bluuebunny/crossref_metadata_2025 huggingface dataset:
200 years of digitalis: the emerging central role of the sodium ion in the control of cardiac force
Authors: Lee C. O.
Published: December 2017

Digitalis has been used therapeutically for two centuries, but the mechanism by which it enhances the ability of cardiac muscle to produce force (the positive inotropic effect) has not been fully elucidated. The major controversy concerns the question of whether the inhibition of the Na+-K+ pump by digitalis, particularly at low (therapeutic) concentrations, increases the intracellular Na+ concentration and thus is causally related to the positive inotropic effect. Na+-selective microelectrodes, introduced recently, have made it possible to measure small changes in intracellular Na+ activity (aiNa) in beating preparations of cardiac muscle and, in particular, to follow the exact time course of change in both aiNa and contractile force during the positive inotropic effect of digitalis. It has been demonstrated that digitalis at low and high concentrations produces a parallel increase in aiNa and in contractile force during the onset of its effect; washout of the drug results in a parallel and complete recovery of aiNa and contractile force. Additional strong evidence for a correlation between the pump inhibition and digitalis inotropy is the fact that the magnitude of increase in aiNa and contractile force produced by digitalis depends on the level of aiNa and therefore on the rate of Na+ extrusion by the Na+-K+ pump. The study on the quantitative relationship between aiNa and contractile force reveals that the force of contraction is a power function of aiNa, such that a small rise in aiNa produces a significant increase in contractile force. Direct measurements of aiNa and intracellular free Ca2+ during digitalis inotropy strongly support the hypothesis that an increase in aiNa raises intracellular Ca2+ via Na+-Ca2+ exchange, thus producing the positive inotropic effect. In conclusion, the recent data available from the simultaneous and continuous measurements of aiNa and contractile force strongly indicate that the inhibition of the Na+-K+ pump is causally related to the positive inotropic effect of digitalis on cardiac muscle.
Radiation inactivation of multimeric enzymes: application to subunit interactions of adenylate cyclase
Authors: Verkman A. S. , Skorecki K. L. , Ausiello D. A.
Published: December 2017

Radiation inactivation has been applied extensively to determine the molecular weight of soluble enzyme and receptor systems from the slope of a linear ln (activity) vs. dose curve. Complex nonlinear inactivation curves are predicted for multimeric enzyme systems, composed of distinct subunits in equilibrium with multimeric complexes. For the system A1 + A2----A1A2, with an active A1A2 complex (associative model), the ln (activity) vs. dose curve is linear for high dissociation constant, K. If a monomer, A1, has all the enzyme activity (dissociative model), the ln (activity) vs. dose curve has an activation hump at low radiation dose if the inactive subunit, A2, has a higher molecular weight than A1 and has upward concavity when A2 is smaller than A1. In general, a radiation inactivation model for a multistep mechanism for enzyme activation fulfills the characteristics of an associative or dissociative model if the reaction step forming active enzyme is an associative or dissociative reaction. Target theory gives the molecular weight of the active enzyme subunit or complex from the limiting slope of the ln (activity) vs. dose curve at high radiation dose. If energy transfer occurs among subunits in the multimer, the ln (activity) vs. dose curve is linear for a single active component and is concave upward for two or more active components. The use of radiation inactivation as a method to determine enzyme size and multimeric subunit assembly is discussed with specific application to the hormone-sensitive adenylate cyclase system. It is shown that the complex inactivation curves presented in the accompanying paper can be used select the best mechanism out of a series of seven proposed mechanisms for the activation of adenylate cyclase by hormone.
Evidence for vasopressin activation of adenylate cyclase by subunit dissociation
Authors: Skorecki K. L. , Verkman A. S. , Jung C. Y. , Ausiello D. A.
Published: December 2017

Radiation inactivation is used to probe the sequence of subunit interactions involved in the activation of adenylate cyclase by vasopressin in cultured renal epithelial cells (LLC-PK1) based on our previous analysis of the radiation inactivation of multimeric enzymes [Verkman et al., Am. J. Physiol. 250 (Cell Physiol. 19): C103-C114, 1986]. For basal adenylate cyclase activity, a concave downward ln(activity) vs. dose relation was observed with limiting slope corresponding to a molecular weight of (169-196) X 10(3). Similar results were obtained with NaF. In contrast, addition of vasopressin, guanylyl imidodiphosphate, or forskolin resulted in transition to a linear ln(activity) vs. dose relation with a slope corresponding to a molecular weight similar to that observed for basal activity. These findings were incorporated into a cyclic dissociation model for the hormonal activation of adenylate cyclase (graph see text) where H is hormone, R is receptor, C is catalytic unit, alpha and beta are subunits of guanyl nucleotide-regulatory protein (G), GTP is guanosine triphosphate, and GDP is guanosine diphosphate. The addition of H favors the dissociation of G into alpha and beta subunits by providing a rapid pathway for addition of GTP to dissociated alpha subunits. The observed target size of the active enzyme species formed corresponds to the composite molecular weights of alpha GTP with C. This model consolidates the radiation inactivation findings as well as the known biochemical characteristics for adenylate cyclase.
Co2+, low Ca2+, and verapamil reduce mechanical activity in rat skeletal muscles
Authors: Kotsias B. A. , Muchnik S. , Obejero Paz C. A.
Published: December 2017

We have studied the effects of Co2+ (5 mM), low-Ca2+ solution [0 added CaCl2, 5 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, 3 mM MgCl2 Ringer], and verapamil (0.1 mM) on mechanical and electrical properties of rat soleus muscle in vitro at 34 degrees C. Muscle fibers had normal resting potentials in Co2+ and verapamil solutions. Low-Ca2+ solution produces a depolarization of approximately 4 mV. The action potentials are smaller and have a slower time course when exposed to test solutions. Iterative generation of action potentials in the presence of Co2+ and low-Ca2+ solution is not modified. In the presence of Co2+ or low-Ca2+ solution, the mechanical output, twitch and tetanus tensions, and caffeine contracture are reduced significantly. Verapamil produces a decrease in the twitch and tetanic tensions but does not modify the caffeine contracture tension. The effect of verapamil on the twitch becomes more manifest when the muscle is stimulated at 3-5 Hz. We suggest that changes in the action potential characteristics or the inhibition of a Ca2+ current are responsible for the mechanical changes observed in the presence of the drugs.
Evidence for two cellular sodium compartments in suspensions of smooth muscle cells
Authors: Scheid C. R. , Jones A. W.
Published: December 2017

Unidirectional transmembrane fluxes of 24Na were examined in suspensions of enzymatically dispersed toad stomach muscle cells. Compartmental analysis of 24Na influx indicated that 24Na exchange consisted of two kinetically distinguishable components in these cells: a large pool (approximately 45 mmol/l cell H2O) with a rapid rate of exchange [half time (t1/2) approximately 5.8 min] and a smaller pool (approximately 26 mmol/l cell H2O) that exchanged more slowly (t1/2 approximately 69 min). Only the latter appeared ouabain sensitive; thus we postulated that the rate of exchange of this pool might be determined by the inward leak of Na+. Based on this assumption, we derived estimates for Na+ influx rate (0.9 X 10(-12) mol . cm-2 . s-1) and Na+ permeability (0.3 X 10(-8) cm/s), which presumably represent upper limits for these parameters in smooth muscle. The location and significance of the rapidly exchanging pool were not resolved. The exchange of both cellular Na+ pools was increased by the ionophore monensin. Thus we speculated that the rapidly exchanging pool was associated with a small population of permeable (high Na+) smooth muscle cells, while the slowly exchanging pool was associated with smooth muscle cells with normal Na+ permeability (low-Na+ cells).
Potassium channel selectivity in mouse pancreatic B cells
Authors: Rosario L. M. , Rojas E.
Published: December 2017

High-resistance microelectrodes were used to measure membrane potential changes in response to increased extracellular K+ concentration ([K+]o; or a test cation X+ such as Li+, Rb+, Cs+, NH+4) in B cells from mouse islets of Langerhans. In the absence of glucose, a sudden increase in [K+]o (or [X+]o), keeping the sum [Na+]o + [K+]o constant (or [Na+]o + [K+]o + [X+]o), induced a rapid depolarization of the membrane. The membrane potential changes were essentially unchanged in the presence of 20 mM tetraethylammonium (TEA). The Goldman-Hodgkin-Katz equation was fitted to the experimental relationship between membrane potential and [K+]o (or [X+]o), and permeability (P) ratios were estimated. In the absence of TEA, P Na/PK was estimated to be approximately 0.046. In the presence of TEA the following ratios were estimated: P Rb/PK = 0.74, P Cs/PK = 0.62, and P NH4/PK = 0.36. From these ratios the following sequence of permeabilities was obtained, PK greater than P Rb greater than P Cs greater than P NH4 greater than P Na. It is proposed that this sequence reflects the selectivity of the intracellular [Ca2+]-activated K+ channel of the pancreatic B cell.
Interaction of dihydropyridine Ca2+ agonist Bay K 8644 with normal and transformed pituitary cells
Authors: Enyeart J. J. , Aizawa T. , Hinkle P. M.
Published: December 2017

The dihydropyridine (DHP) Ca2+ agonist Bay K 8644 produced a dose-dependent increase in 45Ca2+ uptake by GH4C1 rat pituitary tumor cells. For agonist concentrations between 10(-9) and 10(-5) M, the enhanced 45Ca2+ uptake was well correlated with simultaneous increases in prolactin (PRL) secretion. Bay K 8644 combined with depolarizing concentrations of KCl produced more than additive effects on net Ca2+ uptake and hormone release. Nisoldipine, a DHP Ca2+ antagonist, competitively blocked Bay K 8644-stimulated 45Ca2+ uptake. This drug also potently inhibited 45Ca2+ uptake triggered by depolarization with KCl (estimated half-maximal inhibiting concentration: 2 nM). Bay K 8644 enhanced PRL secretion from normal rat pituitaries in culture and in a perifusion system. These results indicate that Bay K 8644 is a potent modulator of voltage-sensitive Ca2+ channels of both normal and transformed pituitary cells. In this respect endocrine cell Ca2+ channels resemble those found in heart, smooth muscle, and neuronal cell bodies.
The amiloride-sensitive sodium channel
Authors: Sariban-Sohraby S. , Benos D. J.
Published: December 2017

Net Na+ movement across the apical membrane of high-electrical resistance epithelia is driven by the electrochemical potential energy gradient. This entry pathway is rate limiting for transepithelial transport, occurs via a channel-type mechanism, and is specifically inhibited by the diuretic drug amiloride. This channel is selective for Na+, Li+, and H+, saturates with increasing extracellular Na+ concentration, and is not affected, at least in frog skin epithelium, by changes in apical membrane surface potential. There also appears to be multiple inhibitory regions associated with each Na+ channel. We discuss the possible implications of a voltage-dependent block by amiloride in terms of macroscopic inhibitory phenomena. We describe the use of cultured epithelial systems, in particular, the toad kidney-derived A6 cell line, and the preparation of apical plasma membrane vesicles to study the Na+ entry process. We discuss experiments in which single, amiloride-sensitive channel activity has been detected and summarize current experimental approaches directed at the biochemical identification of this ubiquitous Na+ transport system.
Variability of functional characteristics of MDCK cells
Authors: Husted R. F. , Welsh M. J. , Stokes J. B.
Published: December 2017

We measured several functional parameters of MDCK cells cultured as monolayers in order to more fully characterize their ion transport properties. Most of the present studies were completed with five groups (A-E) of MDCK cells studied from passage 62 to 78. Each group represents the same subline of MDCK cells after having been frozen, stored, and thawed at passage 62 or 64. The median transmonolayer resistances of the groups were 507, 149, 284, 72, and 126 omega X cm2. Addition of amphotericin B to the apical solution induced a oubain-sensitive transepithelial current. The apical membrane voltage and fractional resistance exhibited a wide range of values in two of the groups studied, with mean values of -32 mV and 0.68 in group B and -40 mV and 0.78 in group E. Neither apical nor basolateral membrane displayed significant Na+ conductance. K+ conductance was present in the basolateral but not in the apical membrane. Acidification or alkalinization of the apical solution was dependent on the conditions used to study the cells. The 4,4'-diisothyocyano-2,2'-disulfonic stilbene inhibited acidification (or induced alkalinization), whereas increasing ambient HCO-3 concentration induced alkalinization. The results of these studies indicate qualitatively similar behavior between five groups of MDCK cells but significant quantitative differences between the groups. Analysis of the variability of the measured parameters indicated that there were no differences as a function of passage number within a group. The factors responsible for functional differences between groups are not known but may be related to the cell storage process.
Calcium release rate in skinned skeletal muscle fibers measured with arsenazo III
Authors: Best P. M. , Fill M.
Published: December 2017

Calcium ion release from the sarcoplasmic reticulum of single skinned (sarcolemma removed) skeletal muscle fibers was studied using the calcium-sensitive dye arsenazo III (Arz III). Isotropic absorption measurements were made differentially to reduce the effect of movement artifacts. The question of dye stoichiometry was addressed by measuring the absorption ratio at 600 and 660 nm at various times during the calcium transient. The results indicate that little change in the proportions of the various calcium-dye species occurs until at least 1 s into the release and, further, that the 1:2 calcium-dye complex is unlikely to be the dominant species present at early times. The relationship between dye concentration and the slope of the early absorption change was found to be linear for all levels of fiber loading. This suggests that the 1:1 rather than the 2:2 complex is the major species formed at early times in skinned fibers, although this conclusion is at odds with in vitro studies of Arz III in solution. Beer's law was used to convert the slope of the absorption transient measured over the first 125 ms of a release to the rate of change of the calcium-dye complex. The average rate at which the calcium-dye complex was formed was found to be 0.6 microM/ms. Two models are considered that allow calculation of a correction factor that is used to convert this value to the rate of calcium release from the sarcoplasmic reticulum. The magnitude of these correction factors was a function of the dye and intrinsic buffer concentrations as well as the stoichiometry of the calcium-dye reaction. After application of the correction factors, the average release rate in our fibers was calculated to range from 0.8 to 13.5 microM/ms.
Phosphagen and intracellular pH changes during contraction of creatine-depleted rat muscle
Authors: Meyer R. A. , Brown T. R. , Krilowicz B. L. , Kushmerick M. J.
Published: December 2017

To evaluate the functional role of phosphocreatine (PCr) and creatine in muscle metabolism, these compounds were depleted by feeding rats the creatine analogue, beta-guanidinopropionate (beta-GPA, 2% of diet). Changes in phosphate metabolites and intracellular pH were monitored in gastrocnemius muscle in situ by phosphorus nuclear magnetic resonance (31P-NMR) at 162 MHz using the surface coil technique. After 3 mo of feeding, 25 mumol/g of phosphorylated beta-GPA (beta-GPAP) had accumulated, and PCr, creatine, and ATP levels were reduced to 6, 17, and 56%, respectively, compared with muscles of control animals. In resting muscle, there was no measurable exchange of phosphate between beta-GPAP and ATP by the NMR saturation transfer method. During muscle stimulation at 1 and 5 Hz, the maximum net rate of beta-GPAP hydrolysis was 10% that of PCr in control muscles, so that after 150 s inorganic phosphate had increased to less than 50% of the level attained in control muscles. At both rates, peak twitch force declined toward a steady state more rapidly in beta-GPA-loaded muscles, but after 100 s force was either not different (1 Hz) or significantly greater (5 Hz) in the beta-GPA-fed animals. Intracellular pH initially decreased more rapidly during stimulation and recovered more rapidly afterward in the beta-GPA-loaded muscles compared with controls. This difference could be explained by the difference in expected proton consumption due to net PCr hydrolysis. However, despite buffering by PCr hydrolysis, pH ultimately decreased more in control muscle (6.1 vs. 6.3 for 5 Hz), indicating greater acid accumulation compared with beta-GPA-loaded muscles. In the superficial, predominantly fast-twitch glycolytic section of muscles clamp-frozen after 5-Hz stimulation for 150 s, lactate accumulation was twofold greater in controls. The results indicate that PCr is not essential for steady-state energy production but that the phosphate from PCr hydrolysis may be important for maximum activation of glycogenolysis and/or glycolysis.
Changes in intercellular electrical coupling of smooth muscle accompanying atrophy and hypertrophy
Authors: Bortoff A. , Sillin L. F.
Published: December 2017

Longitudinal tissue impedance was determined for cat circular intestinal muscle that was either hypertrophied due to volume overloading or atrophied due to defunctionalization. These conditions were produced by bypassing 50 cm of jejuno-ileum in six cats and, 2-6 mo later, removing segments from the proximal jejunum of the hypertrophied functional gut and from the atrophied proximal end of the bypassed loop. Impedances were compared with those of jejunal circular muscle from 15 normal cats. Specific tissue impedance was determined by a modification of the method of Tomita (J. Physiol. Lond. 201: 145-159, 1969), which employs Krebs and Krebs-sucrose solutions; a tissue shrinkage of 5%, empirically found to occur in Krebs-sucrose solution, was corrected for. Impedance values were determined at 20 frequencies between 30 Hz and 30 kHz. The value at 30 kHz was taken to represent the specific myoplasmic resistance (Rmyo) of each tissue, while the difference between the value of 30 Hz and 30 kHz was taken to represent the specific junctional resistance (Rj). Values (in omega X cm) for Rmyo were control 134 +/- 2, functional 128 +/- 5, bypassed 151 +/- 6 (mean of means +/- SE). Corresponding values for Rj were control 173 +/- 15, functional 96 +/- 27, bypassed 340 +/- 75. Calculated values (in microF/cm) for junctional capacitance were control 2.66, functional 6.10, bypassed 1.97. Acid uncoupling by saturating the bathing solutions with 100% CO2 revealed a pH-sensitive resistive component of Rj, assumed to be attributable to gap junctions.(ABSTRACT TRUNCATED AT 250 WORDS)
Cooperation between epithelial cells demonstrated by potassium transfer
Authors: Ledbetter M. L. , Young G. J. , Wright E. R.
Published: December 2017

Junction-mediated communication can be measured in fibroblast cultures by determining the ability of mixed cultures of cells sensitive and resistant to ouabain to concentrate K+ in the presence of ouabain. We now report the extension of this assay procedure to cultured epithelial cells. Hamster kidney (HaK) cells maintain their ability to concentrate K+ in ouabain at levels inhibitory to dog kidney (MDCK) cells. When HaK and MDCK cells were cultured together in ouabain-containing medium, the K+ (measured as 86Rb+) in the mixed population was greater than expected if the cells were not interacting. The degree of enhancement, expressed as "index of cooperation," depended on the numbers of cells in the cultures, their opportunity for cell-to-cell contact, and (above a certain permissive level) the concentration of ouabain. As with other cell types, protein synthesis in MDCK cells depends on maintenance of cell K+. Autoradiography of cells incubated with [3H]leucine demonstrated that MDCK cells in ouabain-treated mixed cultures were able to synthesize proteins only when physically adjacent to HaK cells. The transmission of labeled nucleosides among the cells provides independent evidence of the phenomenon of cooperation, probably mediated by gap junctions. This system offers promise for investigation of stimuli modulating junctional communication.
Coupled sodium-calcium transport in cultured chick heart cells
Authors: Murphy E. , Wheeler D. M. , LeFurgey A. , Jacob R. , Lobaugh L. A. , Lieberman M.
Published: December 2017

In cultured embryonic chick heart cells, alterations of extracellular Na (Nao) and Ca (Cao), intracellular Na (Nai) and Ca, extracellular pH, and membrane potential resulted in changes in Na and Ca contents that were consistent with sarcolemmal Na-Ca exchange. 24Na efflux measurements revealed a large ouabain-insensitive component, one-third of which was inhibited by removal of Cao. Incubating the cells in Na-free solution resulted in a rapid, 1.5- to 2-fold increase in total cell Ca that remained elevated for at least 15 min. Cells exposed for 15 min to Nao less than or equal to 20 mM became maximally loaded with Ca, whereas Ca loading fell off sharply at values of Nao greater than 20 mM. The movement of Na against its electrochemical gradient was shown to be associated with Ca accumulation. During Na-K pump inhibition (in 10(-4) M ouabain), Na initially rose 2- to 3-fold to a level below its equilibrium value; then, lowering Cao for 30 min from 1.25 to 0.75 mM caused a 26% elevation in Nai, whereas raising Cao from 1.25 to 2.7 mM resulted in a 25% fall in Nai against its electrochemical gradient. These data are consistent with Nai being maintained by a Na-Ca exchange during Na-K pump inhibition. In the presence of ouabain (10(-4) M), Ca uptake into intracellular organelles, e.g., mitochondria, was suggested by an increase in total cell Ca as well as the occurrence of mitochondrial matrix granules, which were shown qualitatively by X-ray analysis to contain Ca. Although matrix granules also occurred in mitochondria during Na-free incubation, they did not contain detectable amounts of Ca when examined under identical conditions of fixation and analysis.
Effects of forskolin and cyclic nucleotides on isometric force in rat aorta
Authors: McMahon E. G. , Paul R. J.
Published: December 2017

The present study was undertaken to determine the extent to which cyclic nucleotide-induced relaxation in the intact rat aorta is mediated at the level of the contractile system. The relaxant effects of the cyclic nucleotide analogues [8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) and dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP)] and forskolin were examined in both the intact vessel and a Triton X-100-skinned preparation of rat thoracic aorta. Relaxation of a norepinephrine-induced contraction was essentially complete 30 min after the addition of 50 microM 8-BrcGMP [% relaxation = 87.2 +/- 4.4% (n = 4)], 100 microM DBcAMP [98.2 +/- 1.2% (n = 4)], and 1 microM forskolin [107.0 +/- 3.3% (n = 5)]. These same doses were ineffective in relaxing precontracted skinned rat aortic rings compared with the relaxation achieved in the intact vessel. The largest relaxation in the skinned aortas was achieved with the addition of 1 microM forskolin [17.4 +/- 1.5% (n = 4)]. The addition of catalytic subunit of cAMP-dependent protein kinase had no effect on isometric tension in the precontracted skinned aorta. Preincubation with the cyclic nucleotide analogues or forskolin in a low-Ca2+ solution (pCa less than 8) was also ineffective in inhibiting subsequent isometric tension development. Our results suggest that only a very small fraction of the relaxation with cyclic nucleotides and forskolin in the intact rat aorta is due to the action of these agents at the level of the contractile system.
Activators of protein kinase C inhibit sodium transport in A6 epithelia
Authors: Yanase M. , Handler J. S.
Published: December 2017

To evaluate the role of protein kinase C in sodium transport via amiloride-sensitive sodium channels, we studied the effect of activators of protein kinase C on short-circuit current in epithelia formed by A6 cells in culture. In A6 epithelia, short-circuit current is equivalent to net sodium transport and is blocked by low concentrations of amiloride added to the solution bathing the apical surface. After any of four different activators of protein kinase C [phorbol 12,13-dibutyrate (20 ng/ml), phorbol 12-myristate 13-acetate (20 ng/ml), 1-oleoyl-2-acetylglycerol (50 micrograms/ml), and mezerein (10 ng/ml)] was added to the solution bathing the apical surface, short-circuit current fell, and electrical resistance rose. Nystatin added to the apical solution reversed the inhibition of short-circuit current, indicating that activators of protein kinase C inhibited transport at amiloride-sensitive sodium channels in the apical plasma membrane. Under some conditions, the activators also stimulated amiloride-insensitive short-circuit current. The ion transport represented by the amiloride-insensitive short-circuit current appears to be the result of basal to apical transport of chloride, but this has not been established conclusively.
Membrane ATPase mechanism of K+-return relaxation in arterial muscles of stroke-prone SHR and WKY
Authors: Hermsmeyer K. , Harder D.
Published: December 2017

These studies compared the importance of electrogenic Na+-K+ active (ATP driven) transport, changes in K+ conductance, and passive Ca2+-Na+ countertransport in the large relaxation that occurs in the rat caudal and basilar artery on return to K+ from K+-free solutions. Furthermore, we compared the importance of these three membrane electrical mechanisms in stroke-prone spontaneously hypertensive rats (SP-SHR) versus their normotensive Wistar-Kyoto control rats (WKY) in basilar (cerebral) and caudal arteries. We found that in both basilar and caudal arteries the hyperpolarization and relaxation that occurred on return to K+ after exposure to a 0 K+ (extracellular) solution was consistently greater in SP-SHR than in WKY. The change in membrane potential occurring on transition to 0 K+ in arteries maintained at low temperature (16 degrees C), used as an estimate of the change in K+ conductance during the K+ transition, was not different in either basilar or caudal arteries between SP-SHR and WKY. Thus the hyperpolarization on return to K+ at body temperature would depend primarily on the level of activity of the membrane ATPase, referred to as the Na+ pump. We also sought to compare the passive (but electrogenic) Ca2+-Na+ countertransport mechanism between strains for both arteries, but we were unable to detect any evidence of the predicted hyperpolarization-contraction on transition from 145 to 10 mM extracellular Na+. Furthermore, the return to extracellular Na+ solution failed to show the depolarization-relaxation predicted by the Ca2+-Na+ countertransport mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Dissociation between muramyl dipeptide-induced fever and changes in plasma metal levels
Authors: Riveau G. , Parant M. , Damais C. , Parant F. , Chedid L.
Published: December 2017

A fall in plasma iron level and an increase in copper level were observed in rabbits subsequent with the febrile response induced by an intravenous administration of muramyl dipeptide, AcMur-L-Ala-D-isoGln (MDP). The pyrogenic activity of MDP was due partly to the induction of circulating endogenous pyrogen (EP). EP produced in vitro by activated macrophages also elicited changes in iron and copper levels in rabbits. Nonpyrogenic MDP derivatives murabutide [MDP(Gln)-OnBu] and the stereoisomer of MDP [MDP(D,D)] did not cause any change in blood metal levels. Another adjuvant and nonpyrogenic analogue, murametide [MDP(Gln)-OMe], elicited hypoferremia and hypercupremia. Murametide, which has been previously shown to induce secretion of circulating EP but prevents in vivo fever response, was unable to prevent an EP-induced effect on plasma metal concentrations. Injection of supernatant fluids of macrophages incubated with these different glycopeptides showed that only compounds able to induce EP release were capable of evoking hypoferremia and hypercupremia. The EP-containing fluid was 10-fold more active on change in temperature and in plasma metal levels when it was given intracerebroventricularly compared with intravenously. In contrast, a pyrogenic dose of MDP that can act directly on the central thermoregulatory structures did not modify iron and copper levels when it was injected intracerebroventricularly.
Effect of prostaglandin E1 on DNA synthesis in vascular smooth muscle cells
Authors: Owen N. E.
Published: December 2017

The effect of prostaglandin E1 (PGE1) on DNA synthesis was determined using cultured vascular smooth muscle cells. It was found that when PGE1 was added to synchronous, quiescent (growth-arrested) cells, it enhanced DNA synthesis. This was in contrast to the effects of PGE1 on asynchronous, cycling (growing) cells. In these cells, when PGE1 was added, it functioned as an antiproliferative agent. In both cases the effects of PGE1 could be mimicked by 6 alpha-carbaprostacyclin (stable prostacyclin analogue) or by 8-bromo adenosine 3',5'-cyclic monophosphate [a permeable adenosine 3',5'-cyclic monophosphate (cAMP) analogue]. In addition PGE1 was shown to cause an elevation in cellular cAMP levels. On the basis of these studies it is hypothesized that the ultimate effect of addition of PGE1 to vascular smooth muscle cells is dependent on the phase of the cell cycle in which it is added.
Limited proteolysis of rabbit cardiac procathepsin D in a cell-free system
Authors: Smarel A. M. , Worobec S. W. , Ferguson A. G. , Decker R. S. , Lesch M.
Published: December 2017

Rabbit cardiac cathepsin D is initially synthesized as an inactive, apparent molecular weight (Mr) 53,000, pI 6.6 precursor (procathepsin D) that is proteolytically processed during intracellular transport to produce the Mr 48,000 isoforms of active cathepsin D found in cardiac lysosomes. To examine potential proteases responsible for intracellular proteolytic processing, biosynthetically labeled procathepsin D was isolated from rabbit ventricular tissue perfused for 30 min with [35S]methionine. Procathepsin D was then incubated in vitro (40 degrees C, 1-240 min) with active cathepsin D, papain, and cathepsin B, either singly or sequentially, and the reaction products analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Incubation of 35S-labeled procathepsin D with active cathepsin D produced a single reaction product (Mr 51,000; pI 6.2). This limited proteolysis occurred at pH 3-5 and was inhibited by pepstatin. Incubation of 35S-labeled procathepsin D with papain or cathepsin B produced a major reaction product (Mr 48,000; pI 6.4) and a minor form (Mr 50,000; pI 6.0). These reactions occurred at pH 4-7 and were inhibited by leupeptin but not pepstatin. Only the Mr 48,000, pI 6.4 products of papain and cathepsin B-mediated proteolysis comigrated with the most basic isoform of active cathepsin D found in cardiac tissue. In addition, the Mr 51,000 intermediate produced by cathepsin D was susceptible to further limited proteolysis by cysteine proteases with resultant production of a Mr 48,000 product. Thus the intracellular proteolytic processing of rabbit cardiac procathepsin D does not result solely from autocatalysis but requires at least one other protease, possibly cathepsin B.
Intracellular diffusion gradients of O2 and ATP
Authors: Jones D. P.
Published: December 2017

Endogenous enzymes with different subcellular localizations provide in situ probes to study O2 and ATP concentration at various sites within cells. Results from this approach indicate that substantial intracellular concentration gradients occur under some O2- and ATP-limited conditions. These studies, along with electron microscopic analyses and mathematical modeling, indicate that clustering and distribution of mitochondria are major factors in determining the magnitude and location of the concentration gradients. The mitochondria appear to be clustered in sites of high ATP demand to maximize ATP supply under conditions of limited production. The size of such clusters is limited by the magnitude of the O2 gradient needed to provide adequate O2 concentrations for mitochondrial function within the clusters. Thus microheterogeneity of metabolite concentrations can occur in cells without membranal compartmentation and may be important in determining the rates of various high-flux processes.
The number of chloride-cation cotransport sites on Ehrlich ascites cells measured with [3H]bumetanide
Authors: Hoffmann E. K. , Schiodt M. , Dunham P.
Published: December 2017

Simultaneous measurements were made of net Cl influxes and [3H]bumetanide binding to Ehrlich ascites tumor cells in which the chloride-cation cotransport pathway had been activated by hypertonic challenge. There was a good linear correlation between inhibition of Cl influx during regulatory volume increase and numbers of bumetanide molecules bound per cell, consistent with high specificity of bumetanide binding to cotransport sites. The extrapolation to the number of bumetanide binding sites per cell at maximal inhibition of Cl transport thus gives the number of cotransport sites per cell as 2.0 X 10(6). From this, and the fluxes measured (not necessarily maximum fluxes), the turnover number is calculated at 50 Cl ions per site per second. Unstimulated cells in isotonic medium, with negligible bumetanide-inhibitable fluxes, have the same number of bumetanide binding sites as the activated cells undergoing volume regulation.
Renal transport and metabolism of nicotinic acid
Authors: Schuette S. , Rose R. C.
Published: December 2017

Renal metabolism and brush-border transport of nicotinic acid were studied in renal cortical slices and brush-border membrane vesicles exposed to a physiological concentration of vitamin (2.2-3.5 microM). Vesicle transport of [3H]nicotinic acid was found to be Na+ dependent and concentrative. The presence of a Na+ gradient resulted in a fivefold increase in the rate of nicotinic acid uptake over that observed with mannitol and caused a transient nicotinic acid accumulation two- to fourfold above the equilibrium value. The effects of membrane potential, pH, and elimination of Na+-H+ exchange were also studied. Cortical slices and isolated tubules exposed to 2.2 microM [14C]nicotinic acid took up vitamin and rapidly metabolized most of it to intermediates in the Preiss-Handler (J. Biol. Chem. 233: 488-493, 1958) pathway for NAD biosynthesis; little free nicotinic acid was detectable intracellularly. The replacement of Na+ with Li+ in the bathing medium reduced total accumulation of 14C label primarily as a result of reduced nicotinic acid uptake. Cortical tissue concentrated free nicotinic acid only when the involved metabolic pathways were saturated by levels of nicotinic acid far in excess of what occurs in vivo.
Protein secretion in lacrimal gland: alpha 1-beta-adrenergic synergism
Authors: Mauduit P. , Herman G. , Rossignol B.
Published: December 2017

We have shown the existence of a homogeneous population of specific binding sites for [3H]prazosin in membranes from rat lacrimal glands. The value of the equilibrium dissociation constant was 0.186 +/- 0.07 nM, and the density of specific binding sites was 20.4 +/- 1 fmol/mg protein. Taking into account the potency sequences for adrenergic agonists and antagonists for competition with these [3H]prazosin binding sites, we identified them as alpha 1-adrenergic receptors. Moreover, we have demonstrated that the stimulation of protein discharge evoked by epinephrine could be partly attributed to the occupation of this alpha-adrenergic receptor subtype. However, the inhibition pattern of the epinephrine effect by a beta-adrenergic antagonist, 1-propranolol, and the characteristics of the secretory response observed when selectively stimulating the alpha 1- and beta-adrenergic receptors, either separately or simultaneously, suggest that 1) a simultaneous activation of both receptors is necessary to produce a maximal secretory response to catecholamines; and 2) a synergism may exist between these two routes of stimulation, leading to an amount of protein discharge higher than that expected in the case of additive effects.
Insulin-induced cytoplasmic alkalinization and glucose transport in muscle cells
Authors: Klip A. , Ramlal T. , Cragoe E. J.
Published: December 2017

Insulin stimulates glucose uptake into muscle within minutes, preceding stimulation of glycolysis. Signals involved in stimulation of glycolysis include cytoplasmic alkalinization and specific intracellular proteolytic products. In contrast, the signals that mediate stimulation of glucose transport remain unknown. Here we explore whether the insulin-induced cytoplasmic alkalinization is an early event that precedes activation of sugar uptake, whether such alkalinization is causally related to stimulation of sugar uptake, and whether proteolytic activity mediates stimulation of hexose transport. Cytoplasmic pH (pHi) was measured in suspended skeletal muscle cells of the L6H9 line with the intracellularly trapped fluorescent pH indicator bis(carboxyethyl)carboxy fluorescein. At 37 degrees C, insulin (1 X 10(-7) M) produced an increase in pHi of 0.11 units in 10 min. This increase became apparent 2 min after addition of the hormone, and maximal elevation of pHi was observed after 10 min, remaining elevated for up to 60 min. Removal of the hormone with anti-insulin antiserum did not reverse pHi back to the resting level. The alkalinization was prevented by amiloride, by 5-(N,N'-disubstituted)amiloride analogues, and by isosmotic replacement of Na+ with N-methylglucamine+ or choline+. This suggests that insulin activates Na+-H+ exchange. In contrast, stimulation of 2-deoxy-D-glucose transport by insulin was not affected by replacement of external Na+ or by addition of amiloride. Monensin, an exogenous Na+-H+ exchanger, did not stimulate sugar transport even though it increased pHi. Proteinase inhibitors that block hormonal stimulation of glycolysis were ineffective in preventing stimulation of 2-deoxy-D-glucose transport by insulin.(ABSTRACT TRUNCATED AT 250 WORDS)
Amiloride analogues inhibit proximal tubule metabolism
Authors: Soltoff S. P. , Cragoe E. J. , Mandel L. J.
Published: December 2017

The effects of two amiloride analogues, ethylisopropylamiloride and methylisopropylamiloride, were tested on active Na+ transport and oxidative metabolism of a rabbit proximal renal tubule suspension. These two analogues have been recently reported to inhibit Na+-H+ antiport activity of various tissues with greater potency than amiloride. In proximal tubules, no effects were detected at concentrations less than 10(-4) M. At concentrations greater than 10(-4) M, both of these compounds inhibited the ouabain-sensitive O2 consumption (a direct measure of Na+ pump activity) with a greater potency than amiloride. Investigations into possible metabolic effects revealed that both amiloride analogues inhibited mitochondrial production of ATP at these concentrations, whereas amiloride did not directly affect metabolism. The amiloride analogues inhibited the nystatin-stimulated O2 consumption (which measures Na+-K+-ATPase activity in the intact cells) as well as the ATP content. These results suggest that the primary effects of these analogues on rabbit proximal tubules are to inhibit both the Na+-K+-ATPase and oxidative metabolism.
Calcium transients and resting levels in isolated smooth muscle cells as monitored with quin 2
Authors: Williams D. A. , Fay F. S.
Published: December 2017

The Ca2+-sensitive dye quin 2 was used to monitor Ca2+ levels and to follow Ca2+ transients in suspensions of isolated toad (Bufo marinus) smooth muscle cells, whose contractile activity was monitored with a Coulter counter. Single cells were not utilized to follow Ca2+ changes involved with contraction because of problems of rapid dye bleaching and due to photosensitization of smooth muscle cells loaded with quin 2. High levels of quin 2 loading (2-5 mM) severely prolonged (time course increase greater than 3 times), or completely inhibited, contractile responses to carbachol or potassium depolarization. Lower levels (less than 1 mM) produced adequate fluorescent signals, even at the single cell level, and allowed contractile responses of normal magnitude, although with somewhat prolonged (2-3 times) time course. Resting Ca2+ concentrations determined using quin 2 at these lower levels were 129 +/- 3 nM, a value that closely coincided with that measured in the same cell type using Ca-sensitive microelectrodes, or an alternate, more highly fluorescent dye, Fura-2. Resting Ca2+ was highly dependent on the extracellular Ca2+ concentration that appeared to effect intracellular Ca2+ (Cai2+) both by altering the driving force on Ca2+ cross the membrane, as well as the Ca2+ permeability of the cell itself. A small but significant relaxation was observed in response to the lowering of cytoplasmic Ca2+ below resting levels. After carbachol or K+ addition, fluorescent transients peaked significantly before the onset of contraction, which was also transient. Isoproterenol, a known relaxant of these cells, caused a small decrease in Cai2+ (approximately 40 nM) below rest, when applied in maximal dosage (10(-4) M). Isoproterenol also consistently diminished the Ca2+ transient induced by excitatory stimuli such as carbachol or K+. These results indicate that changes in contractility may be directly linked to changes in free cytoplasmic Ca2+ in smooth muscle cells.
Sulfhydryl substituents of the human erythrocyte hexose transport mechanism
Authors: Abbott R. E. , Schachter D. , Batt E. R. , Flamm M.
Published: December 2017

Sulfhydryl substituents of the hexose transport mechanism of human erythrocyte membranes were studied with membrane-impermeant and -permeant maleimide derivatives. Three sulfhydryl classes have been identified on the basis of their reactivity toward the reagents and their effects on the transport mechanism. Type I sulfhydryl is located at the outer (exofacial) surface of the membrane and bound covalently on treatment of intact cells with the membrane-impermeant glutathione-maleimide. This sulfhydryl is required for the transport, and it is protected from alkylation, i.e., its reactivity toward maleimides is decreased by the presence of D-glucose or cytochalasin B. Type II sulfhydryl is also required for the transport, but it differs from type I in that D-glucose (but not cytochalasin B) increases the reactivity toward maleimides. Further, it is located at the endofacial surface of the membrane, since reaction with glutathione-maleimide occurs only in leaky ghosts and not in intact cells. Alkylation by glutathione-maleimide of type I and type II sulfhydryls increases the half-saturation for the binding of D-glucose to erythrocyte membranes. In contrast, inactivation of type III sulfhydryls by N-ethylmaleimide or dipyridyl disulfide decreases the half-saturation concentration for the binding of D-glucose and other transported hexoses to the membranes; nontransported sugars are not affected similarly. Type III sulfhydryl is not inactivated by the polar reagent glutathione-maleimide and is probably located in a nonpolar domain of the transport mechanism. Inactivation of either type I or II sulfhydryls decreases or eliminates the flux asymmetry of the hexose transport mechanism.
Receptor-independent sequestration of beta-adrenergic ligands by alveolar type II cells
Authors: Fabisiak J. P. , Rannels S. R. , Vesell E. S. , Rannels D. E.
Published: December 2017

Numerous studies indicate that synthesis and secretion of surfactant by type II pneumocytes are modulated by the interaction of beta-adrenergic agonists with specific cell surface receptors. Two 125I-labeled beta-adrenergic ligands, l-iodopindolol (IPIN) and l-iodocyanopindolol (ICYP), were thus employed to investigate the properties of type II cell beta-receptors. Saturable, high-affinity, stereospecific binding to crude membrane fractions from whole rat lungs was exhibited by both ligands (IPIN, KD 283 pM, Bmax 508 fmol/mg protein; ICYP, KD 18 pM, Bmax 404). Type II cell membranes obtained by N2 cavitation also revealed stereospecific, saturable, high-affinity binding. In intact cells (37 degrees C) however, rapid, highly concentrative (cell/media greater than 1000), nonspecific ligand uptake compromised estimates of specific binding (specific/total binding less than 0.1). Total ligand uptake was inhibited at 4 degrees C, by decreasing pH within the physiological range (7-8) and by the lysosomotropic compound chloroquine (50-200 microM), without a detectable change in specific binding. Other basic drugs were also inhibitory at similar concentrations; acidic drugs had no effect. Even at 4 degrees C, specific binding remained low, as IPIN and ICYP were displaced less than 30% by l-alprenolol (1 microM). Physicochemical properties of IPIN and ICYP considered with the above studies suggest that passive ligand entry into intact pneumocytes and subsequent trapping of the protonated species in a cellular compartment of low pH may account for high nonspecific ligand uptake.
Bradykinin and vasopressin stimulate Na+-K+-Cl- cotransport in cultured endothelial cells
Authors: Brock T. A. , Brugnara C. , Canessa M. , Gimbrone M. A.
Published: December 2017

We have characterized a Na+-K+-Cl- cotransporter in vascular endothelial cells (EC) cultured from different blood vessels and species that is inhibited by the diuretics furosemide and bumetanide (50% inhibitory concentration for 86Rb influx approximately 20 microM and 0.5 microM, respectively). Inward 86Rb influx mediated via this pathway is greater than 86Rb influx transported by the Na+-K+ pump in cultured EC from bovine and pig aorta, bovine vena cava, and baboon cephalic vein but not in human umbilical or saphenous vein EC. External Na+ or Cl- -stimulated, ouabain-insensitive 86Rb influx is equal to furosemide or bumetanide-sensitive 86Rb influx. Ouabain-insensitive 22Na influx is also partially inhibited by these drugs and stimulated by increasing external K+ or Cl-. Net Na+ extrusion occurs via the Na+-K+-Cl- cotransporter in the absence of external K+, whereas net Na+ influx occurs at higher external K+ (greater than 1 mM). Maximal concentrations (100 nM) of bradykinin and vasopressin increase the initial rate of bumetanide-sensitive 86Rb influx by approximately 60 and 70% (50% effective concentration approximately 1 and 0.6 nM, respectively). Addition of either ethyleneglycol-bis(beta-aminotethylether)-N,N'-tetraacetic acid or LaCl3 (to block calcium influx) prevents bradykinin-stimulated 86Rb influx. When intracellular calcium is elevated using ionomycin (100 nM), a Ca2+ ionophore, bumetanide-sensitive 86Rb influx increases approximately twofold. In contrast, isoproterenol (100 microM) and forskolin (50 microM), adenylate cyclase stimulators, decrease furosemide-sensitive 86Rb influx.(ABSTRACT TRUNCATED AT 250 WORDS)