bluuebunny/crossref_metadata_2025 huggingface dataset:
Response of slow and fast muscle to hypothyroidism: maximal shortening velocity and myosin isoforms
Authors: Caiozzo V. J. , Herrick R. E. , Baldwin K. M.
Published: December 2017

This study examined both the shortening velocity and myosin isoform distribution of slow- (soleus) and fast-twitch (plantaris) skeletal muscles under hypothyroid conditions. Adult female Sprague-Dawley rats were randomly assigned to one of two groups: control (n = 7) or hypothyroid (n = 7). In both muscles, the relative contents of native slow myosin (SM) and type I myosin heavy chain (MHC) increased in response to the hypothyroid treatment. The effects were such that the hypothyroid soleus muscle expressed only the native SM and type I MHC isoforms while repressing native intermediate myosin and type IIA MHC. In the plantaris, the relative content of native SM and type I MHC isoforms increased from 5 to 13% and from 4 to 10% of the total myosin pool, respectively. Maximal shortening velocity of the soleus and plantaris as measured by the slack test decreased by 32 and 19%, respectively, in response to hypothyroidism. In contrast, maximal shortening velocity as estimated by force-velocity data decreased only in the soleus (-19%). No significant change was observed for the plantaris.
AMP deaminase binding in contracting rat skeletal muscle
Authors: Rundell K. W. , Tullson P. C. , Terjung R. L.
Published: December 2017

AMP deaminase, which hydrolyses AMP to inosine 5'-monophosphate (IMP) and NH3 at high rates during excessive energy demands in skeletal muscle, is activated when bound to myosin in vitro. We evaluated AMP deaminase binding in vivo during muscle contractions to assess whether binding 1) is inherent to deamination and found only with high rates of IMP production or simply coincident with the contractile process and 2) requires cellular acidosis. AMP deaminase activity (mumol.min-1.g-1) was measured in the supernatant (free) and 10(4)-g pellet (bound) homogenate fractions of muscle of anesthetized rats after in situ contractions to determine the percent bound. In resting muscle, nearly all (approximately 90%) AMP deaminase is free (cytosolic). During contractions when energy balance was well maintained, binding did not significantly differ from resting values. However, during intense contraction conditions that lead to increased IMP concentration, binding increased to approximately 60% (P less than 0.001) in fast-twitch and approximately 50% in slow-twitch muscle. Binding increased in an apparent first-order manner and preceded initiation of IMP formation. Further, binding rapidly declined within 1 min after cessation of intense stimulation, even though the cell remained extremely acidotic. Extensive binding during contractions was also evident without cellular acidosis (iodoacetic acid-treated muscle). Thus the in vivo AMP deaminase-myosin complex association/dissociation is not coupled to changes in cellular acidosis. Interestingly, binding remained elevated after contractions, if energy recovery was limited by ischemia. Our results are consistent with myosin binding having a role in AMP deaminase activation and subsequent IMP formation in contracting muscle.
Changes of cytosolic Ca2+ interfere with measurements of cytosolic Mg2+ using mag-fura-2
Authors: Hurley T. W. , Ryan M. P. , Brinck R. W.
Published: December 2017

In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)
Depletion in nuclear spermine during human spermatogenesis, a natural process of cell differentiation
Authors: Quemener V. , Blanchard Y. , Lescoat D. , Havouis R. , Moulinoux J. P.
Published: December 2017

Polyamines (PA), polycations present in all mammalian cells, are essential for cell proliferation and differentiation. In vitro, PA are known to bind to DNA with a high affinity. In vivo, the intimate association of endogenous PA with highly condensed chromatin has been reported. During spermatogenesis, when processes of cell proliferation and differentiation take place, the potential role of polyamines has not been studied in depth. We report here the PA levels measured in human spermatogenic cell nuclei at different stages of differentiation. Cell populations (spermatocytes and round, elongating, or elongated spermatids) were obtained after submitting human testes to a trypsin-deoxyribonuclease digestion, then to a centrifugal elutriation and Percoll gradient centrifugation. A significant and progressive nuclear spermine level decrease was observed from primary spermatocytes to elongated spermatids. This release of spermine from nuclei was concomitant with three major events in mammalian spermiogenesis: the reduction of DNA transcription activity, the replacement of histone proteins by protamines, and the compaction of chromatin. This is the first report arguing a release of nuclear spermine during an in vivo physiological cell differentiation process.
Chloride secretory response to extracellular ATP in human normal and cystic fibrosis nasal epithelia
Authors: Clarke L. L. , Boucher R. C.
Published: December 2017

Chloride secretion across cystic fibrosis (CF) airway epithelia is effectively regulated by pathways associated with intracellular Ca2+ metabolism, but not by mechanisms dependent on protein kinase A or C. In a search for therapeutically useful agonists, we investigated the effects of extracellular ATP on the Cl- secretory process in human normal and CF nasal epithelial cultures with double-barreled Cl- selective microelectrodes. When applied to the basolateral membrane of normal, but not CF, nasal epithelium, extracellular ATP (10(-4) M) stimulated a small increase in Cl- secretion that was primarily associated with a hyperpolarizing conductance in the basolateral membrane. In contrast, ATP applied to the apical (luminal) membrane of either normal or CF nasal epithelium stimulated a greater increase in Cl- secretion that was associated with activation of an apical membrane Cl- conductance. The increases in Cl- current and apical conductance were greater in CF tissues and attained maximal values similar to normal nasal epithelium. We conclude 1) that basolateral application of ATP indirectly induces Cl- secretion by activating a basolateral (K+) conductance and is an effective secretagogue only in normal nasal epithelium and 2) that luminally applied ATP is an effective Cl- secretagogue that activates the apical membrane Cl- conductance of normal and CF nasal epithelia to an equivalent level.
TGF-beta 1 potentiates growth factor-stimulated proliferation of vascular smooth muscle cells in genetic hypertension
Authors: Saltis J. , Agrotis A. , Bobik A.
Published: December 2017

We have examined the interactions between transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF) isoforms PDGF-AB and PDGF-BB on the proliferation of vascular smooth muscle cells isolated from the spontaneously hypertensive rat. TGF-beta 1 alone stimulated [3H]thymidine incorporation approximately twofold without a corresponding increase in cell number. In combination, TGF-beta 1 action was synergistic in further stimulating both DNA synthesis and cell proliferation 100-300% above the responses elicited by each growth factor. To gain further insight into the mechanism responsible for this potentiation, we examined the interaction between TGF-beta 1 and EGF. The synergistic interaction between TGF-beta 1 and EGF on DNA synthesis was independent of initial cell density. This effect of TGF-beta 1 was initiated early in the G1 phase of the cell cycle and did not appear to be mediated through the mobilization of Ca2+ or alterations in c-jun mRNA expression. However, in the presence of both TGF-beta 1 and EGF, there was a sustained elevation of c-myc mRNA levels over a 24-h period. These results suggest that TGF-beta 1 may interact with other growth factors in vivo to enhance their proliferative action on vascular smooth muscle of spontaneously hypertensive rats via mechanisms dependent on c-myc mRNA expression.
5'-Aminolevulinate synthase activity is decreased in skeletal muscle of anemic rats
Authors: McNabney L. A. , Essig D. A.
Published: December 2017

Expression of the rate-limiting heme biosynthetic enzyme 5'-aminolevulinate synthase (ALAS) was investigated in skeletal muscle of 3-wk-old rats fed an iron-deficient diet. After 14 days, ALAS activity had declined 70% relative to control (2.1 +/- 0.2 vs. 0.6 +/- 0.1 nmol.h-1.g-1; P less than 0.005). Similar decreases were observed for blood hemoglobin (11.4 +/- 0.2 vs. 3.9 +/- 0.3 g/dl; P less than 0.005) and muscle cytochrome c (14.5 +/- 1.3 vs. 7.1 +/- 0.6 nmol/g; P less than 0.005). An iron-deficient diet decreased body and skeletal muscle growth by 15 (P less than 0.005) and 10% (P less than 0.05), respectively, whereas concentrations of protein, RNA, ALAS mRNA, and citrate synthase activity in muscle were not different from control. One mechanism by which heme biosynthesis may be slowed in muscle of young anemic rats is a decrease in ALAS activity. At a time when enzyme activity was decreased, ALAS mRNA expression was not affected by an iron-deficient diet, suggesting that steps after transcription of the ALAS gene may regulate the decrease in activity.
Ca2+ influx via Na(+)-Ca2+ exchange in immortalized aortic myocytes. II. Feedback inhibition by [Ca2+]i
Authors: Lyu R. M. , Smith L. , Smith J. B.
Published: December 2017

Depolarization with 50 mM K+ evoked a spike in cytosolic free Ca2+ ([Ca2+]i) and increased 45Ca2+ uptake in immortalized aortic myocytes. The following evidence indicates that the electrogenic Na(+)-Ca2+ exchanger caused the Ca2+ influx that was evoked by K+ depolarization. First, K+ depolarization had no effect on [Ca2+]i and 45Ca2+ uptake in cells with basal Na+ but strikingly increased both in Na(+)-loaded cells. Second, the [Ca2+]i increases produced by K+ depolarization depended hyperbolically on external Ca2+ (50% maximum concentration = 1.5 mM). Third, the increases in [Ca2+]i and 45Ca2+ uptake were greater when external Na+ was replaced with K+ rather than with N-methyl-D-glucamine or choline. A series of K+ depolarizations elicited a sequence of [Ca2+]i spikes, provided there was a short incubation at 5 mM K+ between the depolarizations. A prior K+ depolarization almost abolished the 45Ca2+ uptake response to K+ depolarization. The inhibition of exchange activity by a prior K+ depolarization required external Ca2+ and was completely reversible. A prior incubation with angiotensin II, platelet-derived growth factor, or ionomycin also inhibited exchange activity. Moderate [Ca2+]i increases probably feedback inhibit Ca2+ influx via the exchanger by a kinetic mechanism. Inactivation of the exchanger, together with Ca2+ extrusion or sequestration, causes the rapid decrease in [Ca2+]i from the peak evoked by depolarization.
Increase of apamin receptors in skeletal muscle induced by colchicine: possible role in myotonia
Authors: Behrens M. I. , Vergara C.
Published: December 2017

We have shown an increase of apamin receptors in rat skeletal muscle membranes following the application of colchicine to the sciatic nerve. 125I-apamin binding to partially purified membrane fractions was observed since day 4, reached a maximum around days 6-15, and was negligible at day 35 after the application of colchicine. Control muscles (nerves treated with buffer solution) showed low binding values (11 fmol/mg protein). Maximal 125I-apamin binding values to partially purified muscle membranes of colchicine-treated rats (42 fmol/mg protein) were lower than those obtained in denervated muscle (95 fmol/mg protein). The affinity binding constant values were 37 (colchicine) and 95 pM (denervation). No signs of muscle denervation were observed on histological examination of the nerve submitted to colchicine treatment nor in the muscles innervated by it. Muscle tension developed by indirect stimulation was the same as in controls. We here show also that partially purified membranes of normal untreated muscles have measurable amounts of 125I-apamin binding (13 fmol/mg protein), similar to those obtained in control muscles. Electromyographic recordings of the muscles after colchicine treatment of the nerve showed abnormal repetitive electrical discharges, similar to myotonic discharges, that were present with a similar temporal course as the increase in apamin receptors. The myotonic-like discharges were suppressed by the topical application of apamin to the muscle, whereas the toxin had no effect on anthracene-9-carbolytic acid-induced myotonia. Our results suggest that a neurotrophic factor that travels by axonal flow is involved in the regulation of the expression of apamin receptors in skeletal muscle membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Muscle fatigue in the frog semitendinosus: role of the high-energy phosphates and Pi
Authors: Thompson L. V. , Fitts R. H.
Published: December 2017

The purpose of this study was to determine the concentration of ATP, phosphocreatine (PC), Pi, lactate, and glycogen in single frog skeletal muscle fibers and assess their role in the etiology of muscle fatigue. The frog semitendinosus (ST) muscle was fatigued, quick frozen at selected time points of recovery, and freeze-dried, and single fibers were dissected, weighed, and assayed for ATP, PC, lactate, Pi, and glycogen. The fatigue protocol reduced peak tetanic force (Po) to 8.5% of initial, while ATP and PC decreased from 45.18 to 33.16 and 128.90 to 28.76 mmol/kg dry wt, respectively. Lactate and Pi increased from 29.36 to 100.84 and 33.04 to 142.50 mmol/kg dry wt, respectively. It is doubtful that the small decline in ATP limited cross-bridge force production. Although a significant correlation between the recovery of PC and Po was demonstrated (r = 0.994), the time period showing the fastest rate of force recovery coincided with little change in PC. A significant correlation was demonstrated between the recovery of both total and the H2PO4- form of Pi and Po. In conclusion, the results of this study are incompatible with the hypothesis that the high-energy phosphates (ATP and PC) mediate muscle fatigue. The large increase in Pi with stimulation and the high correlation between the recovery of both total and the H2PO4- form of Pi and Po support a role for Pi in the production of skeletal muscle fatigue.
Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia
Authors: Kemendy A. E. , Kleyman T. R. , Eaton D. C.
Published: December 2017

We used patch-clamp methods to examine the effects of depletion and readdition of aldosterone on single, highly selective, amiloride-blockable sodium channels in the A6 cell line. Single-channel characteristics changed little before 24 h of continuous aldosterone depletion, although there was some reduction in short-circuit current. Thereafter, apical sodium permeability, measured as product of channel number per patch and individual channel open probability (NPo), was reduced between five- and sevenfold, primarily due to a large decrease in channel mean open time. With about the same time course, short-circuit current also decreased approximately fivefold. Readdition of aldosterone to depleted cells produced an increase in NPo within 2 h, primarily through an increase in mean open time. After readdition, channel number per patch increased twofold compared with cells not hormone deprived, with a return to control levels between 24 and 48 h after continuous exposure. The increase in short-circuit current followed a similar time course. The primary effect of aldosterone appears to be modulation of the open time of channels continuously present in the apical membrane, rather than promotion of the appearance or disappearance of channels from the membrane. In particular, it cannot be demonstrated statistically that aldosterone removal reduces the number of channels per patch, and there may actually be up to a twofold increase after a long period of aldosterone depletion.
Immunolocalization of chloride-transporting membrane vesicles in tracheal epithelial cells
Authors: Dubinsky W. P. , Preston C. L. , Calenzo M. A. , White G. J. , Decker E. R.
Published: December 2017

A membrane fraction eluted from a phenyl Sepharose column (MPS) was isolated from renal cortex and bovine tracheal epithelia that is enriched in a single type of Cl- channel [C. L. Preston, M. A. Calenzo, and W. P. Dubinsky. Am. J. Physiol. 263 (Cell Physiol. 32): C879-C887, 1992]. A 200-kDa membrane protein that copurifies with and appears to be specific to this fraction was purified and used to raise antisera for immunological characterization of these membranes. The antisera reacted in immunoblots with a 200-kDa protein in homogenates of bovine trachea, kidney, pancreas, lung, and intestine. There was also cross-reactivity with a 200-kDa protein in immunoblots in rat stomach, pancreas, and lung. There was no cross-reaction with rat skeletal muscle, cardiac muscle, or aorta. Thus this protein appears to be preferentially enriched in epithelial tissues. Examination of each major fraction during the purification of MPS membranes from trachea shows no enrichment of the 200-kDa protein in plasma, mitochondrial, or nuclear membrane fractions. The only significant enrichment was observed in MPS that is purified by hydrophobic chromatography. In frozen sections, antisera and monospecific immunoaffinity-purified antibodies localize the protein primarily to the apical domain of tracheal columnar epithelial cells with small punctate structures throughout the cytoplasmic compartment.
Alpha 1-adrenergic stimulation of Na-H exchange in cardiac myocytes
Authors: Wallert M. A. , Frohlich O.
Published: December 2017

The activation of Na-H exchange in adult rat heart myocytes was characterized in response to a phorbol ester (phorbol 12-myristate 13-acetate) and an alpha 1-adrenergic agonist [6-fluoronorepinephrine (6F-NE)]. Transport activation was assessed by determining the initial rate with which intracellular pH (pHi) was returned from an acid pulse and by following changes in steady-state pHi; pHi was determined by a pH-sensitive fluorescent dye. Both agonists shifted the intracellular pH dependence of Na-H exchange by 0.10-0.15 pH units in the alkaline direction. This shift was prevented by the presence of sphingosine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), inhibitors of protein kinase C. The agonists also alkalinized pHi at steady state. The alkalinization by 6F-NE was blocked by prazosin and H-7. This indicates that the adrenergic stimulation of cardiac Na-H exchange is mediated by an alpha 1-adrenergic mechanism and very likely involves the activation of protein kinase C.
Modulation of Na-H exchange activity by angiotensin II in opossum kidney cells
Authors: Jourdain M. , Amiel C. , Friedlander G.
Published: December 2017

Angiotensin II (ANG II) was shown to modulate transport in the renal proximal tubule through both inhibition of adenylate cyclase and protein kinase C (PKC) activation. We evaluated the effects of ANG II on adenosine 3',5'-cyclic monophosphate (cAMP) content and Na-H exchange activity (amiloride-sensitive Na influx) in two strains of opossum kidney (OK) cells originating from different sources, OK-VD and OK-RR cells. In OK-VD cells, ANG II inhibited basal and parathyroid hormone (PTH)-induced cAMP generation in a pertussis toxin-sensitive manner and reversed PTH inhibition of Na-H exchange. These effects of ANG II were prevented by PD 123319, a selective nonpeptide antagonist of AT2 receptors. In contrast, DuP 753, which antagonizes selectively AT1 receptors, had no effect. In OK-RR cells, ANG II had no effect on cAMP content and decreased Na-H exchange activity. The effect of ANG II persisted in the presence of PTH but was abolished by PKC downregulation and by DuP 753, but not by PD 123319. In conclusion, two types of ANG II receptors, coupled to distinct signaling pathways, were expressed independently in OK cells originating from two different sources and mediated opposite effects of ANG II on Na-H exchange activity. Those models provide a powerful tool for studying the intracellular steps involved in the tubular effects of ANG II and to evaluate the effect of pharmacological inhibitors of ANG II binding to its receptors.
K-Cl cotransport in rabbit red cells: further evidence for regulation by protein phosphatase type 1
Authors: Starke L. C. , Jennings M. L.
Published: December 2017

We have examined inhibition of swelling-induced K-Cl cotransport in rabbit red blood cells by calyculin A, a potent serine-threonine protein phosphatase inhibitor, to determine whether transport is regulated by phosphatase type 1 or type 2A. Calyculin A blocks K(Rb) influx [half-maximal inhibitory concentration (IC50) = 3-6 nM] 10 times more potently than a second phosphatase inhibitor, okadaic acid (IC50 = 40 nM), consistent with earlier pharmacological studies showing that calyculin A inhibits phosphatase type 1 10 times more effectively than does okadaic acid. Calyculin A always inhibits Rb influx when added either before or after cell swelling, indicating that the phosphatase must operate continually to first activate and then maintain high transport rates in swollen cells. Similarly, N-ethylmaleimide (NEM) fails to stimulate K-Cl cotransport only when added to cells pretreated with calyculin A. Therefore, like cell swelling, activation of K-Cl cotransport by NEM involves a phosphatase sensitive to calyculin A. We conclude that cell swelling and NEM activate K-Cl cotransport via a net dephosphorylation that appears to involve protein phosphatase type 1.
Reconstitution of abnormalities in the malignant hyperthermia-susceptible pig ryanodine receptor
Authors: Shomer N. H. , Louis C. F. , Fill M. , Litterer L. A. , Mickelson J. R.
Published: December 2017

Malignant hyperthermia-susceptible (MHS) pigs homozygous for the Cys615 ryanodine receptor allele demonstrate altered sarcoplasmic reticulum (SR) ryanodine binding and Ca2+ release channel regulatory properties when compared with normal pigs homozygous for the Arg615 allele. While solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified MHS and normal ryanodine receptors had a similar dissociation constant (Kd) for ryanodine, maximum binding, and Ca2+ concentration for half-maximal stimulation and inhibition of ryanodine binding (Ca2+(0.5)); however, after reconstitution into proteoliposomes, the purified MHS and normal receptors had Kd values for ryanodine of 75 and 150 nM, respectively, which were significantly different. The purified MHS and normal porcine ryanodine receptors also had similar single-channel Cs+ conductance, optimal cis-Ca2+ for channel opening, and cis-Ca2+(0.5) for channel activation. Significantly, at inactivating levels of cis-Ca2+ (> 0.1 mM), MHS channels had a greater open probability, a higher cis-Ca2+(0.5) for inhibition of channel opening (250 vs. 75 microM for MHS and normal, respectively), longer mean open times, and shorter mean closed times than did normal channels. We conclude that the mutation at residue 615 causes a detectable alteration in ryanodine receptor/Ca2+ channel activity and thus may represent the primary defect responsible for the altered SR Ca2+ regulation characteristic of MHS porcine muscle.
Adaptation of muscle to creatine depletion: effect on GLUT-4 glucose transporter expression
Authors: Ren J. M. , Semenkovich C. F. , Holloszy J. O.
Published: December 2017

Feeding rats beta-guanidinopropionic acid (beta-GPA), a creatine analogue, results in depletion of creatine and phosphocreatine and induces increases in mitochondrial oxidative enzymes and hexokinase in skeletal muscle. Comparisons of different muscle types and studies of the adaptation to exercise suggest that 1) the levels of the insulin-responsive glucose transporter (GLUT-4), mitochondrial oxidative enzymes, and hexokinase may be coregulated and 2) GLUT-4 content can determine maximal glucose transport activity in muscle. To further evaluate these possibilities, we examined the effects of feeding rats 1% beta-GPA in their diet for 6 wk on muscle GLUT-4 expression and glucose transport activity. beta-GPA feeding induced 40-50% increases in cytochrome c concentration, citrate synthase activity, and hexokinase activity in plantaris muscle. GLUT-4 protein concentration was increased approximately 50% in plantaris and epitrochlearis muscles, while GLUT-4 mRNA was increased approximately 40% in plantaris muscles of beta-GPA-fed rats. Glucose transport activity maximally stimulated by insulin was increased in parallel with GLUT-4 protein concentration in the epitrochlearis. These results provide evidence that chronic creatine depletion increases GLUT-4 expression by pretranslational mechanisms. They support the hypothesis that the levels of mitochondrial enzymes, hexokinase, and GLUT-4 protein are coregulated in striated muscles. They also support the concept that the GLUT-4 content of a muscle determines its maximal glucose transport activity when the signaling pathways for glucose transport activation are intact.
Adenosine stimulation of AMP deaminase activity in adult rat cardiac myocytes
Authors: Hu B. , Altschuld R. A. , Hohl C. M.
Published: December 2017

Using an in situ assay for analyzing AMP deaminase activity in isolated adult rat ventricular myocytes, we have shown that IMP production is stimulated approximately twofold in cardiac cells incubated with 10 microM adenosine. This effect of adenosine was not blocked by the adenosine A1-receptor antagonist 8-cyclophenyl-1,3-dipropylaxanthine (0.01-1 microM) except at a concentration (100 microM) that may inhibit adenosine transport. Similarly, in situ AMP deaminase activity was not enhanced by treatment with the specific adenosine A1-receptor agonists N6-phenylisopropyl adenosine or cyclopentyladenosine, nor was it sensitive to prior treatment of cells with pertussis toxin. The nucleoside transport blockers S-4-nitrobenzyl-6-thioinosine, dipyridamole, and papaverine inhibited adenosine-induced increases in IMP production by 75-85%, suggesting an intracellular site of action. Modulation of enzyme activity via the transmethylation pathway could not be implicated since incubation of cardiac cells under conditions known to elevate intracellular S-adenosyl-L-homocysteine had no demonstrable effect on AMP deaminase. Furthermore, a direct allosteric effect of adenosine on the partially purified rat cardiac enzyme was not observed. The results indicate that intracellular adenosine modulates rat cardiac AMP deaminase by an unknown mechanism.
PDGF-induced mitogenic signaling is not mediated through protein kinase C and c-fos pathway in VSM cells
Authors: Sharma R. V. , Bhalla R. C.
Published: December 2017

This study examines the role of protein kinase C (PKC) in platelet-derived growth factor (PDGF)-induced vascular smooth muscle (VSM) cell proliferation and initial signaling events. A 24-h pretreatment of VSM cells with 200 nM phorbol 12-myristate 13-acetate (PMA) completely abolished immunologically reactive PKC activity. Depletion of PKC activity from VSM cells did not attenuate PDGF-stimulated [3H]thymidine incorporation compared with control cells. Similarly, acute activation of PKC by treatment with 200 nM PMA for 10 min had no effect on PDGF-mediated [3H]thymidine incorporation. Both PMA and PDGF increased c-fos induction to the same magnitude; however, treatment with PMA did not induce DNA synthesis in these cells. In PKC-depleted cells PDGF-mediated c-fos induction was reduced by 50-60%, while DNA synthesis in response to PDGF stimulation was not reduced. PKC depletion did not alter PDGF-stimulated increase in cytosolic calcium levels, 125I-PDGF binding, or receptor autophosphorylation. On the basis of these results, we conclude that PKC activation and c-fos induction do not play a significant role in PDGF-mediated mitogenesis in VSM cells.
Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic β-cells
Authors: Sjöholm Aring;.
Published: December 2017

Pages C116 and C118: Å. Sjőholm. “Differential effects of cytokines on long-term mitogenic and secretory responses of fetal rat pancreatic β-cells.” Page C116, Table 1, the units for [Glucose] should read mM, and page C118, Table 3, the units for [IFN-ggr] should read U/ml, as shown in the corrected tables. (The errors were inadvertently introduced by the printer at the final stage of proof.)

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G protein activation inhibits amiloride-blockable highly selective sodium channels in A6 cells
Authors: Ohara A. , Matsunaga H. , Eaton D. C.
Published: December 2017

Single-channel methods were used to examine the regulation of amiloride-blockable highly selective sodium channels by guanine nucleotide-binding proteins (G proteins). A6 cells (a renal cell line derived from Xenopus laevis kidney) were cultured on permeable collagen films, and patch recordings were made from the apical membranes of confluent cells. The predominant channel in the apical membranes is a highly selective, 4-pS, amiloride-blockable sodium channel (the Na(+)-to-K+ permeability ratio is > 30). In inside-out patches, application to the cytosolic surface of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which deactivates G proteins, increased sodium channel activity. GDP beta S produced a sevenfold increase in channel activity. In contrast, GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) decreased sodium channel activity to about one-twentieth of the untreated value. The effect of GTP (but not GTP gamma S) was reversible. In cell-attached patches, a 3- to 4-h exposure of the apical membrane to pertussis toxin (PTX) increased the mean open time of sodium channels approximately 2.7 times and the open probability approximately 1.6-fold, but pretreatment of apical membranes with cholera toxin (250 ng/ml) for 3-4 h had no effect on open probability or mean open time. These results imply that a PTX-sensitive G protein regulates amiloride-blockable highly selective sodium channels in the apical membranes of A6 cells and that the G protein in a GTP-bound, activated state inhibits sodium channel activity.
ANG II- or AVP-induced increases in protein synthesis are not dependent on autocrine secretion of PDGF-AA
Authors: Stouffer G. A. , Shimizu R. T. , Turla M. B. , Owens G. K.
Published: December 2017

Previous studies have demonstrated that angiotensin II (ANG II) and arginine vasopressin (AVP) stimulate increased protein synthesis and cellular hypertrophy in cultured rat aortic smooth muscle cells (SMC). The aim of this study was to explore the hypothesis that ANG II- and/or AVP-induced increases in protein synthesis are mediated by autocrine secretion of platelet-derived growth factor (PDGF)-AA. Results demonstrated that ANG II or AVP increased expression of PDGF-A, but not -B, chain mRNA. Additionally, conditioned media from ANG II- and AVP-treated SMC had increased mitogenic activity for Swiss 3T3 cells, which could be inhibited with a neutralizing antibody to PDGF-AA. However, PDGF-AA-neutralizing antibodies did not inhibit ANG II- or AVP-induced increases in protein synthesis, and exogenous PDGF-AA did not stimulate increased protein synthesis. Furthermore, no PDGF-alpha receptors were evident based on 125I-labeled PDGF-AA binding studies. In summary, results indicate that ANG II- or AVP-induced increases in protein synthesis were not dependent on autocrine secretion of PDGF-AA.
Spatial arrangement and metabolic capacity of fiber types in self-reinnervated cat muscle
Authors: Nemeth P. A. , Cope T. C. , Kushner S. , Nemeth P. M.
Published: December 2017

The recovery potential of skeletal muscle was explored by examining cat muscle between 10 and 33 mo after complete transection and immediate surgical reunion of its own nerve. Biochemical analysis of single muscle fibers showed that the activities of key enzymes in energy metabolism (malate and lactate dehydrogenase and adenylokinase) were similar to normal for their respective fiber types, suggesting that incomplete recovery of the ability to sustain submaximal contraction in reinnervated muscles (T.C. Cope, C.B. Webb, and B.R. Botterman. J. Neurophysiol. 65: 648-656, 1991) is explained in some other way. Two independent statistical procedures for assessing the randomness of adjacencies of histochemically identified fiber types showed type grouping in some areas, but there were also many regions with randomly distributed fiber types. These findings demonstrate the potential for substantial recovery of both energy metabolism and dispersion of fiber types after self-reinnervation.
Calcitonin gene-related peptide stimulates active Na(+)-K+ transport in rat soleus muscle
Authors: Andersen S. L. , Clausen T.
Published: December 2017

Calcitonin gene-related peptide (CGRP) is found in a wide variety of tissues, including sensory and motor nerve endings in skeletal muscle. After intense electrical stimulation or K(+)-induced depolarization, CGRP can be released from nerve terminals and bound to receptors on sarcolemma. We show here that CGRP (rat and human) and salmon calcitonin stimulate 22Na extrusion and the influx of 86Rb and 42K in isolated rat soleus muscle. This leads to a pronounced (up to 56%) decrease in intracellular Na+, a minor increase in intracellular K+, and hyperpolarization. All these effects were blocked by ouabain or cooling, indicating that they reflect an acute stimulation of active electrogenic Na(+)-K+ transport. Capsaicin, which induces release of CGRP from sensory nerve endings, was found to exert similar effects on Na(+)-K+ transport. Various Na(+)-K+ pump-stimulating agents have been shown to counteract the inhibitory effect of a high extracellular concentration of K+ ([K+]o) on muscle contractility (4, 20). CGRP and capsaicin were likewise found to improve contractile performance of muscles inhibited by high [K+]o, and these effects were blocked by ouabain. CGRP might play a role in the maintenance of Na(+)-K+ gradients and excitability during intensive muscle work, known to be associated with an acute rise in the interstitial K+ concentration.
Inhibition of renal ornithine decarboxylase activity prevents kidney hypertrophy in experimental diabetes
Authors: Pedersen S. B. , Flyvbjerg A. , Richelsen B.
Published: December 2017

The selective ornithine decarboxylase (ODC) inhibitor difluoromethyl ornithine (DFMO) was used to investigate the role of polyamines in initial diabetic renal enlargement. ODC activity in kidneys from diabetic animals was increased (fivefold) 24 h after diabetes induction (P < 0.05), and throughout the study (7 days) the activity remained 2- to 3-fold elevated (P < 0.05). Insulin treatment normalized renal ODC activity, whereas DFMO treatment totally inhibited the kidney ODC activity. The kidney weight in diabetic rats was 21% higher than that of control rats (1,074 +/- 35 mg and 889 +/- 16 mg, P < 0.001). Insulin treatment normalized kidney weight (847 +/- 13 mg). Despite unaltered diabetic metabolic aberrations the kidney weight in DFMO-treated diabetic rats was normalized (911 +/- 7 mg). In conclusion, the ODC activity in diabetic kidneys undergoing hypertrophy was increased. Insulin treatment normalized both kidney weight and kidney ODC activity. Finally, selective inhibition of ODC activity by DFMO resulted in kidneys of normal size, despite unaltered diabetic metabolic aberrations. These findings support the hypothesis that polyamines play an important role in initial diabetic renal enlargement.
Correlation between magnesium and potassium contents in muscle: role of Na(+)-K+ pump
Authors: Dorup I. , Clausen T.
Published: December 2017

In young rats fed a Mg(2+)-deficient diet for 3 wk, Mg2+ and K+ contents in soleus and extensor digitorum longus muscles were significantly reduced and closely correlated. In isolated soleus muscles, Mg2+ depletion induced an even more pronounced loss of K+, and Mg2+ and K+ contents were correlated over a wide range (r = 0.95, P < 0.001). Extracellular Mg2+ (0-1.2 mM) caused no change in total or ouabain-suppressible 86Rb influx. After long-term incubation in Ca(2+)-Mg(2+)-free buffer with EDTA and EGTA, cellular Mg2+ and K+ contents were reduced by 35 and 15%, respectively, without any reduction in ATP and total or ouabain-suppressible 86Rb influx. In Mg(2+)-depleted muscles 42K efflux was increased by up to 42%, and repletion with Mg2+ produced a graded decrease. We conclude that Mg2+ and K+ contents are closely correlated in muscles Mg2+ depleted in vivo or in vitro and that neither extracellular nor moderate intracellular Mg2+ depletion affects total or Na(+)-K+ pump-mediated K+ influx. The reduced K+ content may rather be related to increased K+ efflux from the muscles.
Calcium-dependent regulation of cation transport in cultured human nonpigmented ciliary epithelial cells
Authors: Mito T. , Delamere N. A. , Coca-Prados M.
Published: December 2017

We performed 86Rb flux studies to examine Na-K-adenosinetriphosphatase (ATPase), Na-K-2Cl cotransporter, and potassium channel activity in an established cell line derived from human nonpigmented ciliary epithelium (ODM2). The elevation of intracellular calcium by A23187 (3 microM) or thapsigargin (200 nM) increased both ouabain-sensitive potassium (86Rb) uptake (Na-K-ATPase mediated) and ouabain-insensitive potassium (86Rb) uptake. The ouabain-insensitive component could be inhibited substantially by bumetanide (0.1 mM), suggesting the involvement of a Na-K-2Cl cotransporter. The increase of potassium (86Rb) uptake caused by thapsigargin could be prevented by the intracellular calcium buffer 1,2-bis(2-amino-phenoxy)ethane N,N,N',N'-tetraacetic acetoxymethyl ester (BAPTA/AM); in BAPTA/AM-treated cells, the thapsigargin stimulation of the bumetanide-sensitive portion of 86Rb uptake was abolished. After A23187 (5 microM), the 86Rb efflux rate was significantly increased; the increase could be blocked partially by quinidine (0.1 mM) and partially by bumetanide, suggesting that potassium channels and the Na-K-2Cl cotransporter contribute to the effect. We propose that the cell potassium loss after activation of quinidine-sensitive potassium channels is involved in the calcium-induced activation of Na-K-ATPase because 0.1 mM quinidine and 100 mM external potassium both markedly inhibited the A23187-induced increases of the ouabain-sensitive component of potassium (86Rb) uptake. Calcium-induced stimulation of the Na-K-2Cl cotransporter may not be linked to channel activation.
Regulation of the mitochondrial ATP-Mg/Pi carrier in isolated hepatocytes
Authors: Dransfield D. T. , Aprille J. R.
Published: December 2017

This study investigated the cellular regulation of net adenine nucleotide movements between the cytoplasm and mitochondria in intact cells. Such movements are presumed to occur primarily by ATP-Mg exchange with Pi via the mitochondrial ATP-Mg/Pi carrier. Vasopressin, A23187, and thapsigargin all elevate intracellular free [Ca2+] and all caused dose-dependent increases in the mitochondrial adenine nucleotide content (29, 63, and 39%, respectively). Phorbol 12-myristate 13-acetate had no effect. The effect of vasopressin was abolished when cytoplasmic [ATP] was decreased (by 43%) and [Pi] was increased (3-fold) by addition of carboxyatractyloside. The effect of thapsigargin was abolished by addition of xylulose to deplete cytoplasmic [ATP] (by 50%) and [Pi] (> 4-fold). The results indicate that in intact cells Ca2+ activates the mitochondrial ATP-Mg/Pi carrier to enable changes in the subcellular distribution of adenine nucleotides and that the relative [ATP] and [Pi] gradients govern the direction and magnitude of net adenine nucleotide movements between the cytoplasm and mitochondria.
ATP synthase activity is required for fructose to protect cultured hepatocytes from the toxicity of cyanide
Authors: Snyder J. W. , Pastorino J. G. , Thomas A. P. , Hoek J. B. , Farber J. L.
Published: December 2017

The contributions of the loss of the mitochondrial membrane potential (MMP) and a depletion of ATP to the genesis of lethal injury were evaluated in the killing of cultured hepatocytes by cyanide (CN). The glycolytic production of ATP from fructose (Fru) maintained the MMP and prevented the killing by CN. Inhibition of the mitochondrial ATP synthase by 0.1 micrograms/ml oligomycin (Oligo) reduced ATP stores at the same rate and to the same extent as did 1 mM CN. With Oligo there was no loss of the MMP, and the hepatocytes maintained viability over the 6 h during which CN killed all of the cells. Oligo had no effect on the rate of killing by CN. However, Oligo reversed the protective effect of Fru on CN-induced killing, a result that correlated with the loss of MMP but not with the depletion of ATP. Neither Fru nor Oligo affected the intracellular acidosis achieved with CN alone. Fru also prevented toxicity of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), a result that correlated with the preservation of MMP. Oligo potentiated the toxicity of CCCP. It is concluded that a functioning mitochondrial ATP synthase is required for the production of ATP from Fru to prevent the killing of hepatocytes by CN. The extent of killing correlated closely with changes in the MMP but not with changes in the content of ATP.
Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells
Authors: Rich A. , Kenyon J. L. , Hume J. R. , Overturf K. , Horowitz B. , Sanders K. M.
Published: December 2017

Experiments were performed to identify and characterize the types of calcium channels that regulate inward calcium current in canine colonic smooth muscle. Freshly dispersed smooth muscle cells from the circular layer of the canine proximal colon were used. Single-channel currents were measured with 80 mM Ba2+ as the charge carrier. Small-conductance (10 +/- 2 pS, EBa = 46 +/- 11 mV, n = 9) and large-conductance (21 +/- 1 pS, EBa = 52 +/- 3 mV, n = 19) single-channel currents were observed during depolarizing voltage steps positive to -30 mV. Both types of single-channel currents were inhibited by the addition of 10(-6) M nifedipine to the bath solution. The smaller current was infrequently observed and therefore was not further characterized. Open probability (P(o)) of the larger current amplitude was strongly dependent on voltage. Activation curves were well described by a Boltzmann function with half activation occurring at 4 mV, and a 5-mV increase in membrane potential resulted in an e-fold increase in P(o). BAY K 8644 (1 microM) shifted the activation curve to the left while nifedipine (1 microM) resulted in a right shift. Molecular analysis showed that only the C class of Ca2+ channel alpha 1-subunit is expressed in this tissue. Furthermore, only a single splice variant (rbc-II) was observed. The results suggest that a single class of dihydropyridine-sensitive calcium channels regulates inward calcium current in canine colonic smooth muscle cells.
Increase in cell size underlies neuron-specific temperature acclimation in Aplysia
Authors: Treistman S. N. , Grant A. J.
Published: December 2017

Aplysia californica with similar genetic and environmental backgrounds were raised under mariculture conditions at either 10 or 20 degrees C. Determinations of cell surface area using capacitance and visual measurements indicated that there was an increase in the surface area of identified neurons. However, this increase was cell specific, occurring only in certain identified neurons. The amplitude of the early potassium current (IA) was monitored in identified neurons from the cold- and warm-reared groups during acute changes in temperature. The response to acute temperature change differed in the two groups, indicative of partial temperature acclimation of IA. Cold rearing shifted the acute temperature-response curve for IA amplitude to color temperatures, compared with the temperature-response curve for animals reared at warmer temperatures. This acclimation was related to the increase in surface area previously noted. Cells showing an increase in IA amplitude in cold-reared animals also showed an increase in membrane surface area, presumably associated with an increased number of A-channels, while channel density was unchanged. There was a small but significant shift in the steady-stage voltage dependency of inactivation, whereas kinetic properties were unaffected by rearing temperature.
Stable expression of the cardiac sodium-calcium exchanger in CHO cells
Authors: Pijuan V. , Zhuang Y. , Smith L. , Kroupis C. , Condrescu M. , Aceto J. F. , Reeves J. P. , Smith J. B.
Published: December 2017

A line of Chinese hamster ovary (CHO) cells called CK1.4 was produced by transfection with the gene for the bovine cardiac Na(+)-Ca2+ exchanger. CK1.4 cells stably expressed substantial exchange activity and exchanger protein as shown by immunoprecipitation. Exchange activity was quantified as 45Ca2+ influx that depended on both increasing intracellular Na+ and lowering the concentration of external Na+. Replacing external Na+ with K+ slightly increased 45Ca2+ uptake by CK1.4 cells with basal Na+ and greatly increased 45Ca2+ uptake by Na(+)-loaded cells. Neither exchange activity nor exchanger protein was detected in the nontransfected parental line. By contrast to CK1.4 cells, replacing external Na+ with K+ decreased 45Ca2+ uptake in the nontransfected cells whether or not they were Na+ loaded. Changes in cytosolic free Ca2+ determined with fura-2 were consistent with the 45Ca2+ uptake data. Analysis of poly(A)(+)-RNA by Northern blot confirmed that CK1.4 cells, but not the parental line, expressed the exchangerx. Expression of the exchanger was also observed in aortic myocytes and a renal epithelial cell line (LLC-MK2) but not in other lines of renal epithelial cells (MDCK, LLC-PK1) or human dermal fibroblasts. The cardiac exchanger produced substantial 45Ca2+ efflux from CK1.4 cells in response to hormone-evoked release of stored Ca2+. CK1.4 cells are an attractive model for studies of the regulation of the cardiac exchanger because they stably express sufficient exchanger for biochemical and immunological analysis.