A diel rhythm of the short-circuit current expressed by the opercular epithelium of the killifish, Fundulus heteroclitus.

The inner opercular epithelium of the killifish, Fundulus heteroclitus, may be separated as a single epithelial sheet and mounted in an Ussing-style chamber with the short-circuit current (Isc) approximating chloride secretion. Steady state opercular Isc values in seawater-adapted killifish were higher in tissues removed from fish in the afternoon than those observed in the morning. Opercular conductance was also higher in the afternoon than the morning. The opercular steady state Isc from killifish on a 13:11 light:dark cycle exhibited a diel variation with the peak in the middle of the light cycle and a nadir near the end of the dark phase. Temperature and salinity remained constant during the experimental period. Daily variation cued by the photoperiod should be considered when using this preparation as an experimental model for ion transport in the gill of a seawater-adapted fish.

Glycogen synthesis from lactate in a chronically active muscle.

In response to neural overactivity (pseudomyotonia), gastrocnemius muscle fibers from C57Bl/6Jdy2J/dy2J mice have different metabolic profiles compared with normal mice. A population of fibers in the fast-twitch superficial region of the dy2J gastrocnemius stores unusually high amounts of glycogen, leading to an increased glycogen storage in the whole muscle. The dy2J muscle also contains twice as much lactate as normal muscle. A [14C]lactate intraperitoneal injection leads to preferential 14C incorporation into glycogen in the dy2J muscle compared with normal muscle. To determine whether skeletal muscles were incorporating lactate into glycogen without body organ (liver, kidney) input, gastrocnemius muscles were bathed in 10 mM [14C]lactate with intact neural and arterial supply but with impeded venous return. The contralateral gastrocnemius serves as a control for body organ input. By using this in situ procedure, we demonstrate that under conditions of high lactate both normal and dy2J muscle can directly synthesize glycogen from lactate. In this case, normal whole muscle incorporates [14C] lactate into glycogen at a higher rate than dy2J whole muscle. Autoradiography, however, suggests that the high-glycogen-containing muscle fibers in the dy2J muscle incorporate lactate into glycogen at nearly four times the rate of normal or surrounding muscle fibers.

Cellular permeation pathways in a leaky epithelium: the human amniochorion.

The presence of cellular permeation pathways in human fetal membranes at term was evaluated. Electrical parameters (transepithelial potential [TEP], and conductance [Gt], and intracellular potentials [cell PD]), and water and urea diffusional coefficients (Pdw, Pdu), were determined in Ussing-like chambers. In amniochorion, the TEP was practically 0 (0.1 +/- 0.03 mV), and the Gt very high (144 +/- 14 mS/cm2). The Cell PD of amnion cells was -37 +/- 3 mV. Increasing the [K+] of the amniotic perfusate between 5.8 and 125.8 mM depolarized the cells with a slope of 23 mV. The deletion of Na+ hyperpolarized the cells, whereas amiloride and ouabain depolarized them. The Pdw and Pdu were determined in intact amnion and chorion and in their epithelial cell layers. The Pdw/Pdu ratio in amnion was 4.0, and 7.0 in its cell layer; the ratio in chorion was 2.5, and 3.3 in its cell layer. The amniochorion is a leaky structure, but its cellular layers possess definite transcellular permeation pathways. The ionic conductances in amnion cells are complex, with the Cell PD being determined by at least K+ and Na+ conductances, and ouabain- and amiloride-sensitive pathways. The amnion is a more effective diffusional barrier to water and urea than chorion is; its diffusional characteristics are comparable to those of nystatin-treated lecithin: cholesterol bilayers and the membranes of human erythrocytes.

Intracellular voltage recordings in the opercular epithelium of Fundus heteroclitus.

Opercular epithelial cells of Fundus heteroclitus were investigated using conventional microelectrodes. The area of interest was the cells lining the inside of the opercular epithelium closest to the gill arches, an area with a high density of chloride cells. Only one cell type could be discerned from the values of 60 opercular cells measured with the opercular epithelium in open circuit conditions. A mean apical voltage of -18.0 +/- 0.6 mV was observed with intracellular values ranging from -10 to -30 mV. The predicted intracellular chloride content was 59 mM/liter. Apical fractional resistance (faR) was 0.78 +/- 0.02. The intracellular potential measurements were typically difficult to maintain for extended periods (longer than 3 min). The opercular cells depolarize with serosal isoproterenol treatment (10(-6) M) corresponding to the increase in opercular transepithelial potential. The opercular cell apical fR decreased with isoproterenol treatment. These data indicate the observed opercular cells were involved in opercular chloride transport.

Effect of pH on chloride absorption in the flounder intestine.

Chloride absorption in the small intestine of the winter flounder, Pseudopleuronectes americanus, is reported to be sensitive to ambient pH. We studied this sensitivity in isolated stripped intestinal mucosa mounted in modified Ussing chambers. Unidirectional 36Cl fluxes (JClm----s, JCls----m) were measured under short-circuited conditions in bathing solutions containing various combinations of HCO3- (0-20 mM), partial pressure of CO2 (0-36 mmHg), and pH (6.77-7.85). We found that JClm----s, net 36Cl flux (JClnet), and short-circuit current (Isc) increased and JCls----m decreased predominately in response to increases in bathing solution pH. There was a linear relationship between pH and both JClnet (r = 0.92, P less than 0.01) and Isc (r = 0.96, P less than 0.005) between pH 6.77 and 7.74. The pH effect was completely reversible, did not require either CO2 or HCO3-, and was not affected by the presence of mucosal barium at 1 mM. Mucosal bumetanide (0.1 mM) completely inhibited the pH effect. These data suggest that the process by which Cl- is absorbed in the flounder intestine is sensitive to pH. The data do not indicate whether pH affects Na+-K+-2Cl- cotransport or a Cl- transport pathway in series with this process. The direction of Cl- absorption in response to pH contrasts with inverse relation of pH and Cl- absorption in mammalian small intestine.

Effect of a chloride channel blocker on bullfrog corneal epithelium.

The effect of the chloride channel blocker, diphenylamine-2-carboxylate (DPC), was investigated on the bullfrog cornea epithelium using microelectrodes. Perfusion of 2 X 10(-4) M DPC on the tear side resulted in a 44% reduction of the corneal transepithelial potential difference (TEP) and a 9% reduction in corneal conductance. The cornea epithelial cells hyperpolarized 7.2 +/- 1.5 mV, and the apical fractional resistance increased 35% (n = 8 tissues). The basolateral potential difference depolarized 5.5 +/- 1.8 mV. The relative apical conductance decreased 29%, whereas relative changes of basolateral membranes increased 56% with prolonged DPC exposure, indicating that the DPC inhibition was acting at the apical cell membrane. DPC-treated corneas could be stimulated with isoproterenol (10(-6) M), indicating that the DPC inhibition could be reversed with the stimulation of chloride conductance. The DPC inhibitory effect was dependent on basolateral chloride. Intracellular chloride activity increases with DPC, indicating that the integrity of the basolateral chloride entry mechanism was not altered. The DPC inhibitory action was due to a reduction in apical chloride conductance.

Effect of atriopeptin II on isolated opercular epithelium of Fundulus heteroclitus.

The effect of atriopeptin II (ANF) on the in vitro opercular epithelium was investigated by use of short-circuit current techniques. Serosal addition of ANF stimulates chloride secretion (short-circuit current) 19% above control values with a 7% increase in tissue conductance. Mucosal addition of ANF to the opercular epithelium was without effect. The ANF stimulation of the current was dose dependent with a maximum at 10(-7) M. The addition of ANF had no effect on the current or the conductance of opercular epithelia bathed in Cl--free Ringer. The opercular current could be stimulated above the ANF response by isoproterenol (10(-6) M). Pretreatment of the opercular epithelium with propranolol (10(-5) M) did not inhibit the stimulation of the short-circuit current by ANF but did inhibit the isoproterenol response indicating that the ANF stimulatory activity was independent of the beta-adrenergic receptors. The ANF-stimulated short-circuit current was found in operculi pretreated with tetrodotoxin (10(-6) or 10(-5) M) or diltiazem (10(-4) M) indicating the ANF response was not due to nerve stimulation. Pretreatment of opercular tissue with dibutyryl adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate (10(-4) M) had no effect on the ANF stimulatory response. Opercular epithelia from short-term freshwater-adapted killifish also showed the ANF-induced response. The stimulation of chloride secretion in Fundulus heteroclitus chloride cells by ANF may have a role in teleost ion regulation.