The effect of calcium on the respiratory responses of corn mitochondria.

Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca2+ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage.

A standard of comparison for acute surgical necrotizing enterocolitis.

We reviewed 187 cases of documented neonatal necrotizing enterocolitis (NEC) from 1976 to 1988. Of these patients, 111 infants underwent celiotomy for acute surgical complications. The following protocol of operative indications was employed: pneumoperitoneum, localized mass, abdominal wall erythema, portal venous air, and clinical deterioration, singly or in any combination. Clinical deterioration was defined as falling platelet count, rising or falling white blood cell count, left shift in the myeloid series, persistently or progressively low pH, and increasing frequency of apnea or bradycardia. Overall mortality was 15% (28 of 187). For the patients who underwent celiotomy, all had histologic confirmation of NEC. Ninety-five had localized disease, and 16 had diffuse disease. All of the former had resection and diverting enterostomy with 85 (89.5%) surviving; none with diffuse disease survived, P less than 0.0001. Forty-one infants with NEC weighed less than 1,000 g; 25 underwent surgery and 15 (60%) survived. Fifty-one of the 159 surviving neonates (32%) developed intestinal strictures. All neonates with strictures have had resection and successful reconstruction of their gastrointestinal tract. These indications and surgical principles resulted in a high degree of diagnostic accuracy and a low degree of surgical mortality.

Classification of otitis media and surgical principles.

Otitis media is an important disease of children and adults and is caused by multiple interrelated factors, including infection, eustachian tube dysfunction, allergy, and barotrauma. This article includes a pertinent review of the literature regarding otitis media. The pathogenesis, classification, and treatment of otitis media in children and adults are also reviewed in this article. Additionally, therapy is discussed with emphasis on the surgical options appropriate at each stage.

Application of the chemiosmotic hypothesis to ion transport across the root.

The evidence on how ions accumulated in the root symplasm are released to the xylem vessels is examined. It is suggested that Mitchell's chemiosmotic hypothesis as applied to ion transport might account for the process. A model based on this hypothesis shows the symplasm as an osmotic unit connecting two isolated solutions, but with no significant difference in proton motive force across the unit. If it is assumed that the resistance to transport by plasmalemma uniports and antiports differs in the cortical and stelar ends of the symplasm, the model will provide for an influx of ions to the xylem. The reported properties of isolated steles suggests that the porters (carriers) do have properties in accord with the model.

Ion transport induced by polycations and its relationship to loose coupling of corn mitochondria.

Treatment of corn mitochondria (Zea mays L., WF9 (Tms) x M14) with polycations (protamine, pancreatic ribonuclease, or polylysine) releases acceptorless respiration if phosphate is present. Concurrently, there is extensive active swelling which is reversed when respiration is uncoupled or stopped. Mersalyl, the phosphate transport inhibitor, blocks both the release of respiration and the active swelling. Diversion of energy into phosphate transport lowers respiratory control and ADP: O ratios. This response is termed "loose coupling" in distinction to "uncoupling" in which energy is made unavailable for either transport or ATP formation. Corn mitochondria as used here are endogenously loose coupled to some extent, and show state 4 respiration linked to active transport.The action of polycations can be partially mimicked by lowering pH of the suspending medium; both give swelling in sucrose medium and increased light absorbancy. Triton X-100, a nonionic detergent, will uncouple and accentuate active swelling, but unlike polycations it will not release state 4 respiration. Calcium ion acts something like polycation in activating phosphate transport and releasing respiration, and polycation appears to block entry of Ca(2+).It is speculated that neutralization of certain negatively charged acid groups on the membranes by polycations increases permeability to solutes and decreases coulombic repulsion of phosphate in approaching transport sites. In consequence, respiration rates and active transport rates increase.

The effect of 2,4-dichlorophenoxyacetic acid upon the metabolism and composition of soybean hypocotyl mitochondria.

Dark-grown, 3-day-old soybean seedlings were sprayed with 1 mM 2,4-dichlorophenoxyacetic acid 24 hours before harvest. Mitochondria from 2,4-D-treated lower hypocotyls were found to be larger and showed greater incorporation in vivo, of amino acids into protein and phosphate into phospholipids and RNA, than mitochondria from untreated tissue. Mitochondria isolated from 2,4-D-treated hypocotyls showed an enhanced energy-dependent incorporation of amino acids into protein, although the incorporation of nucleoside triphosphates into the RNA of isolated mitochondria was not affected. No effect of 2,4-D, applied in vitro, was noted, and no enhancement of mitochondrial respiratory efficiency followed auxin treatment. A method of isolating mitochondria with a very low level of bacterial contamination is reported.

Uncoupling of energy-linked functions of corn mitochondria by linoleic Acid and monomethyldecenylsuccinic Acid.

Linoleic acid and monomethyldecenylsuccinic acid were tested as uncoupling agents for energy linked functions of corn mitochondria. 2,4-dinitrophenol was used as a standard for comparison. Both compounds uncoupled oxidative phosphorylation, released oligomycin-blocked respiration, and accelerated adenosine triphosphatase. Linoleic acid uncoupled calcium-activated phosphate accumulation and the increase in light scattering that accompanies the accumulation. Unlike dinitrophenol, linoleic acid at 0.1 mm had a destructive effect on membrane semipermeability. Kinetic studies indicated that dinitrophenol and linoleic acid compete with phosphate for active sites in oxidative phosphorylation.Some linoleic acid is taken up by respiring mitochondria and a major share of the uptake is incorporated into phospholipids. Calcium ion and oligomycin promote the uptake, but coenzyme A does not. It is deduced that fatty acid probably attacks the non-phosphorylated intermediate, I approximately X, producing X approximately acyl. Uncoupling results from breakdown of X approximately acyl, but sufficient X approximately acyl is maintained to serve as a source of activated fatty acid.

Swelling and contraction of corn mitochondria.

A survey has been made of the properties of corn mitochondria in swelling and contraction. The mitochondria swell spontaneously in KCl but not in sucrose. Aged mitochondria will swell rapidly in sucrose if treated with citrate or EDTA. Swelling does not impair oxidative phosphorylation if bovine serum albumin is present.Contraction can be maintained or initiated with ATP + Mg or an oxidizable substrate, contraction being more rapid with the substrate. Magnesium is not required for substrate powered contraction. Contraction powered by ATP is accompanied by the release of phosphate. Oligomycin inhibits both ATP-powered contraction and the release of phosphate. However, it does not affect substrate-powered contraction. Substrate powered contraction is inhibited by electron-transport inhibitors. The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone, accelerates swelling and inhibits both ATP-and substrate-powered contraction. However, the concentrations required are well in excess of those required to produce uncoupling and to accelerate adenosine triphosphatase; the concentrations required inhibit respiration in a phosphorylating medium.Phosphate is a very effective inhibitor of succinate-powered contraction. Neither oligomycin nor Mg affects the phosphate inhibition. Phosphate is less inhibitory with the ATP-powered contraction.The results are discussed in terms of a hypothesis that contraction is associated with a nonphosphorylated high energy intermediate of oxidative phosphorylation.

Calcium-activated phosphate uptake in contracting corn mitochondria.

The phosphate inhibition of succinate-powered contraction in corn mitochondria can be reversed with calcium. Associated with this reversal is an accumulation of phosphate and calcium. Both ions are essential for accumulation, although strontium will partially substitute for calcium. Arsenate does not substitute for phosphate except in producing the inhibition of contraction.The antibiotics oligomycin and aurovertin do not block the phosphate inhibition of contraction or the calcium-activated phosphate uptake associated with the release of the inhibition. Dinitrophenol uncouples the phosphate uptake but permits full contraction.Calcium promotes inorganic phosphate accumulation in root tissue as well as in mitochondria.The results are discussed from the viewpoint of theories of calcium reaction with high energy intermediates of oxidative phosphorylation. It is concluded that calcium probably reacts with X approximately P in corn mitochondria, rather than with X approximately I as with animal mitochondria.

Contracted state as an energy source for ca binding and ca + inorganic phosphate accumulation by corn mitochondria.

An investigation has been made of the possibility of utilizing the potential energy of the contracted state of corn mitochondria to drive Ca + inorganic phosphate accumulation. Contraction was obtained with succinate or NADH oxidation. In the succinate experiments the mitochondria were contracted in buffered KCl layered over sucrose in centrifuge tubes and centrifuged down through distinct wash, reactive and isotope exchange layers. In the NADH experiments, ion accumulation was initiated upon exhaustion of the substrate. The results show that mitochondria in the contracted state will actively bind some (45)Ca, but no real accumulation occurs until inorganic phosphate is available. Substrate powered contraction in the presence of inorganic phosphate also provides a potential for accumulation upon subsequent reaction of the mitochondria with Ca. It is deducted that contraction is due to X approximately I formation, to which Ca will bind. Subsequent reaction with inorganic phosphate produces CaX approximately P, which is the transport moiety. When X approximately P is formed first, Ca also reacts to produce CaX approximately P. Hence it is immaterial which ion reacts first with the contracted state. Contraction is believed to result from the action of a mechanoenzyme, presumably I approximately . The stability of CaX approximately I must be low for the mitochondria swell very rapidly upon exhaustion of NADH or blocking of succinate oxidation by cyanide.

Controls on na influx in corn roots.

We have investigated the effects of hyperpolarization and depolarization, and the presence of K(+) and/or Ca(2+), on (22)Na(+) influx into corn (Zea mays L.) root segments. In freshly excised root tissue which is injured, Na(+) influx is unaffected by hyperpolarization with fusicoccin, or depolarization with uncoupler (protonophore), or by addition of K(+). However, added Ca(2+) suppresses Na(+) influx by 60%. In washed tissue which has recovered, Na(+) influx is doubled over that of freshly excised tissue, and the influx is increased by fusicoccin and suppressed by uncoupler. This energy-linked component of Na(+) influx is completely eliminated by low concentrations of K(+), leaving the same level and kind of Na(+) influx seen in freshly excised roots. The K(+)-sensitive energy linkage appears to be by the carrier for active K(+) influx. Calcium is equally inhibitory to Na(+) influx in washed as in fresh tissue. Other divalent cations are only slightly less effective. Net Na(+) uptake was about 25% of (22)Na(+) influx, but proportionately the response to K(+) and Ca(2+) was about the same.The constancy of K(+)-insensitive Na(+) influx under conditions known to hyperpolarize and depolarize suggests that if Na(+) transport is by means of a voltage-sensitive channel, the rise or fall of channel resistance must be proportional to the rise or fall in potential difference. The alternative is a passive electroneutral exchange of (22)Na(+) for endogenous Na(+). The data suggest that an inwardly directed Na(+) current is largely offset by an efflux current, giving both a small net uptake and isotopic exchange.

Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria.

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O(2) uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K(+) permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V(max), probably reflecting a diffusion-limited process.The results indicate that rapid malate and succinate oxidation require the operation of both the phosphate and dicarboxylate transporters, which in turn depend on maintenance of a proton motive force across the inner membrane. In addition, phosphate can stimulate acceptorless malate oxidation by reaction with the coupling mechanism, and in uncoupled mitochondria which are depleted of substrate there is a slow rate of oxidation which appears to be limited by diffusive entry.

Pyruvate and malate transport and oxidation in corn mitochondria.

Pyruvate oxidation and swelling in pyruvate solutions by corn (Zea mays) mitochondria were inhibited by alpha-cyano-4-hydroxy-cinnamic acid, an inhibitor of pyruvate transport in animal mitochondria; however, there was no inhibition of pyruvate dehydrogenase activity, and malate and NADH oxidation were not affected. These results suggest the presence of a pyruvate(-)-OH(-) exchange transporter which supplies the mitochondrion with oxidizable substrate. Lactate appears to be transported also, but not dicarboxylate anions or inorganic phosphate. The rate of pyruvate transport was much slower than that of malate, however, and valinomycin was required to elicit appreciable swelling in potassium pyruvate.Malate oxidation contributed significantly to respiration supported by pyruvate plus malate, and malate did not act solely as a "sparker" for pyruvate oxidation. NAD(+)-malic enzyme activity was found in sonicated preparations, and comparison of O(2) consumption with CO(2) released from 1-(14)C-pyruvate indicated that transported malate was being converted to pyruvate, particularly as the malate to pyruvate ratio increased. The results suggest that pyruvate transport becomes limiting under conditions of high energy demand, but that rapid malate transport makes up the difference, supplying pyruvate via malic enzyme and replenishing losses of tricarboxylic acid cycle intermediates.

Activation of endogenous respiration and anion transport in corn mitochondria by acidification of the medium.

Acidification of the suspending medium of corn mitochondria (Zea mays L., WF9 x Mo17) from pH 7.5 to pH 6.8 to 6.4 initiates osmotic swelling with the transportable anions citrate, sulfate, and phosphate. Swelling becomes pronounced with a combination of citrate plus sulfate or phosphate. Acidification proves to activate endogenous respiration, which is essentially zero at pH 7.5. The endogenous respiration transports citrate (in the presence of sulfate or phosphate) which then contributes to respiration and the accelerated osmotic swelling. Mersalyl will inhibit the swelling and antimycin inhibits the endogenous respiration. Magnesium appears to reduce the permeability of the membranes under the acid conditions.

Cell potentials, cell resistance, and proton fluxes in corn root tissue: effects of dithioerythritol.

Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H(+) and K(+) fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents.We deduce that dithioerythritol activates a passive H(+)/K(+) antiport, driven in these experiments by the outwardly directed electrochemical gradient of K(+). The net effect on H(+) and K(+) fluxes is believed to reside with the combined activity of a polarized H(+)/K(+) exchanging ATPase and the passive H(+)/K(+) antiport. A model is presented to show how the combined system might produce stable potential differences and K(+) content.

The action of valinomycin in uncoupling corn mitochondria.

Valinomycin in the presence of potassium is a potent uncoupler of corn (Zea mays L.) mitochondria, eliminating respiratory control. Valinomycin produces higher steady state potassium phosphate swelling which can be reversed to give active shrinkage if mersalyl is added to block the Pi(-)/OH(-) antiporter. Respiration declines concurrently. Uncouplers accelerate the shrinkage and restore the respiration. The same results can be obtained with sodium phosphate if gramicidin D is substituted as ionophore.It is concluded that valinomycin uncoupling is the result of cyclic salt transport, with influx pumping of potassium phosphate via the Pi(-)/OH(-) antiporter and efflux pumping via a K(+)/H(+) antiporter. The result is a higher level of steady state swelling, rapid turnover of the proton gradient, and uncoupled respiration rates. The level of steady state swelling can be manipulated by varying the valinomycin or K(+) concentrations, with high concentrations favoring activation of the efflux pump.A mosaic membrane model with high resistance for proton and monovalent cation penetration to the cation(+)/H(+) antiporter is used to explain the results.