Cation Pump Research Articles

18] Active cation pumping of Na+, K+-ATPase and sarcoplasmic reticulum Ca2+-ATPase induced by an electric field

To explain these results, we have proposed that the applied electric field induces a shift in the enzyme conformational equilibria through coulombic interaction. When the electric field is removed, or when it reverses its sign, the system returns to its initial state, and in the process of returning the free energy absorbed from the electric field is released. This released energy is used to drive active transport of an ion or to synthesize ATP. Models based on the concept of the "electroconformational coupling" have been analyzed and shown to work as predicted. Most cells maintain a steady-state transmembrane potential. This is an energy-consuming process, and as such, it must serve certain functions or purposes. Experiments discussed here would suggest that activation or regulation of ion-transport system is one of these function. Other experiments have shown that electric fields can stimulate ATP synthesis, cell proliferation, membrane fusion, DNA and RNA biosynthesis, etc. (for review see, e.g., Refs. 5 and 12). Apparently, the activation of ATPases is the most fundamental because this class of enzymes can transduce energy from on form to another, including transduction of electric energy to chemical bond energy of ATP and chemical potential energy of cations.

Effects of chlorpromazine on Na+-K+-ATPase pumping and solute transport in rat hepatocytes

Inhibition of Na+-K+-ATPase and sodium-dependent bile acid transport has been suggested as a mechanism for the cholestasis produced by certain drugs such as chlorpromazine. We examined the effects of chlorpromazine (and in selected studies, two of its metabolites) on Na+-K+-ATPase cation pumping (ouabain-suppressible 86Rb uptake), exchangeable intracellular sodium content, membrane potential (assessed by 36Cl- distribution), and sodium-dependent transport of taurocholate and alanine in primary cultures of rat hepatocytes. Chlorpromazine (10-300 microM), 7,8-dihydroxychlorpromazine (10-300 microM), and ouabain (0.1-2 mM), but not chlorpromazine sulfoxide, produced a concentration-dependent decrease in Na+-K+-ATPase cation pumping and an increase in intracellular sodium content. Chlorpromazine (100 microM) and ouabain (0.75 mM) also modestly decreased hepatocyte membrane potential. In further studies, chlorpromazine (75 and 100 microM) and ouabain (0.1, 0.5, and 0.75 mM) decreased initial sodium-dependent uptake rates of taurocholate and alanine by 18-63%. Although the steady-state intracellular content of alanine was decreased 25-53% by both agents, chlorpromazine increased the steady-state content of taurocholate by 171% and decreased taurocholate efflux, apparently related to partitioning of taurocholate into a large, slowly turning over intracellular pool. These studies provide direct evidence that chlorpromazine inhibits Na+-K+-ATPase cation pumping in intact cells and that partial inhibition of Na+-K+-ATPase cation pumping is associated with a reduction of both the electrochemical sodium gradient and sodium-dependent solute transport. These effects of chlorpromazine may contribute to chlorpromazine-induced cholestasis in animals and humans.

Peroxide-induced effects on lens cation transport following inhibition of glutathione reductase activity in vitro

Previous studies from this laboratory have shown that the normal lens can tolerate exposure to 0.05 mM H2O2 without apparent damage and that this is due in part to an active glutathione redox cycle. The present studies were designed to investigate the role of glutathione reductase in protecting cation transport systems in the lens against potentially damaging effects of peroxide. Pre-treatment of rabbit lenses with 0.5 mM 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a relatively specific inhibitor of glutathione reductase, brought about a 71% inhibition of the enzyme in the capsule-epithelia of the lenses. Subsequent exposure of the lenses for 3 hr to a constant level of 0.05 mM H2O2 in culture medium produced significant accumulation of oxidized glutathione (GSSG) in the lens epithelium and severe effects on the electrolyte balance in the lens, on the activity of Na, K-ATPase and on the accumulation and efflux of 86Rb. The effects included a 35% decrease in activity of Na, K-ATPase, a 10 mM increase in the concentration of Na+ and an 8 mM decrease in K+. BCNU-H2O2 treatment also resulted in loss of transparency of the lenses in the form of vacuoles present in the anterior, subcapsular region, encircling the entire periphery of the organ near the germinative zone of the epithelium. Treatment with either BCNU or 0.05 mM H2O2 alone had only minimal effects on accumulation of GSSG in the epithelium, on lens transparency and on the parameters of cation transport which were investigated. When lenses were treated with 0.05 mM H2O2 alone and then placed in normal medium to measure the accumulation of 86Rb it was found that the cation pump was stimulated 20% above the normal level of activity. Levels of H2O2 higher than 0.05 mM without BCNU pre-treatment produced significant inhibition of Na, K-ATPase and the effects of 0.3 mM H2O2 on cation transport and GSSG accumulation were comparable to those of BCNU-0.05 mM H2O2. While inhibition of the activities of glutathione reductase and Na, K-ATPase in the lenses was found to be irreversible, a partial recovery of the Na+ level and nearly complete recovery of the K+ level were observed when treated lenses were cultured in normal medium for an additional 6 hr. In addition, the rate of efflux of 86Rb which was significantly faster from the BCNU-H2O2-treated lenses compared with the controls, was found to return to the control value during the recovery period.(ABSTRACT TRUNCATED AT 400 WORDS)

Diurnal variation of cation pump enzyme activity in pineal and seven other rat brain regions.

Adult female Long-Evans rats were maintained on an automatically regulated artificial lighting schedule of light:dark (L:D) 14.5:9.5 for 12 wk. After sacrifice at 0630, 1130, 1600, 1800, 2000, 2200, 0230, or 0400, the pineals were removed, weighed, and assayed for N-acetyltransferase (NAT), melatonin, Mg++-paranitrophenylphosphatase (pNPPase), and K-pNPPase activity. The brains were quickly dissected into the following areas: cerebellum, superior colliculi, inferior colliculi, visual cortex, auditory cortex, sensorimotor cortex, and the hypothalamic area around the suprachiasmatic nucleus. These regions were weighed and 10% sucrose homogenates were prepared for determinations of protein, Mg++-pNPPase, and K+-pNPPase activity. Pineal melatonin rose over six-fold from 144 +/- 70 pg/gland at 1130 to 981 +/- 173 pg/gland at 0230. Similarly, pineal NAT activity rose over 11-fold, from 119 +/- 12 pmol/gland/h to 1315 +/- 232 pmol/gland/h at the same times. K+-pNPPase activity rose by about two-thirds, from 133 +/- 12.8 nmol/gland/h to 224 +/- 22.3 nmol/gland/h from 1600 to 0230. However, when expressed per mg protein, these differences in pNPPase activity were not significant. There were no significant daily rhythms discernible in any of the seven other brain regions across these times. We conclude that cation pump enzyme activity varies only slightly with time in the rat brain and pineal gland, in spite of definite daily rhythms of pineal melatonin and NAT activity.

Effect of stress on cardiomyocyte transmembrane potential of the working heart and its recovery after hypothermia

Emotional-painful stress in rats was shown to decrease insignificantly transmembrane cardiomyocyte potential (TCP) measured in isolated hearts. The recovery of TCP following its depression due to the preparation cooling was twice slower in stress-exposed than in control animals. This is in keeping with the data on stress-induced disturbances of Na, K-ATPase activity (an enzyme playing a leading role in TCP maintenance). It is suggested that the disturbance in cation pump function activity plays a certain role in the onset of arrhythmias and cardiac fibrillation during stress.

Na+‐dependent amino acid transport is a major factor determining the rate of (Na+, K+)‐ATPase mediated cation transport in intact HeLa cells

Little is known concerning the effects of Na+-coupled solute transport on (Na+,K+)-ATPase mediated cation pumping in the intact cell. We investigated the effect of amino acid transport and growth factor addition on the short term regulation of (Na+,K+)-ATPase cation transport in HeLa cells. The level of pump activity in the presence of amino acids or growth factors was compared to the level measured in phosphate buffered saline. These rates were further related to the maximal pump capacity, operationally defined as ouabain inhibitable 86Rb+ influx in the presence of 15 microM monensin. Of the growth factors tested, only insulin was found to moderately (22%) increase (Na+,K+)-ATPase cation transport. The major determinant of pump activity was found to be the transport of amino acids. Minimal essential medium (MEM) amino acids increased ouabain inhibitable 86Rb+ influx to a level close to that obtained with monensin, indicating that the (Na+,K+)-ATPase is operating near maximal capacity during amino acid transport. This situation may apply to tissue culture conditions and consequently measurements of (Na+,K+)-ATPase activity in buffer solutions alone may yield little information about cation pumping under culture conditions. This finding applies especially to cells having high rates of amino acid transport. Furthermore, rates of amino acid transport may be directly or indirectly involved in the long-term regulation of the number of (Na+,K+)-ATPase molecules in the plasma membrane.

Studies on the eye lens in poikilothermal animals. I. Comparative studies on cation maintenance systems in rainbow trout and rat lenses

The response of the poikilothermal lens to various incubation temperatures in vitro was compared with that of the homothermal lens. The rainbow trout lens was used as the poikilothermal lens and the rat lens as the homothermal lens. In contrast to rat lenses, cataract developed at 37°C in rainbow trout lenses, which was called ‘warm cataract’. Warm cataract developed not only when lenses were incubated in vitro but also when rainbow trout were kept in water at 37°C. Water, Na +, Ca 2+ and insoluble protein increased and K + and Mg 2+ decreased in warm cataract lenses, but GSH and soluble protein sulfhydryl levels did not change. This cataract was irreversible after only 5 min incubation at 37°C. On the other hand, rainbow trout lenses remained transparent without the change of cation balance at 0–25°C while cold cataract developed in rat lenses. Na,K-ATPase activity was detected at 0°C in rainbow trout lens homogenates, but not in rat lens homogenates. Na +K + ratio (Na +/K +) increased when the rainbow trout lens was treated with ouabain at 0°C. In the rainbow trout lens, lactic acid was produced continuously for 30 days at 0°C while it was not in the rat lens between 1 hr and 10 days after. These results strongly suggest that Na,K-ATPase acts as a cation pump at 0°C and that ATP is supplied by glycolysis in the rainbow trout lens in order to maintain the transparency. The above results also suggest that enzymes and membrane structures in rainbow trout lens are adapted to a cold-temperature habitat and that Na,K-ATPase and anaerobic glycolysis are important for the maintenance of lens transparency at low temperatures.

Ion Transport in the Midgut of the Cockroaches Leucophaea Maderae And Blabera Gigantea

ABSTRACT The presence of a cation pump on the apical membrane of epithelial cells in insects, responsible for the extrusion of K or Na, has been proposed for Malpighian tubules (Maddrell, 1977) and salivary glands (Berridge, Lindley & Prince, 1976). An essential role of the midgut in K homeostasis has been established only for the phytophagous larvae of Lepidoptera (Harvey & Nedergaard, 1964). The main features of the rheogenic, luminally-directed potassium pump of this tissue have been extensively studied (Zerahn, 1978; Harvey, Cioffi, Dow & Wolfersberger, 1983). Since the midgut of these animals is also peculiar in that it does not actively absorb Na, the presence of the K pump could be the sole example in insect gut physiology. Na absorption in the midgut has been demonstrated in several species of insects (O’Riordan, 1969; Sauer, Schlenz True & Mills, 1969; Farmer, Maddrell & Spring, 1981 ; Dow, 1981a; Koefoed & Zerahn, 1982) while K secretion has been suggested for Periplaneta americana (Sauer & Mills, 1969), Leucophaea maderae (Sacchi & Giordana, 1979) and Schistocerca gregaria (Dow, 1981b).

Effect of glutathione deprivation on lens metabolism

1-Chloro-2,4-dinitrobenzene (CDNB) was used to conjugate glutathione (GSH) through the catalysis of lens glutathione S-transferase without the untoward oxidative damage to the lens mediated by GSH oxidants. A 2 hr treatment of the rat lens with 1 m m CDNB resulted in a nearly total depletion of lens GSH with neither formation of GSSG nor glutathione-protein mixed disulfides. Rubidium uptake was found to decrease linearly with the loss of GSH; nevertheless, ionic imbalance did not commence until more than 30% cation pump activity was lost. Glycolytic rate dropped following CDNB treatment, due probably to a decline in demand for ATP by the deactivated cation pump. 31P-NMR studies confirmed the irreversible loss of ATP. CDNB depletion of GSH resulted in a two-fold increase in 14CO 2 production from [ 14C]-1-glucose. Whereas oxidative stress resulted in a six-fold increase in glucose utilization through the hexose monophosphate shunt (HMPS), CDNB-treated lenses showed no such stimulation. This indicated that the residual GSH following CDNB treatment was insufficient for the activation of the glutathione peroxidase-reductase-HMPS mechanism and raised the possibility that the increased glucose utilization might be due to mechanisms other than the HMPS. These results indicate an intimate correlation between the GSH content and major metabolic functions in the lens.

Secretion by the Malpighian tubules of Rhodnius prolixus stal: electrical events.

Transepithelial and intracellular potentials have been simultaneously recorded from Rhodnius upper Malpighian tubules before and after stimulation of fluid secretion. The transepithelial electrical response to the diuretic hormone mimic 5-hydroxytryptamine (5-HT) was triphasic; recordings of intracellular potential changes indicated that the three phases represented successive events at the apical membrane. Depolarizations produced by increasing the bathing medium potassium concentration indicated that the basal membrane was much more permeable to potassium than to sodium. Electrical responses to chloride-free saline were inconsistent with a significant basal membrane chloride permeability. Chloride movements across the basal membrane were opposed by an electrical gradient of about 65 mV. The results of experiments in which tubules were exposed to chloride-free saline or sodium-free saline suggested that chloride entry into the cells was linked to the entry of Na+ and K+. The effects of furosemide and bumetanide upon secretion and potential changes suggested that chloride crossed the basal membrane through co-transport with Na+ and K+. Chloride probably crosses the apical membrane into the lumen passively in response to a favourable electrical gradient of about 35 mV. Cations must be actively pumped into the lumen against an electrical gradient of 35 mV. Our results support previous evidence for an apical cation pump which actively transports Na and K into the lumen. A tentative model of ionic movements during fluid secretion is presented. It is suggested that the apical cation pump maintains sodium at low intracellular concentrations, thereby maintaining a favourable gradient for entry of Na+ through the proposed basal co-transport step. The suggested stoichiometry is Na+:K+:2 Cl-.

(Na,K)-ATPase-mediated cation pumping in cultured rat hepatocytes. Rapid modulation by alanine and taurocholate transport and characterization of its relationship to intracellular sodium concentration.

(Na,K)-ATPase is thought to maintain the transmembrane electrochemical sodium gradient which powers secondary active sodium-coupled transport of a variety of solutes including amino acids and bile acids. However, little is known regarding the effect of sodium-coupled solute transport on intracellular sodium concentration ( [Na]ic) and on (Na,K)-ATPase-mediated cation pumping in the intact cell. In order to address this question, we have measured 22Na uptake rate, steady state 22Na content, and ouabain-suppressible 86Rb uptake rate in primary cultures of adult rat hepatocytes under a variety of conditions. Compared with control conditions (sodium uptake rate = 6.00 +/- 0.40 nmol X min-1 X mg-1; [Na]ic = 11.96 +/- 0.54 mM; cation pumping = 2.53 +/- 0.18 nmol X min-1 X mg-1), cation pumping was increased by taurocholate (less than or equal to 158%), alanine (less than or equal to 246%), monensin (less than or equal to 400%), and cold exposure (less than or equal to 525%), and this increase was accompanied by increases in Na uptake and [Na]ic. In contrast, preincubation in low sodium medium decreased all three variables. These changes in cation pumping were blocked in the absence of extracellular sodium and were not accompanied by changes in ouabain-suppressible ATP hydrolysis measured in cell homogenate. An overall plot of cation pumping versus [Na]ic yielded a sigmoid-shaped curve. Values for KNa (17.8 +/- 1.4 mM) and Vmax (8.98 +/- 0.62 nmol X min-1 X mg-1) for cation pumping were estimated assuming three sodium sites per pump unit. These findings indicate that: 1) uptake of alanine and taurocholate is associated with a rapid increase in (Na,K)-ATPase cation pumping; 2) this increase probably results from an increase in pumping per pump unit rather than an increase in the total number of pump units, and it appears to be mediated via an increase in sodium influx and [Na]ic; 3) [Na]ic under control conditions is close to the apparent KNa of cation pumping, implying that substrate availability may be the mechanism whereby sodium uptake is tightly linked to (Na,K)-ATPase cation pumping in intact hepatocytes.

Response of cochlear potentials to presumed alterations of ionic conductance: endolymphatic perfusion of barium, valinomycin and nystatin.

Two models ('single-pump' and 'two-pump') of transepithelial potassium movement by the marginal cells of the stria vascularis have been proposed in the literature. Their validity was considered by exposing the endolymphatic (luminal) surface to agents (barium, valinomycin and nystatin) which are known to alter specific cellular membrane conductances in other tissues. This was accomplished by the use either of injections or of a relatively satisfactory technique for perfusion of scala media, which is described. Injection of barium caused the endocochlear potential (EP) to increase in normal animals and had no effect on the EP of deaf, Waltzing guinea pigs. Perfusion of the ionophores caused a decline in the EP in both normal and Waltzing guinea pigs. Only the 'two-pump' model (Na/K-ATPase-mediated cation pump on the basolateral membrane and rheogenic K transporter at the luminal membrane) is consistent with the results. The cellular heterogeneity of the cochlear duct, however, introduces a measure of uncertainty into this interpretation.

Calmodulin Levels and Divalent Cation Pump Activities in Kidneys of Streptozotocin-Diabetic Rats

Levels of calmodulin and activities of calcium and magnesium ATPases were determined in renal tissues of age-matched male rats after increasing durations of the following conditions: untreated streptozotocin (STZ) induced diabetes; STZ-diabetic rats which received daily insulin (NPH) treatment beginning 24 hours after STZ administration; and STZ-diabetic rats which began receiving NPH after having endured untreated diabetes for 1, 2, and 3 weeks. Results were compared with those of age-matched control animals. Calmodulin levels were the same in renal tissues from all groups of rats as were magnesium-ATPase activities. Calcium-ATPase activities were significantly higher than control activities after 2 weeks of untreated STZ-diabetes and in tissues from all rats that received insulin treatment which was instituted after 1, 2 and 3 weeks of STZ-diabetes. Calcium ATPase activities of tissues from rats which received insulin treatment 24 hrs after STZ injections were no different from control values. The data indicate that prolonged deficiency of insulin results in insufficient passive transport of calcium from urine into tubular cells. Thus, the increased activity of calcium-ATPase on the contraluminal membrane of kidney cells represents a futile attempt to conserve calcium.

Clinical and time factors of various forms of senile cataract. (Prospective study)

This paper presents the results of vision and slit-lamp examinations of supranuclear senile cataracts in patients whom the author has been examining for several years with the same instrument. The supranuclear, gray senile cataract (water cleft-spokes, lamellar fissures, wedge-shaped opacities) is the one with the slowest rate of development, often changing only little or not at all for several years (6 or more). The primary gray nuclear cataract also develops very slowly and frequently causes a deterioration in vision only after several years, by producing a lens with a double focal point and increasing myopia. In very long-standing cases (more than 7 years) a black cataract develops. As a senile cataract, the subcapsular cataract (permeability cataract) deteriorates rapidly within months. Subcapsular colored glints, vacuoles and granular opacities occur, more often posteriorly than anteriorly. In some cases a secondary gray nuclear opacity develops very rapidly (in contrast to the primary variety), which does not lead to myopia or black cataract. Cation pump and glutathione content remain normal in the supranuclear gray senile cataract and the primary nuclear cataract for some time; however, they are disturbed immediately and to a great extent in the subcapsular cataract.(ABSTRACT TRUNCATED AT 250 WORDS)

Response of the lens to oxidative-osmotic stress

Both aldose reductase and glutathione reductase share a common cofactor, NADPH. Glutathione reductase is preferentially activated due to its higher affinity for the cofactor. Since NADPH is primarily consumed by glutathione reductase, which in conjunction with glutathione peroxidase detoxifies H 2O 2 present in the aqueous humor, the cataractogenic role of sorbitol-induced osmotic pressure must therefore depend on the availability of NADPH for aldose reductase activity. We examined the response of the lens to an oxidative-osmotic double stress and found that the lens indeed produced 79% less sorbitol and 45% less fructose than a lens subjected to the osmotic stress alone. Morphological studies showed that photo-oxidation damaged the epithelium where the cation pump resided. However, with additional osmotic stress, the swelling of lens fibers in the posterior pole region became more pronounced, and cell nuclei deep in the lens nuclear bow were dislodged to the posterior pole. This could be explained by the slight but significant loss of K + in the lenses under the double stress. Apparently, the slightly decreased 86Rb uptake (26% loss), caused by photooxidation could not maintain adequate ionic balance even though the stress from accumulation of sorbitol + fructose was sub-maximal. No disturbance in the glycolytic activity or to the 86Rb efflux was found in these lenses, however.

Electrogenic systems of a cell and membrane permeability

The equation of membrane potential (Δψ) for electrogenic cation pump operation has been developed. The expression obtained can be equally used for the description of Δψ created by any electrogenerator that transduces light or chemical energy into electrochemical potential difference. The equation reflects the asymmetric manner of charge transfer across such systems. On the basis of the equation and of the equivalent electrical circuit the simple qualitative analysis of interplay between electrogenic and electro-utilizing systems is considered. The conclusion is that the Δψ cannot exceed the definite value for the following reasons: 1. The metabolic energy which is employed for Δψ generation is not more than 40–50 kJ. In accordance with the first law of thermodynamics the Δψ will also have a limiting value since a further increase of Δψ would lead to self-blocking of the electrogenerator. 2. The occurrence of secondary transport and diffusion systems which utilize the energy of the electrical field permanently decrease Δψ. 3. The electrical field causes microruptures inside the membrane and makes for the formation of novel leakage channels.

Active and passive rubidium influx in normal human lenses and in senile cataracts

The aim of this study was to investigate the mechanisms responsible for the osmotic imbalance which occurs in a majority of human senile cataracts. Active and passive influx of rubidium has been determined in vitro in human lenses both normal and cataractous. It is concluded that the active transport of cations is on the average normal in senile cataractous lenses. It is possible that the activity of the cation pump is defective in a few cataractous lenses but no direct direct evidence of this can be given. The results indicate that cryo-extracted lenses may be utilized in this type of study provided that cryo-treatment during surgery is kept at the lowest possible level.

Electrogenic systems of a cell and membrane permeability

Summary The equation of membrane potential (Δψ) for electrogenic cation pump operation has been developed. The expression obtained can be equally used for the description of Δψ created by any electrogenerator that transduces light or chemical energy into electrochemical potential difference. The equation reflects the asymmetric manner of charge transfer across such systems. On the basis of the equation and of the equivalent electrical circuit the simple qualitative analysis of interplay between electrogenic and electro-utilizing systems is considered. The conclusion is that the Δψ cannot exceed the definite value for the following reasons: o (1) The metabolic energy which is employed for Δψ generation is not more than 40–50 kJ. In accordance with the first law of thermodynamics the Δψ will also have a limiting value since a further increase of Δψ would lead to self-blocking of the electrogenerator. (2) The occurrence of secondary transport and diffusion systems which utilize the energy of the electrical field permanently decrease Δψ. (3) The electrical field causes microruptures inside the membrane and makes for the formation of novel leakage channels.

Ouabain-binding and 86rubidium-uptake in lymphocytes of normal and borderline hypertensive subjects.

In borderline hypertensives cellular sodium concentration seems to be increased, indicating that cellular abnormalities are present in the early course of essential hypertension. In order to study the mechanisms underlying this finding the number of sodium/potassium pump sites and the cation pump activity were studied in lymphocytes of nine borderline hypertensives (27 (20-36) years) and nine controls (28 (20-36) years). Maximum 3H-ouabain binding and 86Rb-uptake were taken as measures of the number of pump sites and cation pump activity, respectively. The median number of sodium/potassium pump sites was 49.6 X 10(3) molecules/cell in the BH group compared to 32.4 X 10(3) in the control group (P less than 0.01). Median 90 min 86Rb-uptakes were 54.0 pmol/10(6) cells in BH subjects and 39.4 in controls (P less than 0.10). The increased number of sodium/potassium pump sites and the tendency to increased cation pump activity in lymphocytes of BH subjects in vitro may be interpreted as an adaptive change possibly induced by a circulating natriuretic substance.

Active Potassium Transport by the Isolated Lepidopteran Larval Midgut: Stimulation of Net Potassium Flux and Elimination of the Slower Phase Decline of the Short-Circuit Current

ABSTRACT Adenosine 3’:5’-cyclic monophosphate (cyclic AMP), Antibiotic A23187, caffeine, cholera toxin, dibutyryladenosine 3’ :5’-cyclic monophosphate (dbcAMP), 5-hydroxytryptamine (5-HT), larval blood, and theophylline were tested for their effects on active potassium transport, as measured by the short-circuit current (SCC), in isolated Manduca sexta larval midgut. None of the tested materials affected the short-circuit current during the rapid phase of its usual exponential decline, and only dbcAMP and larval blood affected the short-circuit current during the slower phase of its exponential decline. Dibutyryl-cyclic AMP stimulated an increase in short-circuit current and net potassium flux (measured using 42K). Both unidirectional potassium fluxes increased but there was no effect on the flux ratio or on the rate at which net potassium transport declined with time. A dialysable extract of fifth instar larval blood effected potassium transport in a manner similar to that of dbcAMP, indicating that larval blood contains natural transport-stimulating factors. A dialysable extract of fourth instar larval blood not only stimulated potassium transport but also prevented the slow phase of decline in the short-circuit current. Therefore, in addition to transport-stimulating factors present in fifth instar blood, fourth instar blood appears to contain substances which prevent degeneration of at least the transport related functions of midgut in vitro. It is argued that the action of the transport-stimulating factor in larval blood on the electrogenic cation pump in larval midgut cells is mediated by cyclic AMP without involving a change in intracellular calcium ion concentration.