β-Cyclodextrin and permeability to water in the bladder of Bufo arenarum

We measured the effect of β-cyclodextrin (BCD, a cholesterol scavenger) on water flow across the isolated toad bladder exposed to an osmotic gradient (Jw) by a gravimetric technique. BCD, when present in the solution bathing the apical side of the bladder, inhibited the increase in Jw caused by nystatin, a polyene antibiotic that acts by directly binding apical membrane cholesterol. When present in the basolateral bath, BCD inhibited the increase in Jw caused by basolateral exposure to oxytocin (which binds membrane receptors and stimulates the synthesis of cAMP), but did not alter the response to theophylline (which inhibits hydrolysis of cAMP by cyclic nucleotide phosphodiesterase). The present data are consistent with the notion that agents that increase Jw by interacting with membrane receptors, which appear to be clustered in cholesterol-rich domains of the basolateral membrane, are altered by cholesterol depletion, whereas agents that do not interact with receptors or other basolateral membrane components are not affected by this treatment. In either case, cholesterol depletion of the apical membrane does not affect the increase in Jw brought about by an increase in intracellular cAMP concentration.

Olfactory response termination involves Ca2+-ATPase in vertebrate olfactory receptor neuron cilia

In vertebrate olfactory receptor neurons (ORNs), odorant-induced activation of the transduction cascade culminates in production of cyclic AMP, which opens cyclic nucleotide–gated channels in the ciliary membrane enabling Ca2+ influx. The ensuing elevation of the intraciliary Ca2+ concentration opens Ca2+-activated Cl− channels, which mediate an excitatory Cl− efflux from the cilia. In order for the response to terminate, the Cl− channel must close, which requires that the intraciliary Ca2+ concentration return to basal levels. Hitherto, the extrusion of Ca2+ from the cilia has been thought to depend principally on a Na+–Ca2+ exchanger. In this study, we show using simultaneous suction pipette recording and Ca2+-sensitive dye fluorescence measurements that in fire salamander ORNs, withdrawal of external Na+ from the solution bathing the cilia, which incapacitates Na+–Ca2+exchange, has only a modest effect on the recovery of the electrical response and the accompanying decay of intraciliary Ca2+ concentration. In contrast, exposure of the cilia to vanadate or carboxyeosin, a manipulation designed to block Ca2+-ATPase, has a substantial effect on response recovery kinetics. Therefore, we conclude that Ca2+-ATPase contributes to Ca2+ extrusion in ORNs, and that Na+–Ca2+exchange makes only a modest contribution to Ca2+ homeostasis in this species.

Carbon dioxide and pH effects on temperature-sensitive and -insensitive hypothalamic neurons

The preoptic-anterior hypothalamus (POAH) controls body temperature, and thermoregulatory responses are impaired during hypercapnia. If increased CO2 or its accompanying acidosis inhibits warm-sensitive POAH neurons, this could provide an explanation for thermoregulatory impairment during hypercapnia. To test this possibility, extracellular electrophysiological recordings determined the effects of CO2 and pH on the firing rates of both temperature-sensitive and -insensitive neurons in hypothalamic tissue slices from 89 male Sprague-Dawley rats. Firing rate activity was recorded in 121 hypothalamic neurons before, during, and after changing the CO2 concentration aerating the tissue slice chamber or changing the pH of the solution bathing the tissue slices. Increasing the aeration CO2 concentration from 5% (control) to 10% (hypercapnic) had no effect on most (i.e., 69%) POAH temperature-insensitive neurons; however, this hypercapnia inhibited the majority (i.e., 59%) of warm-sensitive neurons. CO2 affected similar proportions of (non-POAH) neurons in other hypothalamic regions. These CO2 effects appear to be due to changes in pH since the CO2-affected neurons responded similarly to isocapnic acidosis (i.e., normal CO2 and decreased pH) but were not responsive to isohydric hypercapnia (i.e., increased CO2 and normal pH). These findings may offer a neural explanation for some heat-related illnesses (e.g., exertional heat stroke) where impaired heat loss is associated with acidosis.

Water Transfer by The Toad Bladder

ABSTRACT. Studies have been made on the isolated urinary bladder of the toad, Bufo marinus, in an attempt to investigate the effect of vasopressin on the permeability of water from mucosal surface to serosal surface of the toad bladder. The method adapted was that described by Bentley ( 1 ). The bilobed bladder of the toad is devided into two separate sacs. Each of the sacs is filled with a dilute Ringers solution and then immersed in aerated isotonic Ringers solution. The rate of water loss along the imposed osmotic gradient is estimated by weighing the sacs in air at 30 minute intervals and nothing the weight loss in that time period. In most studies one bladder sac serves as a control for the contra lateral experimental obtained from the same animal. Osmotic flow of water is negligible in both sacs during the initial control periods. However, the addition of vasopressin to the solution bathing the serosal surface of the membrane result in a market increase in net water movement. The effect is readily reversed by rinsing the bladder and adding hormone free Ringgers solution to the serosal surface. Characteristically no response is elicited by addition of hormone to the mucosal bathing solution.

Modification of rhizosphere pH by the symbiotic legume Aspalathus linearis growing in a sandy acidic soil

Aspalathus linearis is a N2-fixing legume used for tea production, and grows in highly acidic soils (pH 3–5.3) of the Cederberg mountains in South Africa. Field and glasshouse studies revealed significantly higher pH in rhizosphere than non-rhizosphere soils. However, when six non-legume species were studied in adjacent fields, there were no differences in pH between rhizosphere and non-rhizosphere soils. The culture of A. linearis plants in sterile Leonard jars similarly showed a marked increase of 2.8 pH units in the nutrient solution bathing the roots of inoculated (nodulated) plants, compared to 1.5 pH units in uninoculated control. The uptake and reduction of NO3– by plants fed 2 mM NO3– also raised the rhizosphere pH by 3.5 units, a value comparable to that of the nodulated plants. The use of titrimetric methods showed that OH– and HCO3– were the components of alkalinity in the nutrient solution bathing roots of A. linearis, and were directly responsible for the increase in rhizosphere pH. These findings suggest that the ability to raise rhizosphere pH is an adaptative feature of this legume symbiosis that overcomes the adverse effects of low pH in enhancing nutrient acquisition and reducing trace element toxicity.

Colocalization of glycolytic enzyme activity and KATP channels in basolateral membrane of Necturusenterocytes

86Rb fluxes through ATP-regulated K+(KATP) channels in membrane vesicles derived from basolateral membranes of Necturus small intestinal epithelial cells as well as the activity of single KATP channels reconstituted into planar phospholipid bilayers are inhibited by the presence of ADP plus phospho enolpyruvate in the solution bathing the inner surface of these channels. This inhibition can be prevented by pretreatment of the membranes with 2,3-butanedione, an irreversible inhibitor of pyruvate kinase (PK) and reversed by the addition of 2-deoxyglucose plus hexokinase. The results of additional studies indicate that PK activity appears to be tightly associated with this membrane fraction. These results, together with considerations of the possible ratio of Na+-K+pumps to KATP channels in the basolateral membrane, raise the possibility that “cross talk” between those channels and pumps (i.e., the “pump-leak parallelism”) may be mediated by local, functionally compartmentalized ATP-to-ADP ratios that differ from those in the bulk cytoplasm.

K+-neutral amino acid symport of Bombyx mori larval midgut: a system operative in extreme conditions

The K+-dependent symporter for leucine and other neutral amino acids expressed along the midgut of the silkworm Bombyx mori operates with best efficiency in the presence of a steep pH gradient across the brush-border membrane, with external alkaline pH values up to 11, and an electrical potential difference (Δψ) of ∼200 mV. Careful determinations of leucine kinetics as a function of external amino acid concentrations between 50 and 1,000 μM, performed with brush-border membrane vesicles (BBMV) obtained from the middle and posterior midgut regions, revealed that the kinetic parameter affected by the presence of a ΔpH was the maximal rate of transport. The addition of Δψ caused a further marked increase of the translocation rate. At nonsaturating leucine concentrations in the solution bathing the external side of the brush-border membrane, leucine accumulation within BBMV and midgut cells was not only driven by the gradient of the driver cation K+ and Δψ but occurred also in the absence of K+. The ability of the symporter to translocate the substrate in its binary form allows the intracellular accumulation of leucine in the absence of K+, provided that a pH gradient, with alkaline outside, is present. The mechanisms involved in this accumulation are discussed.

The kinetics of inactivation of the rod phototransduction cascade with constant Ca2+i.

A rich variety of mechanisms govern the inactivation of the rod phototransduction cascade. These include rhodopsin phosphorylation and subsequent binding of arrestin; modulation of rhodopsin kinase by S-modulin (recoverin); regulation of G-protein and phosphodiesterase inactivation by GTPase-activating factors; and modulation of guanylyl cyclase by a high-affinity Ca(2+)-binding protein. The dependence of several of the inactivation mechanisms on Ca2+i makes it difficult to assess the contributions of these mechanisms to the recovery kinetics in situ, where Ca2+i is dynamically modulated during the photoresponse. We recorded the circulating currents of salamander rods, the inner segments of which are held in suction electrodes in Ringer's solution. We characterized the response kinetics to flashes under two conditions: when the outer segments are in Ringer's solution, and when they are in low-Ca2+ choline solutions, which we show clamp Ca2+i very near its resting level. At T = 20-22 degrees C, the recovery phases of responses to saturating flashes producing 10(2.5)-10(4.5) photoisomerizations under both conditions are characterized by a dominant time constant, tau c = 2.4 +/- 0.4 s, the value of which is not dependent on the solution bathing the outer segment and therefore not dependent on Ca2+i. We extended a successful model of activation by incorporating into it a first-order inactivation of R*, and a first-order, simultaneous inactivation of G-protein (G*) and phosphodiesterase (PDE*). We demonstrated that the inactivation kinetics of families of responses obtained with Ca2+i clamped to rest are well characterized by this model, having one of the two inactivation time constants (tau r* or tau PDE*) equal to tau c, and the other time constant equal to 0.4 +/- 0.06 s.

Reconstitution of a KATP channel from basolateral membranes of Necturus enterocytes

We have previously reported that basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells and incorporated into planar phospholipid bilayers display a highly selective "maxi"-conductance K+ channel whose open-time probability is affected by voltage. We now report that this channel is inhibited by MgATP in the solution bathing the intracellular face of the channel but not by Mg2+ or the Na+ or K+ salts of ATP; the effects of MgATP can be prevented or reversed by MgADP. The channel is also inhibited by the nonhydrolyzable ATP analogue magnesium adenosine 5'-O-(3-thiotriphosphate) and the sulfonylurea derivatives tolbutamide and glibenclamide; all of these agents are effective in the intracellular compartment but not when added to the extracellular compartment alone. Channel activity is stimulated by the "K+ channel opener," diazoxide, which also reverses the effect of glibenclamide but not of MgATP. The possible role of this channel as a mediator of the parallelism between basolateral membrane Na(+)-K+ pump activity and the macroscopic K+ conductance of that barrier is discussed.