was well within 1 mV for 12 hr with the glass microelectrode but more than 10 mV with the Sb-microelectrode. One the other hand, the 95% response time was about 1 min with the glass microelectrode, whereas it was less than a few seconds with the Sb-microelectrode. Blood pH was measured with the two electrodes and reasonable values were obtained with both methods, although the values by use of the Sb-microelectrode were slightly higher than those of the other. In view of the practical ease of manufacturing, maintenance and durability, the use of the Sb-microelectrode in biological measurement should also be re-evaluated, but several restrictions must be imposed on it.
1) Antimony (Sb) microelectrodes with tips of 2 to 5 micrometers in outside diameter were constructed, and their electromotive forces (EMF) were tested in response to the pH of several calibration buffers. The data were compared with those of glass pH electrodes. 2) Increasing ionic strength (I) caused a significant deviation in pH readings of the Sb-microelectrode (delta pHSb-Glass). The above salt effect was empirically given by delta pHSb-Glass = 0.017--0.125 I (I = 0.09--0.22) 3) Even with the correction for ionic strength, the values of delta pHSb-Glass were still dependent on the nature of calibration buffers employed. Among various buffers, Tris buffer [Tris(hydroxymethyl)-aminomethane, (THAM)] had the least disturbance on the Sb-microelectrode, and bicarbonate had also relatively minor effect. 4) The pH value according to the Sb-electrode was caused to deviate considerably by phosphate. The effect of phosphate within the range of 1 to 67 mM can be predicted by 1/delta pHSb-Glass = 1 + (0.174/[PO4]) 5) Like phosphates, BES [N, N-Bis (2-hydroxyethyl)-2-aminoethane sulfonic acid] also had a significant effect on the EMF of the Sb-microelectrode. 6) Upon consideration of the above facts, measurements of several biological fluids, such as those of the in vivo pH of intratubular fluid of bullfrog kidney were carried out with the Sb-microelectrode calibrated by 90 mM Tris buffer. 7) Thus, it was concluded that the Sb-microelectrode when properly calibrated could be used for the pH measurement of ultraminute amounts of biological samples.
With regard to pH measurement of biological fluids in vivo with metal-metal oxide microelectrodes, the effect of temperature and partial pressure of oxygen on antimony (Sb) microelectrodes was examined, and pH of blood was estimated in the bullfrog. The temperature coefficient (dE/dt) of electromotive force (EMF) of Sb-microelectrodes in the range of 7 to 37 degrees C was -1.18 +/- 0.113 mV/degrees C (mean +/- SEM) in Ringer solution, whereas that of the pH glass electrode in the same solution was -0.43 +/- 0.035 mV/degrees C. When estimated in Tris buffer solution, it was -0.06 +/- 0.063 mV/degrees C for Sb-microelectrodes and 1.05 + 0.036 mV/degrees C for glass electrodes. The change of slope constant (alpha in -mV/pH) in the Sb-microelectrode due to temperature change could be predicted empirically from: alpha = 0.40 (t-25) + 55.3, where t represents the measuring temperature in degrees C. The resultant deviation of pH reading between Sb and glass electrodes, delta pHSb-Glass, may be expressed by: delta pHSb-Glass = 0.00183 (t-25) +0.016. In the range of 45 to 760 mmHg of oxygen partial pressure it fixed pH, the EMF increased linearly with the increase of Po2, the slope (dE/dlog(Po2)) being 11.7 +/- 0.42 (SEM) mV (n = 13, t = 25 degrees C). In consideration of the above effects, the blood pH of bullfrog was estimated to be 7.697 +/- 0.092 (SD) and 7.729 +/- 0.111 with glass and Sb-microelectrodes respectively, the difference between the two being relatively minor.
In order to examine the effect of protein on the Sb-microelectrode used for the pH measurement of biological fluids, bovine serum albumin (BSA) and plasma protein were used for test samples. It was found that, BSA, probably by binding to components in the electrode system, produced a marked deviation of both the electromotive force and slope constant of the microelectrode, resulting in an alkaline shift of pH of 0.1-0.7. But the shift of the pH reading could be empirically predicated within an error of +/- 0.03 from the cubic function of protein concentration. Use of Tris standard solution as the calibration buffer, together with corrections for the effects of temperature, ionic strength, Po2, etc. on the Sb-microelectrode, would reduce the alkaline shift to approximately less than 0.08 and 0.04 pH units in the pH measurement of blood plasma and whole blood, respectively.
Effects of extracellular diffusible ions, such as K+, Cl- and HCO3- (pH), on the peritubular membrane potential (EM) and intracellular activities of K+, (K)i, or Cl-, (Cl)i, were studied in the perfused proximal tubule of bullfrog kidneys with K+ or Cl- -selective microelectrodes. In steady-state conditions, in which both the peritubular and luminal sides were perfused with control Ringer solutions, the K+ equilibrium potential (EK) always exceeded the EM by approximately 19 mV and correlated well with the EM (correlation coefficient r = 0.78), whereas no correlation was recognized between the equilibrium potential of Cl-(ECl) and the EM. In the quick peritubular perfusion experiments, in which the extracellular diffusible ions were changed, the (K)i and (Cl)i were maintained relatively stable. The following facts were observed: (1) At constant EK, decreasing the peritubular chloride (Cl)e produced a small degree of hyperpolarization of the EM instead of depolarization. (2) At constant ECl, increasing the (K)e depolarized the EM. (3) At constant PCO2, the EM was depolarized with low HCO3- (acid) perfusions, while it was hyperpolarized with high HCO3- (alkaline) perfusions. These results are in agreement with the views that, 1) intracellular K+ in the proximal tubule is maintained by an uphill uptake mechanism on the peritubular cell membrane, (2) the ionic conductance of peritubular membrane is relatively high to K+, but low to Cl-, and (3) the pH gradient across the peritubular membrane can modulate the passive permeability to Na+ or K+.
A triple-barreled Na+, K+-selective microelectrode was constructed with liquid ion exchangers for Na+ (monensin) and K+ (Corning #477317) to measure the intracellular Na+ and K+ activities ((Na)i and (K)i) of a single cell and its membrane potential (EM), simultaneously. The tip of the triple-barreled assembly was made less than 0.6 micron in outside diameter. Prior to in vivo measurements, some physiochemical properties of microelectrodes were examined in vitro for the slope constant, selectivity coefficient, electrical resistance, and pH effect, as well as measurements of the activity coefficient on ions in blood serum and Ringer solution. Carrying out direct micropunctures on single cells of the sartorius muscle and renal proximal tubule of bullfrogs in vivo, we obtained the following results: (1) In sartorius muscle, the average (Na)i was 14.8 mEq/liter, the (K)i 64.5 mEq/liter, and the EM -86.2 mV. (2) In proximal tubule cells, the average (Na)i, (K)i and EM were 16.8, 63.0 mEq/liter and -65.9 mV, respectively. (3) There were significant correlations in the proximal tubule between (K)i and EM, and inversely between (Na)i and EM, and between (Na)i and (K)i. These facts may somehow be related to both the activity of Na+-K+ exchange pump and the osmotic equilibrium of water across the membrane. Further, several problems inherent in the multibarreled microelectrode were discussed from the practical point of view.
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A micropuncture study was performed on the bullfrog kidney proximal tubular cells utilizing double-barreled ion-selective microelectrodes. The intracellular of Na+, K+, Cl-, HCO3(-) and pH were determined to be 21.6 mEq/L, 67.4 mEq/L, 9.9 mEq/L, 20.2 mEq/L, and 7.49 pH units, respectively. In the extracellular fluid the following activities were found: Na+, 87.4 mEq/L; K+, 2.64 mEq/L; Cl-, 72.5 mEq/L; HCO3(-), 17.9 mEq/L; and pH, 7.66. The membrane potential difference was 68.4 mV and 60.4 mV across the peritubular and brush borders, respectively. The electrochemical potential differences across the individual borders of the proximal tubule cells were separately calculated by setting the intracellular level of both electrical and chemical potentials at zero for convenience. From these analyses, the following interpretations are made. (1) In the net reabsorption of Na+, luminal Na+ enters the cell along a 95-mV gradient across the luminal border and is pumped out to the interstitium against a 104 mV gradient. In the reabsorption of bicarbonate, an uphill pump of about 69 mV (about 70% of the Na+ entry gradient) must exist on the luminal border, of which about 55 mV (80% of the bicarbonate gradient) is accounted for by the H+ secretory pump. (2) In the net reabsorption of K+, a significant K+ uptake pump must exist on the luminal border in addition to the powerful peritubular Na+-K+ exchange pump. The reabsorption of Cl- by the epithelium may take place in two ways: (a) transmembrane transport involving an uphill step of several millivolts, and (b) paracellular leakage through the tight junction. It is thought that the Na+ pump located on the basolateral border of the proximal tubule cell plays a primary role in the regulation of the movement of other ions and water. The regulatory mechanisms of these substances may involve some electrochemical feedback mechanism that works across the proximal tubular epithelium.
A pencil-type antimony microelectrode of double-barreled design with a tip of less than 1 to 2 microns in outside diameter was constructed and used to measure intracellular pH(pHi) on frog sartorius muscle and renal tubular cells. Simultaneous observations of membrane potential difference (EM) were made. The results obtained were as follows: (1) The in vivo pHi of frog sartorius muscle was 7.12 +/- 0.07 (SD) (n = 144); the simultaneously measured EM was -51.1 +/- 7.9 mV. The in vivo pHi of frog proximal tubule was 7.49 +/- 0.07 (n = 221) and the EM Peri across the peritubular membrane was -50.2 +/- 9.0 mV. (2) In proximal tubule in vivo, there was a negative correlation between pHi and EM (r = -.62, p < .05). On the other hand, in sartorius muscle in vivo, a positive correlation between the two was found (r = .85, p < .001). (3) In in vitro sartorius muscle, the pHi was 7.03 +/- 0.14 (n = 9) and EM was -62.4 +/- 4.4 mV within one hour after isolation. (4) Increasing the external potassium concentration in the preparations to 75 mM caused a progressive depolarization by 43.3 +/- 15.9 (m = 4) mV, while pHi changed in the alkaline direction by 0.22 +/- 0.03 pH unit. (5) These results indicate that the pHi in both tissues does not obey the Donnan rule.
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Potential difference across the cell membrane and intracellular activity of the potassium ion in rat liver cells were measured simultaneously using double-barreled potassium ion-selective microelectrodes. Both potential difference across the membrane and K+ activity in liver cells were depressed after treatment with the antineoplastic agents mitomycin C and 5-Flourouracil Dry Syrup, suggesting that these drugs would induce disturbances of cellular energy metabolism in liver cells. When the antineoplastic agents were used in combination with coenzyme Q10, the depression of potential difference across the membrane and K+ activity and the hypofunction of liver cells in energy metabolism were significantly prevented.
The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.