Physiological basis of a steady endogenous current in rat lumbrical muscle

In an attempt to determine the mechanism by which rat skeletal muscle endplates generate a steady outward current, we measured the effects of several drugs (furosemide, bumetanide, 9-anthracene carboxylic acid [9-AC]) and changes in external ion concentration (Na+, K+, Cl-, Ba++) on resting membrane potential (Vm) and on the steady outward current. Each of the following treatments caused a 10-15-mV hyperpolarization of the membrane: replacement of extracellular Cl- with isethionate, addition of furosemide or bumetanide, and addition of 9-AC. These results suggest that Cl- is actively accumulated by the muscle fibers and that the equilibrium potential of Cl- is more positive than the membrane potential. Removal of external Na+ also caused a large hyperpolarization and is consistent with evidence in other tissues that active Cl- accumulation requires external Na+. The same treatments greatly reduced or abolished the steady outward current, with a time course that paralleled the changes in Vm. These results cannot be explained by a model in which the steady outward current is assumed to arise as a result of a nonuniform distribution of Na+ conductance, but they are consistent with models in which the steady current is produced by a nonuniform distribution of GCl or GK. Other treatments (Na+-free and K+-free solutions, and 50 microM BaCl2) caused a temporary reversal of the steady current. Parallel measurements of Vm suggested that in none of these cases did the electrochemical driving force for K+ change sign, which makes it unlikely that the steady current arises as a result of a nonuniform distribution of GK. All of the results, however, are consistent with a model in which the steady outward current arises as a result of a nonuniform distribution of Cl- conductance, with GCl lower near the endplate than in extrajunctional regions.     

Development of a steady electric current in neonatal rat lumbrical muscle

We have used a vibrating probe and intracellular recording techniques to study the development of a steady electric current generated by rat lumbrical muscle. In adult animals, previous work has revealed a steady outward current generated at the end plate region. In the present study, we show that at birth muscles generate a steady inward, not outward current. The inward current declines with age, disappearing about 5 days after birth. At about the same time, the steady outward current appears, and reaches adult amplitude by 2-3 weeks after birth. The two currents are generated by completely different mechanisms. The inward current is blocked by alpha-bungarotoxin and apparently results from activation of acetylcholine-gated channels at the end plate. The outward current, on the other hand, is not affected by alpha-bungarotoxin but is blocked by agents which interfere with chloride movements across the membrane, as in the adult.     

Identification of an endogenous activator of calpain in rat skeletal muscle

An additional component of the regulatory system of rat skeletal muscle calpain has been identified. It exerts a potent activating effect on calpain activity and is a heat stable small molecular weight protein. Of the two calpain isozymes present in muscle, the activator is specific for calpain II, being uneffective with calpain I. It promotes activation of the proteinase by reducing 50 fold, from 1 mM to of 20 microM, the requirement of Ca2+ for maximum catalytic activity of the proteinase. However in the presence of the activator calpain II expresses a consistent fraction of the maximum activity even at significantly lower concentrations of Ca2+ (below 5 microM Ca2+). The activator effect follows kinetics that are consistent with the presence of specific binding sites on the calpain molecules. The activator not only removes in a dose dependent fashion the inhibition of calpain by calpastatin, but also prevents inhibition of the proteinase upon the addition of calpastatin. Competition experiments revealed that the proteinase contains distinct sites for the activator and the inhibitor, and that both ligands can bind to calpain with the formation of an almost fully active ternary complex.     

Contractions of dysgenic skeletal muscle triggered by a potentiated, endogenous calcium current

The dihydropyridine (DHP) receptor of normal skeletal muscle is hypothesized to function as the voltage sensor for excitation-contraction (E-C) coupling, and also as the calcium channel underlying a slowly activating, DHP-sensitive current (termed ICa-s). Skeletal muscle from mice with muscular dysgenesis lacks both E-C coupling and ICa-s. However, dysgenic skeletal muscle does express a small DHP-sensitive calcium current (termed ICa-dvs) which is kinetically and pharmacologically distinct from ICa-s. We have examined the ability of ICa-dys, or the DHP receptor underlying it, to couple depolarization and contraction. Under most conditions ICa-dys is small (approximately 1 pA/pF) and dysgenic myotubes do not contract in response to sarcolemmal depolarization. However, in the combined presence of the DHP agonist Bay K 8644 (1 microM) and elevated external calcium (10 mM), ICa-dys is strongly potentiated and some dysgenic myotubes contract in response to direct electrical stimulation. These contractions are blocked by removing external calcium, by adding 0.5 mM cadmium to the bath, or by replacing Bay K 8644 with the DHP antagonist (+)-PN 200-110. Only myotubes having a density of ICa-dys greater than approximately 4 pA/pF produce detectible contractions, and the strength of contraction is positively correlated with the density of ICa-dys. Thus, unlike the contractions of normal myotubes, the contractions of dysgenic myotubes require calcium entry. These results demonstrate that the DHP receptor underlying ICa-dys is unable to function as a "voltage sensor" that directly couples membrane depolarization to calcium release from the sarcoplasmic reticulum.     

Externalization of an endogenous chicken muscle lectin with in vivo development

Chicken-lactose-lectin-I (CLL-I), an endogenous lectin that is developmentally regulated in embryonic muscle, was localized by immunohistochemical techniques in tissue samples taken at various stages of in vivo development and in primary muscle cultures. Lectin, which was diffusely distributed in myoblasts, became localized in myotubes in a distribution similar to that of the sarcoplasmic reticulum and T tubules. Later in development, lectin was predominantly extracellular. This sequence suggests that externalization may have occurred by migration in the T tubules, which are continuous with the extracellular space, although alternative explanations are possible. Only traces of lectin were found in the adult. These studies did not reveal the function of CLL-I in muscle development. However, we infer that it acts by organizing complementary glycoconjugates in the intracellular tubular network, on the muscle surface, and/or in extracellular materials.     

Isolation of an endogenous clonidine-displacing substance from rat brain

An endogenous substance which specifically displaces clonidine, yohimbine and rauwolscine from rat brain alpha 2-adrenergic receptors, has been isolated. The new compound, designed clonidine-displacing-substance (CDS), has been partially purified by ion exchange chromatography, zone electrophoresis and high performance liquid chromatography (HPLC). CDS binds specifically to alpha 2-adrenergic receptors by competing with either alpha 2-adrenergic agonists or alpha 2-antagonists, but has no effect on the specific binding of [3H]prazosin to alpha 1-adrenergic receptors in rat brain membranes. In the course of isolation, CDS was shown to be neither the endogenous neurotransmitter (-)norepinephrine (NE) nor the guanyl nucleotide GTP which lowers the specific binding of alpha 2-agonists to the alpha 2-adrenergic receptors.     

Occurrence of an endogenous inhibitor of platelet-activating factor in rat liver

Hepatocytes obtained from livers derived from fed rats perfused with a collagenase-containing mixture were found to contain significant levels of platelet-activating factor activity as isolated by Silica Gel G thin layer chromatography. However, when soybean trypsin inhibitor was included in the collagenase-containing perfusion medium for hepatocyte preparation, platelet-activating factor activity could not be detected on Silica Gel G chromatograms. Examination of the lipids extracted from freeze clamped perfused rat livers revealed low, but detectable, levels of platelet-activating factor. Further investigation of these observations indicated that a lipid-like inhibitor was present in freeze-clamped perfused livers as well as in hepatocytes isolated in the presence of soybean trypsin inhibitor. In each instance platelet-activating factor and this newly discovered inhibitor, which comigrated at the same RF value on Silica Gel G thin layer chromatography plates, could be separated by further chromatography on high performance thin layer plates. The present study shows that platelet-activating factor is present in unstimulated liver and that its detection is masked by an endogenous lipid-like inhibitor.     

Presence of an endogenous inhibitor of dipeptidyl carboxypeptidase in rat brain

Whole rat brain dipeptidyl carboxypeptidase (E.C. 3.4.15.1) was heterogeneously distributed among 10 brain regions studied. In corpus striatum, the enzyme was enriched in the P₂ pellet, a subfraction high in myelin and nerve terminals. Using [³H]benzoylphenylalanyl-alanyl-proline as a substrate, dipeptidyl carboxypeptidase manifested a different anion requirement than has been reported for other substrates. Endogenous inhibitors of the enzyme were found in corpus striatum and could be removed by dialysis or Sephadex G25 chromatography. Boiled striatal cytosol inhibited membrane-bound enzyme activity in a concentration-dependent manner and confirmed the presence of an endogenous soluble, heat-stable inhibitor in rat brain. The inhibitor apparently could be degraded by a component of the striatal P₂ membranes. The inhibitor was present in all 10 brain regions studied and its levels did not appear to be related to the specific activity of dipeptidyl carboxypeptidase. Potential mechanisms for biological regulation of dipeptidyl carboxypeptidase activity are discussed in light of the above findings.     

Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout

Hydrogen sulfide (H(2)S) is an endogenous vasodilator in mammals, but its presence and function in other vertebrates is unknown. We generated H(2)S from NaHS and examined the effects on isolated efferent branchial arteries from steelhead (stEBA) or rainbow (rtEBA) trout. H(2)S concentration was measured colorimetrically (CM) and with ion-selective electrodes (ISE) in rainbow trout plasma. NaHS produced a triphasic response consisting of a relaxation (phase 1), constriction (phase 2), and relaxation (phase 3) in both unstimulated vessels and in stEBA precontracted with carbachol (Carb). Phase 1 and phase 3 in stEBA were decreased and phase 2 increased in unstimulated vessels by K(+)(ATP) channel inhibition (glibenclamide), or a cocktail of inhibitors of cyclooxygenase, lipoxygenase, and cytochrome P-450 (indomethacin, esculetin, and clotrimazole). Inhibition of soluble guanylate cyclase with ODQ o NS-2028 inhibited phase 3 in stEBA, although NaHS decreased cGMP production by tEBA. stEBA phase 2 contractions were partially inhibited by the myosin light chain kinase inhibitor, ML-9, but unaffected by L-type calcium channel inhibition (methoxyverapamil), whereas contraction in tEBA was partially inhibited by nifedipine or removal of extracellular calcium. Phase 3 relaxations were more pronounced in stEBA precontracted with Carb and no epinephrine (NE) than those cont acted by KCl or K(2)SO(4). stEBA phase 2 and phase 3 responses were dose dependent (EC(50) = 1.1 +/- 1.2 x 10(-3) M and 6.7 +/- 0.9 x 10(-5) M, respectively; n = 7). NaHS was also vasoactive in steelhead bulbus arteriosus, celiac mesenteric arteries, and anterior cardinal veins. Rainbow trout plasma sulfide concentration was 4.0 +/- 0.3 x 10(-5) M, n = 4 (CM) and 3.8 +/- 0.4 x 10(-5) M, n = 9 (ISE); similar to phase 3 EC(50). Because NaHS has substantial vasoactive effects at physiological plasma concentrations, we propose that its soluble derivative, H(2)S, is a tonically active endogenous vasoregulator in trout.     

Cyclic GMP-dependent phosphorylation of an endogenous protein from rat heart

An endogenous substrate protein (M_r = 70K) for cGMP-dependent protein kinase (G-PK) was found in the cytosol of the rat heart. This protein was specifically phosphorylated by G-PK, with a K_a for cGMP of 0.08 μM, compared with that for cAMP of 2 μM. At least two substrate proteins (M_r = 110K and 26K) specific for cAMP-dependent protein kinase (A-PK) were also noted. The finding in heart of an endogenous substrate protein for G-PK distinguishable from those for A-PK provides additional evidence suggesting an independent role for cGMP in the regulation of cardiac function.     

Comparison of an endogenous protein kinase C substrate in rat aorta with rat brain MARCKS

We have compared the properties of a rat aorta-derived protein kinase C substrate (p75) with those of 80 kDa kinase C substrates from rat brain (MARCKS) and rabbit aorta (p80). Rat aortic p75 appeared to be closely related to rat brain MARCKS on the basis of: solubility in perchloric acid and trichloroacetic acid, heat stability, isoelectric point (pI 4.2), overall V8 protease phosphopeptide map, and immunocrossreactivity with an antibody directed against the N-terminal domain of MARCKS. However, p75 could be distinguished from rat brain MARCKS and from the rabbit aorta-derived p80 on the basis of its consistently more rapid electrophoretic mobility in SDS-containing gels, and in terms of a unique proteolytic phosphopeptide found in MARCKS but not in aortic p75. We conclude that p75 probably belongs to the family of protein kinase C substrates represented by MARCKS, and that differences in post-translational processing (glycosylation) or mRNA processing may account for the unique properties of the p75 protein in rat aortic tissue.Abbreviations p75 75,000 Da protein - MARCKS Myristoylated Alanine-Rich C Kinase Substrate     

Blockers of potassium current and resting membrane potential in rat muscle fibers.

Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.     

Characteristics of the second inward current in cells isolated from rat ventricular muscle

The second inward current (Isi) in single cells isolated from ventricular muscle of adult rat hearts was measured in response to step depolarizations under voltage-clamp conditions. The major ion carrying this current was Ca, and Isi was reduced or abolished by Mn, Ni, Cd, nifedipine, nimodipine and D600. Sr and B could substitute for Ca as charge carriers, and reduced the rate of apparent inactivation of Isi. These effects of Sr and Ba, together with the relation between the steady level of apparent inactivation and membrane potential in Ca containing solution, were taken as evidence that inactivation was at least in part dependent on internal Ca. The reduction of external Na to 11% of normal caused a reduction in peak Isi when Ca was present in the external solution, but did not reduce Isi when Ca was replaced by Sr. It therefore seems unlikely that Na is a major charge carrier for Isi under the conditions of our experiments. The time-to-peak and rate of apparent inactivation of Isi were faster than in previous studies that used multicellular preparations. Both the kinetics and peak amplitude of Isi were markedly dependent on temperature (Q10 close to 3). Contraction of the cells, which was monitored optically, was initiated within 3 ms of the peak Isi, reached a maximum level after approximately 40-50 ms, and was about 100 ms in duration.     

Active muscle fiber as an equivalent cardiac current generator

Equations are derived describing potentials due to an active muscle fiber in an infinite medium in terms of two surface integrals—one of the propagated action potential and the other of the membrane current density, both integrals being taken over the surface of the muscle. These equations are incorporated into an equivalent cardiac current generator in which the left ventricle (i.e. the current source) is represented by a three-dimensional wedge and the thorax (i.e. the volume conductor), by a homogeneous circular cylinder. Since this current generator expresses the body surface potentials in terms of the membrane current density and the membrane potential at any point on the surface of the electrically active muscle fiber, the calculated ECG can be correlated with theactual sources within the heart. This equivalent cardiac generator possesses many of the physical and physiological properties of cardiac muscle. The equations were evaluated numerically on a digital computer. The results indicate that equivalent cardiac current generators of this type can yield clinically significant results and that further research is necessary to investigate their properties fully.     

Properties of a tonically active, sodium-permeable current in mouse urinary bladder smooth muscle

Urinary bladder smooth muscle (UBSM) elicits depolarizing action potentials, which underlie contractile events of the urinary bladder. The resting membrane potential of UBSM is approximately -40 mV and is critical for action potential generation, with hyperpolarization reducing action potential frequency. We hypothesized that a tonic, depolarizing conductance was present in UBSM, functioning to maintain the membrane potential significantly positive to the equilibrium potential for K(+) (E(K); -85 mV) and thereby facilitate action potentials. Under conditions eliminating the contribution of K(+) and voltage-dependent Ca(2+) channels, and with a clear separation of cation- and Cl(-)-selective conductances, we identified a novel background conductance (I(cat)) in mouse UBSM cells. I(cat) was mediated predominantly by the influx of Na(+), although a small inward Ca(2+) current was detectable with Ca(2+) as the sole cation in the bathing solution. Extracellular Ca(2+), Mg(2+), and Gd(3+) blocked I(cat) in a voltage-dependent manner, with K(i) values at -40 mV of 115, 133, and 1.3 microM, respectively. Although UBSM I(cat) is extensively blocked by physiological extracellular Ca(2+) and Mg(2+), a tonic, depolarizing I(cat) was detected at -40 mV. In addition, inhibition of I(cat) demonstrated a hyperpolarization of the UBSM membrane potential and decreased the amplitude of phasic contractions of isolated UBSM strips. We suggest that I(cat) contributes tonically to the depolarization of the UBSM resting membrane potential, facilitating action potential generation and thereby a maintenance of urinary bladder tone.     

Evidence for an endogenous protein inhibitor of sarcoplasmic reticulum calcium pump in heart muscle

A cytosolic protein fraction, termed CPF-I, derived by (NH₄)₂ SO₄ fractionation of rabbit heart cytosol caused marked inhibition (up to 95%) of ATP-dependent Ca²⁺ uptake by cardiac sarcoplasmic reticulum. The inhibitory effect of CPF-I was concentration-dependent (50% inhibition with ～ 80–100 μg CPF-I) and heat labile. The inhibitor reduced the velocity of Ca²⁺ uptake without altering the apparent affinity of the transport system for Ca²⁺. Concomitant with the inhibition of Ca²⁺ uptake, Ca²⁺-sensitive ATP hydrolysis was also inhibited by CPF-I. The inhibitor did not cause release of Ca²⁺ from Ca²⁺-preloaded membrane vesicles. The inhibitor activity of CPF-I could be adsorbed to a DEAE cellulose column and could be eluted with a linear gradient of KCl. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum calcium pump in cardiac muscle and raises the intriguing possibility of its participation in the regulation of calcium pump $\text{in}\text{vivo}$.