Hypertonic Sucrose Solutions Research Articles

Water relations of gas-vacuolate prokaryotes

Gas vesicles can be used to provide an instantaneous measurement of the hydrostatic pressure that exists inside the cells of prokaryotic organisms that possess them. Such measurements, carried out on the blue-green alga Anabaena flos-aquae , have been used to determine its cell-water relations. The cell turgor pressure is determined as the difference between the mean critical pressure required to collapse gas vesicles in cells' suspended in hypertonic sucrose solution and that for the cells suspended in water. The turgor pressure falls slightly as the gas vesicles are collapsed because the volume of the cell contents decreases. The relation between the cell turgor pressure and volume is described by the volumetric elastic modulus, e, which can be determined by measuring the turgor pressure of cells suspended in sucrose solutions of increasing concentration. As the external osmotic pressure is increased, the turgor pressure, which stretches the cell wall, falls and the cell shrinks by losing water. The cell water volume can be calculated from the internal osmotic pressure, which is equal to the sum of the external osmotic pressure and the turgor pressure. The elastic modulus of the A. flos-aquae . cell is quite low, about 11 bar, and constant over a range of turgor pressures down to 1.3 bar. W hen gas vesicles are collapsed inside cells, the turgor pressure suddenly drops, but it partially recovers as water enters to restore the osmotic balance. The rate of entry can be computed from the time course of gas vesicle collapse in cells held under moderate hydrostatic pressures, and from this the hydraulic conductivity, L p , of the cell wall and membrane can be calculated; it is estimated to be about 0.14 μm s -1 bar -1 in A . flos-aquae , with the assumption that there are no errors owing to delay in the elastic contraction of the cell wall. The permeability, k , of the cell to various solutes can be determined by following the rate of turgor pressure recovery as the solutes penetrate the cells from external solutions of 0.15 osm concentration. Two methods are described in which turgor pressure rise is monitored by gas vesicle collapse, one for slowly penetrating solutes (e.g., mannitol) and one for rapidly penetrating solutes (e.g., glycerol). Passive uptake is indicated by exponential kinetics of turgor pressure recovery to the initial value in water, as for sugar alcohols; active uptake results in turgor pressures exceeding the initial value, as observed for K + and glycine—betaine. The biological significance of cell—water relations in gas-vacuolate organisms is discussed.

Thirst and vasopressin release in the dog: an osmoreceptor or sodium receptor mechanism?

The effects of intravenous infusion of hypertonic NaCl, sucrose, glucose, urea, or isotonic NaCl solution on thirst and plasma arginine vasopressin concentration (AVP) were studied in five conscious dogs. The changes in osmolality and sodium concentration of plasma and cerebrospinal fluid (CSF) were measured at the threshold of drinking, or after 45 min if no drinking occurred. Hypertonic NaCl and sucrose stimulated drinking in all dogs and significantly elevated plasma AVP. Equally hypertonic glucose, urea, or isotonic NaCl failed to stimulate any drinking or vasopressin secretion. All hypertonic solutions caused significant and similar increases in the osmolality and sodium concentration of CSF. Plasma osmolality was increased by the hypertonic solutions. Plasma sodium was increased by hypertonic NaCl, decreased by sucrose and glucose, and not changed by urea. Isotonic NaCl had no effect on either plasma or CSF composition. These data are not consistent with either a sodium or an osmoreceptor mechanism located within the blood-brain barrier (BBB) or with a peripheral sodium receptor mechanism. An intracranial osmoreceptor located on the blood side of the BBB is proposed to explain these results.

Osmotic pressure and angiotensin II stimulation of arginine vasopressin release from a guinea pig hypothalamo-neurohypophyseal complex in organ culture (author's transl)

An organ culture system of a male guinea pig hypothalamo-neurohypophyseal complex (HNC) was established. On day 5 in culture, (Na+ -K+) ATPase activity was 0.83 +/- 0.11 mM Pi/mg prot/hr (mean +/- SEM): that is 67% of that on day 1 in culture. 3H-thymidine incorporated into DNA in the explants of HNC was 1,205 +/- 185 cpm/microgram DNA. The explants responded to the elevated KCl medium and the hypertonic solution of sodium chloride with a 470 +/- 38% and 298 +/- 31% increase in arginine vasopression (AVP) release, respectively. This response was inhibited by the addition of tetrodotoxin to the culture medium. AVP release from the explants in res-onse to angiotensin II increased significantly in a dose dependent manner. [Sar1, Ile8] angiotensin II, however, attenuated the response of the explants to angiotensin II when administered simultaneously with angiotensin II. These results suggest that angiotensin II and its analogue cause the AVP release from the explants in a competitive manner. The concentrations of AVP in the culture media made hypertonic with sodium chloride, sucrose and mannitol were 298 +/- 31% (p less than 0.01), 251 +/- 36% (p less than 0.01) and 255 +/- 59% (p less than 0.05) of their control values, respectively. The hypertonic solutions of sodium chloride, sucrose and mannitol caused AVP release from the explants in vitro, while the hypertonic solutions of glucose and urea were revealed to be poor osmotic stimuli on AVP release. These results support the concept of osmoreceptors to release AVP from the hypothalamo-neurohypophyseal axis.

Evidence for adherence of host plasmalemma to infecting hyphae of both compatible and incompatible races of Phytophthora infestans

Aged discs of potato tuber were inoculated with incompatible race 0 and compatible race 1 of Phytophthora infestans, and then plasmolysed with hypertonic sucrose solution. Both the plasmolysed cells infected by the incompatible and compatible races showed conically shaped protoplasts, parts of which adhered to the intracellular hyphae. We named this phenomenon “tent-type plasmolysis”. Electron microscopic observation showed that many fragments of host plasmalemma adhered to the infecting hyphae, when the host plasmalemma appeared to be detached from the hyphae upon plasmolysis. These fragments adhering to the infecting hyphae seemed to be torn off from the host plasmalemma which was separated from the hyphae by plasmolysis. All these results suggested that the host plasmalemma strongly adhered to infecting hyphae of both compatible and incompatible races from the very earliest time of penetration. Tent-type plasmolyses in infected cells were also observed with petioles of potato leaves.

Relation between leaf senescence and stomatal closure: Senescence in light

Senescence of isolated oat seedling leaves, floating on water or solutions in white light, has been followed by the disappearance of chlorophyll and the liberation of free amino nitrogen. In parallel, measurements of stomatal aperture were made with a diffusion resistance porometer, and borne out also by changes in fresh weight. The stomata open as expected in the light but slowly begin to close after the first day; correspondingly, in the dark they close at once but gradually begin to open on successive days. Abscisic acid causes closure and this is accompanied by senescence. Phenylmercuric nitrate also causes closure and again the reaction is closely paralleled by senescence. Kinetin maintains stomatal opening even more than does light alone, and this is accompanied by complete prevention of chlorophyll loss for at least 5 days. Covering the leaf surface with a film of Vaseline, especially when detergents are added, accelerates senescence. Merely floating the leaf segments on hypertonic solutions of sucrose or mannitol suffices to bring the rate of senescence in light up to the rate in darkness. It is concluded that the effect of light in delaying senescence is primarily due to its effect on the stomatal aperture, and, more generally, that stomatal aperture is the principal controlling agent in leaf senescence.

Effects of phospholipase A 2 from cobra and bee venom on the presynaptic membrane

L. G. Magazanik, I. M. Gotgilf, T. I. Slavnova, A. I. Miroshnikov and U. R. Apsalon. Effects of phospholipase A 2 from cobra and bee venom on the presynaptic membrane. Toxicon 17, 477–488, 1979.—Phospholipases A 2 from bee venom and cobra venom have been isolated and studied. A parallelism was found between enzymatic activity and the ability to block spontaneous miniature end-plate potentials (m.e.p.p.'s) or end-plate potentials (e.p.p.'s) induced by nerve stimulation in the frog sartorius muscle. Different experimental procedures affected both enzymatic activity and blocking ability in qualitatively the same way. Thus, modification of the histidine residue in cobra venom phospholipase by bromophenacyl bromide or the removal of Ca-ions from the medium abolished both activities. Replacement of Ca 2+ by Sr 2+ inhibited both the enzymatic and presynaptic effects of cobra venom phospholipase, but did not inhibit the presynaptic action of bee venom phospholipase and decreased its enzymatic activity only 6-fold. Irreversible binding of cobra and bee venom phospholipase to the presynaptic membrane was found in Ca-free solution but Ca-ions were essential for the presynaptic blocking effect induced by these phospholipases. A reduction in the effect of high K + on m.e.p.p. frequency was observed after cobra venom phospholipase treatment. The similar effects of hypertonic sucrose solution and the mitochondrial poison TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) were changed only slightly by bee and cobra venom phospholipase pretreatment. It is concluded that the mechanism of presynaptic blockade induced by bee venom and cobra venom phospholipase consists mainly of damage to sites of release at the presynaptic membrane. There are also some signs of disturbances of depolarization-secretion coupling and of the process of formation of new quanta. The possible functional role of enzymatic activity in the presynaptic effect is discussed.

Synthesis and metabolism of prostaglandins E 2, F 2α and D 2 by the rat gastrointestinal tract. Stimulation by a hypertonic environment in vitro

Whole cell preparations of rat stomach corpus, jejunum, and colon were incubated and the released prostaglandin E 2 (PGE 2), PGF 2α, PGD 2, 15 keto-13,14 dihydro PGE 2, and 15 keto-13,14 dihydro PGF 2α were measured by combined gas chromatography-mass spectrometry. All regions made PGD 2 and possessed a high capacity for producing 15 keto-13,14 dihydro derivatives of both PGE 2 and PGF 2α. Hypertonic sucrose solutions resulted in concentration-dependent increases in prostaglandin release, particularly of PGE 2 and its metabolite. It is suggested that PG's may play a role in the local effects of luminal hyperosomolarity on digestive tract functions.

Decoupling of the excitation-contraction link by glycerol procedure in volume-clamped muscle fibers.

The role of "osmotic shock" and vesiculation in the mechanism of disconnection of the T-system tubules by the glycerol procedure was studied in volume-clamped muscle fibres of the crayfish. The transient dilatation of fibres during the wash-out of glycerol was prevented by application of hypertonic sucrose solutions of decreasing osmolarity to hold the fibre volume constant. Under these conditions the fibres were decoupled only partially; the twitch tension was decreased to a steady level of about 40% of the control tension. The decay in tension developed without the transient potentiation observed in fibres allowed to dilate. The decoupling could not be reversed by reapplication of glycerol saline. The structure of volume-clamped fibres differs from the dilated ones in two respects; the vesicles appear smaller and are absent in continuous layers (about 4 micrometer in width) just under the surface and the cleft membranes. Electron microscopy of these regions has revealed the presence of normal T-system tubules communicating with the fibre exterior. The results provide evidence for the secondary role of the osmotic factor and the essential role of the vesiculation in the induction of the detubulation by glycerol procedure.

Ultrastructural features of osmotic shock in mussel gill cilia

Terminal gill cilia of the freshwater mussel Unio stop abruptly within 5 min (shock-stop) when distal gill filaments are placed in hypertonic (>217 ± mOsm) salt, urea or sucrose solutions. Electron microscopy of stopped cilia reveals that the axonemal complexes are obscured by an electron-dense matrix, although microtubule protofilaments are visible after fixation with tannic acid—glutaraldehyde. We conclude tubulin was not depolymerized because colchicine and vinblastine, inhibitors of tubulin polymerization, did not prevent stopped cilia from resuming beating in water. The cytosol of stopped cilia which resumed beating in the same solution or in water is less dense, and axonemal complexes have reappeared. Solutions of increasing tonicity retard or stop cilia, possibly due to aggregation of protein-rich cytosol around microtubules in the ciliary stalk because dense cytosol of stopped cilia is digested by pepsin and trypsin.

Cellular and extracellular dehydration, and angiotensin as stimuli to drinking in the common iguana Iguana iguana.

1. After water deprivation, the iguana promptly drank slightly more than enough water to restore the body fluids to isotonicity even under conditions of hypervolaemia. 2. In response to systemic injections of hypertonic solutions of NaCl and sucrose, the iguana drank and retained enough water to dilute the injected load to isotonicity irrespective of whether water was offered immediately or after 3 hr, and irrespective of whether the solute was administered I.V. or I.P. 3. Hypertonic solutions to glucose, urea, sorbitol and KCl caused little drinking. 4. The long latencies to drinking after hypertonic loads, which were not dependent on the nature of the solute, the route of administration or the dosage, were shown not to be a result of slow distribution of the solute throughout the body fluids. 5. Clearance of injected solutes via renal and extra-renal (nasal salt gland) routes was negligible during the 6 hr experimental period. 6. Measurements of plasma [Na], haematocrit, osmotic pressure and inulin space showed that the iguana drank, in response to cellular dehydration, enough water to restore the intracellular fluid volume to normal. 7. We conclude that, in response to substances which dehydrate cells, the iguana regulates its body osmolality precisely and efficiently provided it is able to do so by drinking. In this respect the responses of the iguana are similar to those of the nephrectomized rat since, in the short term, both rely exclusively on drinking to restore cellular water to normal. 8. The iguana also drinks in response to extracellular dehydration produced by hyperoncotic peritoneal dialysis, and in response to I.P. angiotensin II.

Visualization of brain interstitial fluid movement during osmotic disequilibrium

The movement of brain interstitial fluid was visualized in cats with the use of dextran blue 2000. The dextran blue was injected intracerebrally, and the cats were subsequently infused or perfused with anisotonic sucrose solutions. Increasing the serum osmolality by intravenous infusion of a hypertonic sucrose solution or decreasing the ventricular fluid osmolality by perfusion with a hypotonic sucrose solution effectively shuts off CSF flow. Conversely, volume flow is increased by either infusing hypotonic sucrose or perfusing with hypertonic sucrose solutions. Cats which were neither infused nor perfused demonstrated a relatively well-defined dye pattern in cerebral white matter. In the brains of cats infused with hypotonic sucrose the dye was more diffuse and asymmetrical. In the brains of cats infused with hypertonic sucrose the dye pattern was discrete and symmetrical. There was no apparent difference in the dye pattern, as compared to controls, in the brains of those cats whose ventricles were perfused with either hypertonic or hypotonic sucrose solutions. Injection of dye into the brains of dead cats produced dye patterns which were more discrete than those seen in controls. It is concluded from observations made on the various dye patterns that there is a movement of interstitial fluid within the brain when serum osmolality is altered. This fluid may contribute to the increase in CSF volume flow which results when serum osmolality is decreased.

Effect of hypertonic solutions on electrical activity of the isolated frog muscle spindle

Changes in electrical activity of the isolated frog muscle spindle were investigated in hypertonic solutions obtained by adding 400 mM sucrose, glucose, or glycerol to Ringer's solution. The spontaneous firing rate in hypertonic sucrose and glucose solutions increased at first (for 3–5 min) and then fell rapidly to zero; the receptor potential and evoked spike activity diminished under these conditions and disappeared. In the hypertonic solution with glycerol a similar effect was observed but, unlike in the first two media, in this case spike activity returned after its initial increase to the normal level; a second rise in the firing rate was then observed up to a steady value which was higher than normal. After rinsing out the hypertonic sucrose and glucose solutions with ordinary Ringer's solution the spontaneous and evoked activity gradually returned to normal with a small overshoot. During the rinsing out of the hypertonic glycerol solution a sharp and considerable rise in spontaneous activity was first observed, while the changes in frequency of the evoked activity were negligible. The spike activity then returned to normal. The observed changes in electrical activity of the muscle spindle in hypertonic media are attributed to deformation of the sensory terminals and intrafusal muscle fibers (in the glycerol medium), leading to depolarization of the receptor membrane.

Hyperosmolality and pancreatic blood flow

In the cat pancreas, close intra-arterial infusions of hypertonic xylose, glucose and sucrose solutions caused a marked vasodilatation but no secretion. The magnitude of the vasodilator response correlated with the degree of induced glandular hyperosmolality but not with the substance per se. Stimulation of the vagus nerve as well as infusion of secretin and/or CCK evoked a pronounced secretion and a small to moderate increase in pancreatic blood flow, but did not significantly change the plasma osmolality in the venous effluent from the gland. Bradykinin, papaverine and isoprenaline caused large increments of pancreatic blood flow; none of these drugs evoked secretion nor did they change the venous osmolality. It is concluded that blood-bone hyperosmolality is a strong stimulus for pancreatic vasodilatation, but the functional hyperaemia of this gland is due to factors other than regional tissue hyperosmolality.

Effects of method of presentation and fluid deprivation upon drinking of sucrose and saline solutions by pigeons

In a comparison of different measures of sucrose and saline solution drinking in domestic pigeons, three factors (single stimulus vs two-bottle “choice”, test period length, and fluid deprivational state) explain the discrepancies in the shape of the saline intake functions obtained in previous studies but do not account for the failure of domestic pigeons in this experiment to display a sucrose preference. Pigeons can make quantitative discriminations between sucrose solutions differing in concentration and qualitative discriminations between isosmotic saline and sucrose solutions. The rapidity with which the birds can make these distinctions suggests the importance of orosensory cues although postingestive consequences function in the development of the pigeons' aversion to hypertonic sucrose solutions.

Chlorophyllase and Lamellar Structure in Phaeodactylum tricornutum III. Situation of Chlorophyllase in Pigmented Membranes

1. Hypotonic treatment of Phaeodactylum tricornutum cells results in disappearance of their Hill activity; at the same time chlorophyllase activity becomes apparent. Aged cells lyse completely: their pigmented membranes (Whole Membranes, WM) are released. The release of WM ist retarded by Mg". Chlorophyllase activity is associated with these pigmented membranes. Membrane density is not influenced by a hypertonic sucrose solution. Young Phaeodactylum cells do not lyse upon hypotonic treatment. 2. Upon French Press passage of aged or young Phaeodactylum cells WM are fragmentated. Contrary to WM, the fragments are affected by a hypertonic sucrose solution. The pigmented fragments can be differentiated in a heavy fraction with maximum density about 1.18 (Heavy Membrane Fragments, HMF)various light fractions (Light Membrane Fragments, LMF) with maximum density about 1.13. The membrane fragments show different chlorophyllase activities; enzyme activity generally increases with decreasing density of the fragments. No relation was found between pigment composition of various fragmentstheir chlorophyllase activity. 3. The results are interpreted to indicate that chlorophyllase activity is unevenly distributed across the pigmented membranes, the easily dissociable, low density membrane parts showing the highest activity. Also a rather stable long-wave chlorophyll form with its fluorescence maximum at 713 nm is most abundant in a readily split off membrane fraction. 4. A hypothesis about the origin of chlorophyllase molecules is forwarded.

The Effect of Dimethyl Sulfoxide on In Vitro Platelet Function

SummaryAlthough dimethyl sulfoxide (DMSO) has been used extensively as a cryopreservative for platelets there are few studies dealing with the effect of DMSO on platelet function. Using techniques similar to those employed in platelet cryopreservation platelets were incubated with final concentrations of 2-10% DMSO at 25° C. After exposure to 5 and 10% DMSO platelets remained discoid and electron micrographs revealed no structural abnormalities. There was no significant change in platelet count. In terms of injury to platelet membranes, there was no increased availability of platelet factor-3 or leakage of nucleotides, 5 hydroxytryptamine (5HT) or glycosidases with final DMSO concentrations of 2.5, 5 and 10% DMSO. Thrombin stimulated nucleotide and 5HT release was reduced by 10% DMSO. Impairment of thrombin induced glycosidase release was noted at lower DMSO concentrations and was dose related. Similarly, aggregation to ADP was progressively impaired at DMSO concentrations from 1-5% and was dose related. After the platelets exposed to DMSO were washed, however, aggregation and release returned to control values. Platelet aggregation by epinephrine was also inhibited by DMSO and this could not be corrected by washing the platelets. DMSO-plasma solutions are hypertonic but only minimal increases in platelet volume (at 10% DMSO) could be detected. Shrinkage of platelets was seen with hypertonic solutions of sodium chloride or sucrose suggesting that the rapid transmembrane passage of DMSO prevented significant shifts of water. These studies demonstrate that there are minimal irreversible alterations in in vitro platelet function after short-term exposure to DMSO.

Negative inotropic influence of hyperosmotic solutions on cardiac muscle.

In cardiac muscle, moderate degrees of hyperosmolality of the type encountered physiologically or clinically (i.e., less than 200 mosM above control) characteristically exert a positive inotropic effect, which presumably is mediated by increased Ca2+ availability for binding to troponin. In contrast, skeletal muscle displays significant contractile depression on exposure to hyperosmotic solutions, even at mild degrees of hypertonicity. To determine whether a similar potential for hyperosmolarity-induced depression also exists in cardiac muscle, right ventricular papillary muscles from cats were exposed to hypertonic solutions of mannitol or sucrose under circumstances in which positive inotropic effects were precluded by prior exposure to a bathing solution of 4.0 mM Ca2+ and paired electrical stimulation to maximize intracellular Ca2+ before addition of the hyperosmotic substances. In contrast to their usual positive inotropic effects, hypertonic solutions under these conditions caused cardiac depression at all osmolarities tested. Developed tension and its maximal rate of development (dT/dt) decreased by 18% at 50 mosM above control, by 30% at 100 mosM, by 36% at 150 mosM, and by 42% at 200 mosM (P less than 0.01 for all). Time to peak tension and resting tension were not changed significantly. When the muscles were returned to control solutions, tension development also returned toward normal. The data are compatible with the hypothesis that, within the range tested, all degrees of hyperosmolarity exert a significant negative inotropic influence on cardiac muscle, as is true in skeletal muscle; manifestation of this effect of increased tonicity normally would be obscured at low degrees of hyperosmolality, however, by an overriding positive influence that is absent in skeletal muscle.

Aspects of bile secretion in the rabbit.

1. Bile secretion was studied in anaesthetized rabbits from whom hepatic bile was collected by cannulation of the common bile duct. 2. The flow and composition of bile formed by rabbits anaesthetized with urethane differed significantly from that formed by rabbits anaesthetized with pentobarbitone sodium. 3. The I.P. injection of a hypertonic solution of sucrose (3 M) decreased bile flow and produced changes in the ionic composition of bile and of plasma. 4. The infusion of sodium taurodeoxycholate (1-5-20 mumole/min I.V.) gave higher rates of bile flow than did equimolar infusions of sodium taurocholate, and unlike taurocholate, increased the bicarbonate concentration of bile. 5. Acetazolamide (10-100 mg/kg) increased the concentration of bicarbonate both in bile and in plasma, and had little effect on bile flow. 6. The infusion of bromsulphthalein (5 mg/kg I.V.) decreased the excretion of bicarbonate into bile, and was associated with the formation of a hypotonic bile. 7. The implications of these results in relation to the mechanisms of bile secretion are discussed.

Osmotic extraction of hypotonic fluid from the lungs.

After injections of sucrose, NaCl, and urea solutions, the flow of tissue fluid from the lungs amounted to 0.182, 0.216, and 0.152 x 10(-3) ml/s per mosmol/kg of concentration difference between plasma and tissues in each gram of wet tissue weight. The extracted fluid contained less than 20% of the Na(+), K(+) and urea concentrations of the plasma. It was concluded that this fluid was distinctly hypotonic in comparision with the fluids of the plasma and tissue compartments both before and after the injection of hypertonic solutions. The presence of low solute concentrations in the extracted fluid is attributed to the passage of this fluid across cellular membranes, which are relatively impermeable to small hydrophilic solutes. Movement of fluid out of the junctions appears to be less than that through the endothelial cells. It is suggested that the injected solutes rapidly leak into the junctions and consequently induce relatively little movement of water or tissue solutes out of the junctions. Concentrations of tritiated water and [(14)C]antipyrine in the extracted fluid are essentially the same as base-line plasma concentrations when the animals have been primed with these tracers. It is therefore likely that these tracers can readily traverse cellular membranes. Red cell transit through the lungs is impaired by hypertonic solutions of sucrose and NaCl with transient increases in pulmonary arterial hemoglobin concentrations of as much as 35% of base-line values.

Impedance of frog skeletal muscle fibers in various solutions.

The linear circuit parameters of 140 muscle fibers in nine solutions are determined from phase measurements fitted with three circuit models: the disk model, in which the resistance to radial current flow is in the lumen of the tubules; the lumped model, in which the resistance is at the mouth of the tubules; and the hybrid model, in which it is in both places. The lumped model fails to fit the data. The disk and hybrid model fit the data, but the optimal circuit values of the hybrid model seem more reasonable. The circuit values depend on sarcomere length. The conductivity of the lumen of the tubules is less than, and varies in a nonlinear manner with, the conductivity of the bathing solution, suggesting that the tubules are partially occluded by some material like basement membrane which restricts the mobility of ions and has fixed charge. The x2.5 hypertonic sucrose solution used in many voltage clamp experiments produces a large increase in the radial resistance, suggesting that control of the potential across the tubular membranes would be difficult to achieve. Glycerol-treated fibers have 90% of their tubular system insulated from the extracellular solution and 10% connected to the extracellular solution through a high resistance. We discuss the implications of our results for calculations of the nonlinear properties of muscle fibers, including the action potential and the radial spread of contraction.