Manabu Miyamoto

Network synthesis of the epithelial transport system

Abstract The epithelial transport system consists of structural subsystems of three transport barriers (membranes) and three solution compartments. Macroscopically, coupled dissipations in the system occur in barrier subsystems, and coupled free-energy increasing or decreasing processes in the system occur in compartment subsystems. Therefore, the barrier subsystem is assumed to be the dissipating subsystem, and the compartment subsystem is assumed to be the free-energy changing subsystem. These structural subsystems consist also of a set of elemental thermodynamic processes, that is, dissipations, processes of free-energy change and power conversions. Each elemental process can be represented by thermodynamic elements prepared in bond graphs. p]In the experiments described in this paper, thermodynamic elements expressing free-energy changes and couplings in compartment subsystems were gathered and modeled as a capacitive module. The capacitive module behaved as a solution compartment and a simultaneous equation could be derived in which a set of flows determined a set of absolute-potential changes in the solution compartment. Elements were gathered and were modeled as a resistive module. The resistive module behaved as a membrane and a simultaneous transport equation could be derived in which a set of forces determined a set of flows through the membrane. A model of the epithelial transport system was achieved by the block diagram with the above modules. As variables in the module, the force vector and the flow vector expressing sets of power variables were assigned, but not a force nor a flow. This model showed the topology of the system and behaved as the real system. This model and the derived equations were used to simulate the frog skin behavior as an example of an epithelial transport system.

Electro-osmotic flow measurements

Abstract Electro-osmotic flow was evoked by an externally applied oscillating electric potential, and it was measured directly using a photodiode, or indirectly using a pressure transducer. Flow increased in proportion to the amplitude of the applied rectangular electric potential wave, in the ratios 5.2 nl-sec−1-cm−2-V−1 in skin, and 11.5 nl-sec−1-cm−2-V−1 in gastric mucosa. In pH 7.4. Ringer solution, skin behaved as a cation-exchange membrane, but gastric mucosa behaved as an anion-exchange membrane. These behaviours were explained by the electric charge in the aqueous channels, and could be changed by either varying the pH or addition of drugs. Electro-osmotic flow was also measured against the finite osmotic flow induced by a sucrose gradient. The electric potentials which balanced the finite osmotic flow were -241 mV/200 mOsm in skin, and 156 mV/100 mOsm in gastric mucosa. Parameters in the transport equation were also determined. The values of reflection coefficients and hydraulic conductivities were σCl - σNa=0.14, Lp = 2 x 10−7 cm-sec−1 -atm−1 in skin, and σCl - σNa = - 0.11, Lp = 4.5 x 10−7 cm-sec−1 -atm−1 in gastric mucosa. The individual reflection coefficients of Na and Cl in the skin were σCl = 0.96, and σNa = 0.82.

Constitutive Activity of Inwardly Rectifying K+ Channel at Physiological [Ca]i Is Mediated by Ca2+/CaMK II Pathway in Opossum Kidney Proximal Tubule Cells

Using patch-clamp technique, we studied the role of the Ca2+/calmodulin kinase II (CaMK II)-mediated phosphorylation process on the K+ channel with an inward conductance of 90 pS in opossum kidney proximal tubule cells (OKPCs). The intracellular Ca2+ concentration ([Ca]i) was measured by use of the fluorescent dye fura 2. The following results were obtained: (i) In cell-attached patches, the channel activity was inhibited by a decrease in [Ca]i induced by perfusion with low Ca2+ (10(-8) M), La3+ (100 microM), or EGTA/AM (100 microM) contained in the bath solution. The application of KN-62 (10 microM) or KN-93 (5 microM), inhibitors of CaMK II, also inhibited the channel activity. (ii) The membrane potential measured with nystatin-perforated patches was significantly decreased by the fall in [Ca]i induced by the perfusion with EGTA- or La(3+)-containing solution. Also, the application of KN-62 (10 microM) or KN-93 (5 microM) to the bath significantly decreased the membrane potential. (iii) In inside-out patches, the channel activity was significantly stimulated by the application of CaMK II (300 pM) at 10(-7) M Ca2+ in the bath. Furthermore, the application of KN-62 (10 microM) to the bath significantly decreased the channel activity. Our findings show that the constitutive activity of inwardly rectifying K+ channel at physiological [Ca]i is mediated by the Ca2+/CaMK II pathway in OKPCs.

Hydraulic Conductivities of Epithelium in Rat Submandibular Glands during Secretory and Resting Phases Measured by the Pressure-Metric Method.

The hydraulic conductivity and water driving forces of a rat submandibular gland were measured by the pressure-metric method. A polyethylene tube was inserted into a main duct of a rat submandibular gland and connected to a narrow vertical tube. The level of fluid was monitored by a pressure transducer. Differentiation of the pressure curve with respect to time gave the volume flow curve. A part of relationship between flow and pressure was linear, and its slope gave the hydraulic conductivity of a gland. We compared the values of hydraulic conductivity under three conditions : 1) secretion of the perfused gland evoked by acetylcholine, 2) secretion evoked by Phenylephrine, 3) passive absorption of fluid performed with 0.15 MNaCl+Amiloride 10-5 M. The values of hydraulic conductivity were very high with little difference between the stimulating and resting phases, and their values were about 2.4-2.6 × 10-10 1 sec-1 Pa-1 kg-1.