Effects of pulsation frequency and endothelial integrity on enhanced arterial transmural filtration produced by pulsatile pressure

The role of the endothelium in regulating transmural fluid filtration into the artery wall under pulsatile pressure and the effects of changes in pulsatile frequency on filtration have received little attention. Previous experiments (Alberding JP, Baldwin AL, Barton JK, and Wiley E. Am J Physiol Heart Circ Physiol 286: H1827-H1835, 2004) demonstrated significantly increased filtration after initial onset of pulsatile pressure compared with that predicted by using parameters measured under steady pressure. To determine the role of the endothelium in this phenomenon, the following experiments were performed on five New Zealand White rabbits (anesthetized with 30 mg/kg pentobarbital sodium). One of each pair of carotid arteries was deendothelialized, and filtration measurements under steady and pulsatile pressure were compared with those made in intact vessels (Alberding JP, Baldwin AL, Barton JK, and Wiley E. Am J Physiol Heart Circ Physiol 286: H1827-H1835, 2004). To determine the effect of increasing pulsatile frequency on arterial filtration, transmural filtration was measured by using pulsatile pressure frequencies of 1 Hz, followed by 2 Hz, in another set of intact arteries (6 arteries and 3 animals). For deendothelialized vessels, the initial increase in filtration after onset of pulsatility was similar to that observed in intact vessels, but the subsequent reduction in filtration was less abrupt. When pulsatile frequency was increased from 1 to 2 Hz in intact arteries, an initial increase in filtration was observed, similar to that obtained after onset of pulsatile pressure subsequent to a steady pressure. The observed responses of arteries to pulsatile pressure, with and without endothelium, or undergoing a frequency change, suggest a dynamic role for the endothelium in regulating transvascular transport in vivo.     

Coordinated regulation of shrinkage-induced Na/H exchange and swelling- induced [K-Cl] cotransport in dog red cells. Further evidence from activation kinetics and phosphatase inhibition

Hypertonic shrinkage of dog red cells caused rapid activation of Na/H exchange and rapid deactivation of [K-Cl] cotransport. Hypotonic swelling caused delayed deactivation of Na/H exchange and delayed activation of [K-Cl] cotransport. Okadaic acid stimulated shrinkage-induced Na/H exchange and inhibited swelling-induced [K-Cl] cotransport. The data are compatible with the kinetic model of Jennings and Al-Rohil (1990. J. Gen. Physiol. 95:1021-1040) for volume regulation of [K-Cl] cotransport in rabbit red cells and suggest that in dog red cells Na/H exchange and [K-Cl] cotransport are controlled by a common regulatory system. The proposal of Jennings and Schulz (1991. J. Gen. Physiol. 96:799-817) that activation/deactivation of volume-sensitive transport involves phosphorylation/dephosphorylation of a regulatory protein is supported by these observations.     

Reflection coefficients of permeant nonelectrolytes for dog and beef red cell membranes.

The reflection coefficient, sigma, for several small permeant nonelectrolytes was determined for dog and beef red blood cell membranes. Our sigma values were considerably higher than those previously reported for dog cells; e.g., out sigma urea was 87% higher than the sigma urea of Rich, Sha'afi, Barton and Solomon (J. Gen. Physiol. 50: 2391, 1967). Our sigma values for urea were only slightly greater in beef cells than previously reported by Farmer and Macey (Biochim. Biophys. Acta 290: 290, 1972). We found that a trend exists when (1 - sigma) is plotted against the log of the permeability coefficient, omega. This observation is also consistent with our previously reported sigma data for human red cell membranes (Owen & Eyring, J. Gen. Physiol. 66: 241, 1972). This trend suggests that small hydrophilic molecules interact highly with cell membrane water. The exceptions to this trend were lipophilic molecules, indicating they do not interact with water while penetrating the red cell membrane.     

Calculation of the reflection coefficient from measurements of endogenous vascular indicators

The solvent drag reflection coefficient (sigma) for total proteins can be estimated by comparing the relative degrees of concentration of erythrocytes and plasma proteins that occur during fluid filtration in an isolated perfused organ. In this analysis, we evaluated the accuracy of equations proposed by Pilati and Maron [Am. J. Physiol. 247 (Heart Circ. Physiol. 16): H1-H7, 1984] and Wolf et al. [Am. J. Physiol. 253 (Heart Circ. Physiol. 22): H194-H204, 1987] to calculate sigma from these concentration changes. We calculated sigma with each equation using data generated from a mathematical model of fluid and solute flux in membranes with known sigma's. We found that the equation of Wolf et al. provided the closest approximation to the true sigma over the entire range of filtration fractions tested (0.1-0.6), with the differences between the two equations increasing with filtration fraction. At low filtration fractions, the difference in sigma obtained using either approach was found to be inconsequential. At larger filtration fractions, a closer approximation of the true sigma can be obtained using the equation of Wolf et al.     

Measurement of the Permeability of Biological Membranes Application to the glomerular wall

The transport equation describing the flow of solute across a membrane has been modified on the basis of theoretical studies calculating the drag of a sphere moving in a viscous liquid undergoing Poiseuille flow inside a cylinder. It is shown that different frictional resistance terms should be introduced to calculate the contributions of diffusion and convection. New sieving equations are derived to calculate r and A_p/Δx (respectively, the pore radius and the total area of the pores per unit of path length). These equations provide a better agreement than the older formulas between the calculated and the experimental glomerular sieving coefficients for [¹²⁵I]polyvinylpyrrolidone (PVP) fractions with a mean equivalent radius between 19 and 37 Å. From r and A_p/Δx, the mean effective glomerular filtration pressure has been calculated, applying Poiseuille's law. A value of 15.4 mm Hg has been derived from the mean sieving curve obtained from 23 experiments performed on normal anesthetized dogs.     

A model for sieving of macromolecules by the glomerular membrane of the kidney.

The transport of water and of macromolecules across the glomerular membrane of the kidney depends on the membrane parameters (radius, length and number of pores) as well as on the hydrostatic and oncotic pressures on either side of the membrane. The filtration pressure decreases along the capillary loops from afferent to efferent end. Water and solute flows are thus given by a system of two differential equations. The sieving coefficient of the macromolecules is the ratio of solute to water flow. In the program described the differential equations are solved by the Runge-Kutta method (fourth order). Rosenbrock's method of minimization is used to adjust the theoretical to the experimental sieving coefficients. The pore radius, total pore area per unit of path length and conductance of the membrane, as well as the intracapillary hydrostatic pressure and its gradient can thus be determined.     

A comparison of methods for estimating the kinetic parameters of two simple types of transport process

Sets of experimental data, with known characteristics and error structures, have been simulated for the Michaelis-Menten equation plus a second term, either for linear transport or for competitive inhibition. The Michaelis-Menten equation plus linear term was fitted by several methods and the accuracy and the precision of the parameter estimates from the several methods were compared. The model-fitting methods were: three for least-squares non-linear regression, computer versions of two graphical methods and of two non-parametric methods. The most precise and accurate method was that of D.W. Marquardt (J. Soc. Ind. Appl. Math. 11 (1963) 431–441). The Michaelis-Menten equation with competitive inhibition was also fitted by several methods, viz., two for least-squared non-linear regression, a non-parametric method and four variants of the Preston-Schaeffer-Curran plot (Preston, R.L. et al. (1974) J. Gen. Physiol. 64, 443–467). The most precise and accurate of these was the non-linear regression method of W.W. Cleland (Adv. Enzymol. 29 (1967) 1–32). For both these models, the various graphical methods and non-parametric methods gave poor results and are not recommended.     

Analysis of protein fouling during ultrafiltration using a two-layer membrane model

Protein fouling can significantly alter both the flux and retention characteristics of ultrafiltration membranes. There has, however, been considerable controversy over the nature of this fouling layer. In this study, hydraulic permeability and dextran sieving data were obtained both before and after albumin adsorption and/or filtration using polyethersulfone ultrafiltration membranes. The dextran molecular weight distributions were analyzed by gel permeation chromatography to evaluate the sieving characteristics over a broad range of solute size. Protein fouling caused a significant reduction in the dextran sieving coefficients, with very different effects seen for the diffusive and convective contributions to dextran transport. The changes in dextran sieving coefficients and diffusive permeabilities were analyzed using a two-layer membrane model in which a distinct protein layer is assumed to form on the upstream surface of the membrane. The data suggest that the protein layer formed during filtration was more tightly packed than that formed by simple static adsorption. Hydrodynamic calculations indicated that the pore size of the protein layer remained relatively constant throughout the adsorption or filtration, but the thickness of this layer increased with increasing exposure time. These results provide important insights into the nature of protein fouling during ultrafiltration and its effects on membrane transport.     

A model of unsteady-state transvascular fluid and protein transport in the lung

Models of steady-state fluid and solute transport in the microcirculation are used primarily to characterize filtration and permeability properties of the transport barrier. Important transient relationships, such as the rate of fluid accumulation in the tissue, cannot be predicted with steady-state models. In this paper we present three simple models of unsteady-state fluid and protein exchange between blood plasma and interstitial fluid. The first treats the interstitium as a homogeneous well-mixed compliant compartment, the second includes an interstitial gel, and the third allows for both gel and free fluid in the interstitium. Because we are primarily interested in lung transvascular exchange we used the multiple-pore model and pore sizes described by Harris and Roselli (J. Appl. Physiol.: Respirat . Environ. Exercise Physiol. 50: 1-14, 1981) to characterize the microvascular barrier. However, the unsteady-state transport theory presented here should apply to other organ systems and can be used with different conceptual models of the blood-lymph barrier. For a step increase in microvascular pressure we found good agreement between theoretical and experimental lymph flow and lymph concentrations in the sheep lung when the following parameter ranges were used: base-line interstitial volume, 150-190 ml; interstitial compliance, 7-10 ml/Torr; initial interstitial fluid pressure, -1 Torr; pressure in initial lymphatics, -5 to -6 Torr; and conductivity of the interstitium and lymphatic barrier, 4.25 X 10(-4) ml X s-1 X Torr-1. Based on these values the model predicts 50% of the total change in interstitial water volume occurs in the first 45 min after a step change in microvascular pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversal of the gating polarity of gap junctions by negative charge substitutions in the N-terminus of connexin 32

Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V(j)), to which they gate by the separate actions of their serially arranged hemichannels (Harris, A. L., D. C. Spray, and M. V. L. Bennett. 1981. J. Gen. Physiol. 77:95-117). Single channel studies of both intercellular and conductive hemichannels have demonstrated the existence of two separate gating mechanisms, termed "V(j)-gating" and "loop gating" (Trexler, E. B., M. V. L. Bennett, T. A. Bargiello, and V. K. Verselis. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:5836-5841). In Cx32 hemichannels, V(j)-gating occurs at negative V(j) (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Gen. Physiol. 114:339-364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 2000. J. Gen. Physiol. 116:13-31). A negative charge substitution at the second amino acid position in the N-terminus reverses the polarity of V(j)-gating of Cx32 hemichannels (Verselis, V. K., C. S. Ginter, and T. A. Bargiello. 1994. Nature. 368:348-351;. J. Gen. Physiol. 116:13-31). We report that placement of a negative charge at the 5th, 8th, 9th, or 10th position can reverse the polarity of Cx32 hemichannel V(j)-gating. We conclude that the 1st through 10th amino acid residues lie within the transjunctional electric field and within the channel pore, as in this position they could sense changes in V(j) and be largely insensitive to changes in absolute membrane potential (V(m)). Conductive hemichannels formed by Cx32*Cx43E1 containing a negatively charged residue at either the 8th or 10th position display bi-polar V(j)-gating; that is, the open probability of hemichannels formed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichannels with negative charges at either the 2nd or 5th positions are uni-polar, closing only at positive V(j). The simplest interpretation of these data is that the Cx32 hemichannel can adopt at least two different open conformations. The 1st-5th residues are located within the electric field in all open channel conformations, while the 8th and 10th residues lie within the electric field in one conformation and outside the electric field in the other conformation.     

Reduced diffusion of charge-modified, conformationally intact anionic Ficoll relative to neutral Ficoll across the rat glomerular filtration barrier in vivo

The glomerular filtration barrier (GFB) is commonly conceived as a negatively charged sieve to proteins. Recent studies, however, indicate that glomerular charge effects are small for anionic, carboxymethylated (CM) dextran vs. neutral dextran. Furthermore, two studies assessing the glomerular sieving coefficients (θ) for negative CM-Ficoll vs. native Ficoll have demonstrated an increased glomerular permeability for CM-Ficoll (Asgeirsson D, Venturoli D, Rippe B, Rippe C. Am J Physiol Renal Physiol 291: F1083-F1089, 2006; Guimar?es M, Nikolovski J, Pratt L, Greive K, Comper W. Am Physiol Renal Physiol 285: F1118-F1124, 2003.). The CM-Ficoll used, however, showed a larger Stokes-Einstein radius (a(e)) than neutral Ficoll, and it was proposed that the introduction of negative charges in the Ficoll molecule had made it more flexible and permeable. Recently, a negative FITC-labeled CM-Ficoll (CMI-Ficoll) was produced with a conformation identical to that of neutral FITC-Ficoll. Using these probes, we determined their θ:s in anesthetized Wistar rats (259 ± 2.5 g). After blood access had been achieved, the left ureter was cannulated for urine sampling. Either polysaccharide was infused (iv) together with a filtration marker, and urine and plasma were collected. Assessment of θ FITC-Ficoll was achieved by high-performance size-exclusion chromatography (HPSEC). CMI-Ficoll and native Ficoll had identical elugrams on the HPSEC. Diffusion of anionic Ficoll was significantly reduced compared with that of neutral Ficoll across the GFB for molecules of a(e) ～20-35 ?, while there were no charge effects for Ficoll of a(e) = 35-80 ?. The data are consistent with a charge effect present in "small pores," but not in "large pores," of the GFB and mimicked those obtained for anionic membranes in vitro for the same probes.     

A mathematical model of cardiocyte Ca(2+) dynamics with a novel representation of sarcoplasmic reticular Ca(2+) control

Cardiac contraction and relaxation dynamics result from a set of simultaneously interacting Ca(2+) regulatory mechanisms. In this study, cardiocyte Ca(2+) dynamics were modeled using a set of six differential equations that were based on theories, equations, and parameters described in previous studies. Among the unique features of the model was the inclusion of bidirectional modulatory interplay between the sarcoplasmic reticular Ca(2+) release channel (SRRC) and calsequestrin (CSQ) in the SR lumen, where CSQ acted as a dynamic rather than simple Ca(2+) buffer, and acted as a Ca(2+) sensor in the SR lumen as well. The inclusion of this control mechanism was central in overcoming a number of assumptions that would otherwise have to be made about SRRC kinetics, SR Ca(2+) release rates, and SR Ca(2+) release termination when the SR lumen is assumed to act as a simple, buffered Ca(2+) sink. The model was sufficient to reproduce a graded Ca(2+)-induced Ca(2+) release (CICR) response, CICR with high gain, and a system with reasonable stability. As constructed, the model successfully replicated the results of several previously published experiments that dealt with the Ca(2+) dependence of the SRRC (, J. Gen. Physiol. 85:247-289), the refractoriness of the SRRC (, Am. J. Physiol. 270:C148-C159), the SR Ca(2+) load dependence of SR Ca(2+) release (, Am. J. Physiol. 268:C1313-C1329;, J. Biol. Chem. 267:20850-20856), SR Ca(2+) leak (, J. Physiol. (Lond.). 474:463-471;, Biophys. J. 68:2015-2022), SR Ca(2+) load regulation by leak and uptake (, J. Gen. Physiol. 111:491-504), the effect of Ca(2+) trigger duration on SR Ca(2+) release (, Am. J. Physiol. 258:C944-C954), the apparent relationship that exists between sarcoplasmic and sarcoplasmic reticular calcium concentrations (, Biophys. J. 73:1524-1531), and a variety of contraction frequency-dependent alterations in sarcoplasmic [Ca(2+)] dynamics that are normally observed in the laboratory, including rest potentiation, a negative frequency-[Ca(2+)] relationship, and extrasystolic potentiation. Furthermore, under the condition of a simulated Ca(2+) overload, an alternans-like state was produced. In summary, the current model of cardiocyte Ca(2+) dynamics provides an integrated theoretical framework of fundamental cellular Ca(2+) regulatory processes that is sufficient to predict a broad array of observable experimental outcomes.     

The epithelial Na(+) channel (ENaC) is related to the hypertonicity-induced Na(+) conductance in rat hepatocytes

The epithelial Na(+) channel (ENaC) is composed of the subunits alpha, beta, and gamma [Canessa et al., Nature 367 (1994) 463-467] and typically exhibits a high affinity to amiloride [Canessa et al., Nature 361 (1993) 467-470]. When expressed in Xenopus oocytes, conflicting results were reported concerning the osmo-sensitivity of the channel [Ji et al., Am. J. Physiol. 275 (1998) C1182-C1190; Hawayda and Subramanyam, J. Gen. Physiol. 112 (1998) 97-111; Rossier, J. Gen. Physiol. 112 (1998) 95-96]. Rat hepatocytes were the first system in which amiloride-sensitive sodium currents in response to hypertonic stress were reported [Wehner et al., J. Gen. Physiol. 105 (1995) 507-535; Wehner et al., Physiologist 40 (1997) A-4]. Moreover, all three ENaC subunits are expressed in these cells [B?hmer et al., Cell. Physiol. Biochem. 10 (2000) 187-194]. Here, we injected specific antisense oligonucleotides directed against alpha-rENaC into single rat hepatocytes in confluent primary culture and found an inhibition of hypertonicity-induced Na(+) currents by 70%. This is the first direct evidence for a role of the ENaC in cell volume regulation.     

Filtration,diffusion, and molecular sieving through porous cellulose membranes

1. A study has been made of the diffusion and filtration of a graded series of molecules (including tritium-labelled water, urea, glucose, antipyrine, sucrose, raffinose, and hemoglobin) in aqueous solution through porous cellulose membranes of three degrees of porosity. 2. Experimental results were in close agreement with predictions based on the membrane pore theory of Pappenheimer et al. (1,2). Restriction to molecular diffusion is a function of pore radius and molecular radius described by equation (11) in the text. Molecular sieving during ultrafiltration is a function of total pore area per unit path length, pore radius, molecular radius, and filtration rate given by equations (16) and (19). 3. Estimates of average pore radius made by means of this theory were considerably larger than estimates made by the method of Elford and Ferry (3) (Table II). Sources of error in the latter method are discussed and a new method of membrane calibration is proposed in which the total cross-sectional area of the pores is measured by direct diffusion of isotope-labelled water. 4. Steady-state osmotic pressures of solutions of sucrose and raffinose measured during molecular sieving through cellulose membranes were found to be close to the "ideal" osmotic pressures calculated by van't Hoff's law. Thus the present experimental data support the methods used by Pappenheimer et al. in their studies on living capillary walls as well as their theory of membrane pore permeability.     

Haloarcula marismortui (Volcani) sp. nov., nom. rev., an extremely halophilic bacterium from the Dead Sea

An extremely halophilic red archaebacterium isolated from the Dead Sea (Ginzburg et al., J. Gen. Physiol. 55: 187-207, 1970) belongs to the genus Haloarcula and differs sufficiently from the previously described species of the genus to be designated a new species; we propose the name Haloarcula marismortui (Volcani) sp. nov., nom. rev. because of the close resemblance of this organism to "Halobacterium marismortui," which was first described by Volcani in 1940. The type strain is strain ATCC 43049.     

Influence of calcium on guanosine 3'',5''-cyclic monophosphate levels in frog rod outer segments

In the presence of 10(-9) M calcium, rod outer segments freshly detached from dark-adapted frog retinas contain between 0.01 and 0.02 moles of guanosine 3',5'-cyclic monophosphate (cyclic GMP) per mole of rhodopsin. The dark level of cyclic GMP is reduced approximately 50% by illumination that bleaches 5 x 10(5) rhodopsin molecules/outer segments. The dark levels of cyclic GMP also can be suppressed to approximately 0.007 mol/mol of rhodopsin by increasing the concentration of calcium from 10(-9) M to 2 x 10(-9) M, and they remain at this level as calcium concentration is raised to 10(-3) M. The final level to which illumination reduces cyclic GMP in unaffected by the calcium concentration between 10(-9) and 10(-3) M. The maximal light-induced decrease in cyclic GMP occurs within 1 s from the onset of illumination at all calcium concentrations. The magnitude and time-course of the light-induced decrease in cyclic GMP measured in these experiments are comparable to values obtained previously (Woodruff et al. 1977. J. Gen. Physiol. 69:677-679; Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). The data are consistent with a role for cyclic GMP in visual transduction irrespective of the calcium concentration.     

Effect of phloretin on chloride permeability: A structure-activity study

On the basis of data obtained with thin lipid membranes, phloretin's inhibition of chloride, urea and glucose transport in biological membranes has been suggested to be due to the effects of interfacial dipole fields on the translocator of these molecules (Andersen, O.S., Finkelstein, A., Katz,I. and Cass, A. (1976) J. Gen. Physiol. 67, 749–771).From the systematic analysis made in the present paper it effectively appears that the ability of phloretin analogs to inhibit chloride permeability in red-cell membrane depends on the capacity they have to alter the interfacial dipole potential: the magnitude of the potential change depending on the dipole moment of the molecule and its membrane concentration, it follows that the inhibitory capacity of a phloretin analog is a function of the dipole moment and the lipid solubility of the compound.     

Effects of strychnine on the potassium conductance of the frog node of Ranvier

The nature of the block of potassium conductance by strychnine in frog node of Ranvier was investigated. The block is voltage-dependent and reaches a steady level with a relaxation time of 1 to several ms. Block is increased by depolarization or a reduction in [K+]O as well as by increasing strychnine concentration. A quaternary derivative of strychnine produces a similar block only when applied intracellularly. In general and in detail, strychnine block resembles that produced by intracellular application of the substituted tetraethylammonium compounds extensively studied by C.M. Armstrong (1969. J. Gen Physiol. 54:553-575. 1971. J. Gen. Physiol. 58:413-437). The kinetics, voltage dependence, and dependence on [K+]O of strychnine block are of the same form. It is concluded that tertiary strychnine must cross the axon membrane and block from the axoplasmic side in the same fashion as these quaternary amines.     

BK channels and the expanding role for PIP2-mediated regulation

Commentary to: Vaithianathan T, Bukiya A, Liu J et al. Direct regulation of BK channels by phosphatidylinositol 4,5-bisphosphate as a novel signaling pathway. J Gen Physiol 2008; 132:13-28.     

Myoplasmic calcium transients monitored with purpurate indicator dyes injected into intact frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after microinjection with tetramethylmurexide (TMX) or purpurate-3,3' diacetic acid (PDAA), two compounds from the purpurate family of absorbance Ca2+ indicators previously used in cut muscle fibers (Maylie, J., M. Irving, N. L. Sizto, G. Boyarsky, and W. K. Chandler. 1987. J. Gen. Physiol. 89:145-176; Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631.) The apparent longitudinal diffusion constant of PDAA (mol wt 380) in myoplasm was 0.99 (+/- 0.04, SEM) x 10(-6) cm2 s-1 (16-17 degrees C), a value which suggests that 24-43% of the PDAA molecules were bound to myoplasmic constituents of large molecular weight. The corresponding values for TMX (mol wt 322) were 0.98 (+/- 0.05) x 10(-6) cm2 s-1 and 44-50%, respectively. Muscle membranes (surface and/or transverse-tubular) appear to be permeable to TMX and, to a lesser extent, to PDAA, since the total amount of indicator contained within a fiber decreased with time after injection. The average time constants for disappearance of indicator were 46 (+/- 7, SEM) min for TMX and 338 (+/- 82) min for PDAA. The fraction of indicator in the Ca2(+)-bound state in resting fibers was significantly different from zero for TMX (0.070 +/- 0.008) but not for PDAA (0.026 +/- 0.009). In in vitro calibrations PDAA but not TMX appeared to react with Ca2+ with 1:1 stoichiometry. In agreement with Hirota et al. (Hirota, A., W. K. Chandler, P. L. Southwick, and A. S. Waggoner. 1989. J. Gen. Physiol. 94:597-631), we conclude that PDAA is probably a more reliable myoplasmic Ca2+ indicator than TMX. In fibers that contained PDAA and were stimulated by a single action potential, the calibrated peak value of the myoplasmic free [Ca2+] transient (delta[Ca2+]) averaged 9.4 (+/- 0.6) microM, a value about fivefold larger than that calibrated with antipyrylazo III under otherwise identical conditions (Baylor, S. M., and S. Hollingworth. 1988. J. Physiol. 403:151-192). The fivefold difference is similar to that previously reported in cut fibers with antipyrylazo III and PDAA. Since in both intact and cut fibers the percentage of PDAA bound to myoplasmic constituents is considerably smaller than that found for antipyrylazo III, the PDAA calibration of delta[Ca2+] is likely to be more accurate. Interestingly, in intact fibers the peak value of delta[Ca2+] calibrated with either PDAA or antipyrylazo III is about half that calibrated in cut fibers.(ABSTRACT TRUNCATED AT 400 WORDS)