Effect of Adenosine and Intracellular GTP on KATP Channels of Mammalian Skeletal Muscle

We investigated the action of adenosine and GTP on K_ATP channels, using inside-out patch clamp recordings from dissociated single fibers of rat flexor digitorum brevis (FDB) skeletal muscle. In excised patches, K_ATP channels could be activated by a combination of an extracellular adenosine agonist and intracellular Mg²⁺-ATP and GTP or GTP-γ-S. The activation required hydrolyzable ATP and could be partially reversed with Mg²⁺, suggesting that it may involve a G-protein dependent phosphorylation of K_ATP channels. We found that K_ATP channels of the rat FDB could not be activated by Mg²⁺-ATP alone or by Mg²⁺-ATP in the presence of extracellular adenosine. Patches whose channel activity had been `rundown' by Ca²⁺ could not be recovered by adenosine, GTP or Mg²⁺-ATP. K_ATP channels activated by adenosine receptor agonists had a similar ATP sensitivity to those under control conditions; but adenosine appears to be able to switch these K_ATP channels from an inactive to an active mode. Received: 29 December 1995/Revised: 22 March 1996     

Regulation of Ryanodine Receptor Calcium Release Channels by Diadenosine Polyphosphates

Abstract: [³H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca²⁺ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (Ap_nAs; n = 2–6 phosphate groups) on [³H]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [³H]ryanodine binding to brain membranes was significantly increased by 171% with 333 µMP¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) and by 209% with the same concentration of the metabolism-resistant ATP analogue βγ-methyleneadenosine 5′-triphosphate (AMP-PCP) compared with control values for [³H]ryanodine binding of 9.6 ± 1.8 fmol/mg of protein. Dose-related increases in [³H]ryanodine binding were observed for all five Ap_nAs tested [P¹,P²-di(adenosine-5′) pyrophosphate (Ap₂A), P¹,P³-di(adenosine-5′) triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A), Ap₅A, and P¹,P⁶-di(adenosine-5′) hexaphosphate (Ap₆A)] as well as AMP-PCP; oxidized salts of Ap_nAs stimulated [³H]ryanodine binding to a greater degree than did nonoxidized Ap_nAs. The apparent rank order for the capacity of these agents to increase [³H]-ryanodine binding was oxidized Ap₄A = oxidized Ap₅A > oxidized Ap₃A > Ap₆A > AMP-PCP > Ap₅A > Ap₂A. Addition of the approximate EC₅₀ dose of oxidized Ap₄A (37 µM) increased the affinity (K_D) of ryanodine receptors from 34 ± 7 to 12 ± 2 nM; the apparent binding site density (B_max) was not significantly different from control values of 107 ± 33 fmol/mg of protein. Increases in [³H]-ryanodine binding by either oxidized Ap₄A or nonoxidized Ap₅A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the Ap_nAs and AMP-PCP. [³H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap₄A, Ap₅A, and AMP-PCP. Oxidized Ap₄A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that Ap_nAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca²⁺ release channels.     

Neurotrophin-4: The odd one out in the neurotrophin family

Neurotrophin-4 (NT-4) is a member of a family of neurotrophic factors, the neurotrophins, that control survival and differentiation of vertebrate neurons (2–4). Besides being the most recently discovered neurotrophin in mammals, and the least well understood, several aspects distinguish NT-4 from other members of the neurotrophin family. It is the most divergent member and, in contrast to the other neurotrophins, its expression is ubiquitous and appears to be less influenced by environmental signals. NT-4 seems to have the unique requirement of binding to the lowaffinity neurotrophin receptor (p75^LNGFR) for efficient signalling and retrograde transport in neurons. Moreover, while all other neurotrophin knock-outs have proven lethal during early postnatal development, mice deficient in NT-4 have so far only shown minor cellular deficits and develop normally to adulthood. Is NT-4 a recent addition to the neurotrophic factor repertoire in search of a crucial function, or is it an evolutionary relic, a kind of wisdom tooth of the neurotrophin family?     

The association of proctolin with a ventral abdominal muscle of Locusta migratoria

The association of proctolin with the external ventral protractor muscle of the VIIth abdominal segment (M234) of Locusta migratoria was investigated using immunohistochemistry and RP-HPLC in conjunction with the sensitive locust oviduct bioassay. Immunohistochemistry of whole-mount tissues revealed two proctolin-like immunoreactive axons in N2B_2b1 (the nerve branch which innervates M234) as well as immunoreactive processes and varicosities on the surface of M234. Immunogold staining of M234 demonstrated that the proctolin-like immunoreactivity was present in electron-dense granules in its motor terminals. A material indistinguishable from proctolin and with proctolin-like bioactivity co-eluted with authentic proctolin on two different RP-HPLC systems. Bath application of proctolin at concentrations greater than 10^-11 M resulted in a dose-dependent increase in the neurally-evoked fast twitch amplitude and duration of M234. Concentrations greater than 10^-9 M resulted in a dose-dependent increase in basal tonus of M234. These results indicate that proctolin, or a peptide very similar to proctolin, is present in the motor innervation of M234 and acts as a cotransmitter and/or neuromodulator at this typical fast skeletal muscle.Abbreviations M234 external ventral protractor muscle of the Vllth abdominal segment - RP-HPLC reverse-phase high pressure liquid chromatography - TFA trifluoroacetic acid     

丝裂素活化蛋白激酶参与内皮素刺激的兔主动脉平滑肌细胞增生

内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）是已知的体内活性最强的缩血管物质,其缩血管作用由Ｇ蛋白偶联受体所介导。但ＥＴ强大的促血管平滑肌细胞（ＶＳＭＣ）增生效应的机理尚未完全阐明。本研究选用培养的兔胸主动脉ＶＳＭＣ,探讨丝裂素活化蛋白激酶（ＭＡＰＫ）在ＥＴ促细胞增生中的作用。结果表明：ＥＴ－１呈时间和浓度依赖性地促进细胞摄取￣３Ｈ－ＴｄＲ和激活ＭＡＰＫ,此作用可被蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ,ＰＫＣ）抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴＰ）,Ｈ－７和ＥＴ＿Ａ受体拮抗剂ＢＱ１２３所抑制,但不被酪氨酸激酶抑制剂ＨｅｒｂｉｍｙｃｉｎＡ（Ｈｅｒｂ）所抑制,用ＰＫＣ激动剂ＰＭＡ（Ｐｈｏｒｂｏｌｍｙｒｉｓｔａｔｅａｃｅｔａｔｅ）预处理ＶＳＭＣ,使其ＰＫＣ活性下调,可显著减弱ＥＴ－１对ＭＡＰＫ的激活能力。本结果提示：（１）ＭＡＰＫ参与ＥＴ－１所致的ＶＳＭＣ增生;（２）ＥＴ－１促细胞增生与激活ＭＡＰＫ的作用是由ＥＴ＿Ａ受体和ＰＫＣ介导的。     

Comparison of a Bone Criterion and a dental Criterion for estimation of age at death in the study of an Ancient Cemetery

Two methods, a visual method based on the appearance of the auricular surface of the ilium and a metric method based on two dental criteria, were used in conjunction for estimation of skeletal age at death in paleodemographic study of an ancient cemetery, and were found to be coherent. However, the paleodemographic profiles differed according to sex, indicating a sexual difference in the evolution of the sacroiliac joint.     

Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts

A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC₅₀ = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC₅₀ = 8 μM), and APN by amastatin (IC₅₀ = 50 nM) and bestatin (IC₅₀ = 100 μM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall