Charybdotoxin block of Ca2+-activated K+ channels in colonic muscle depends on membrane potential dynamics

Charybdotoxin(ChTX) is a specific blocker ofCa²⁺-activatedK⁺ channels. The voltage- andtime-dependent dynamics of ChTX block were investigated using caninecolonic myocytes and the whole cell patch-clamp technique with step andramp depolarization protocols. During prolonged step depolarizations,K⁺ current slowly increased in thecontinued presence of ChTX (100 nM). The rate of increase depended onmembrane potential with an e-foldchange for every 60 mV. During ramp depolarizations, the effectivenessof ChTX block depended significantly on the rate of the ramp (50% at0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanismin which ChTX slowly "unbinds" in a voltage-dependent manner. Asimple kinetic model was developed in which ChTX binds to both open andclosed states. Slow unbinding is consistent with ChTX having littleeffect on electrical slow waves recorded from circular muscle whilecausing depolarization and contraction of longitudinal muscle, whichdisplays more rapid "spikes." Resting membrane potential andmembrane potential dynamics are important determinants of ChTX action.

     

Interstitial cells of Cajal: mediators of communication between circular and longitudinal muscle layers of canine colon

The network of interstitial cells of Cajal associated with Auerbach’s (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach’s plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach’s plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities. Received: 21 July 1995 / Accepted: 22 April 1998     

Phalloidin-rhodamine preparations of Macrostomum hystricinum marinum (Plathelminthes): morphology and postembryonic development of the musculature

Summary A whole-mount fluorescence technique using rhodamine-labeled phalloidin was used to demonstrate for the first time the whole muscle system of a free-living plathelminth, Macrostomum hystricinum marinum. As expected, the body-wall musculature consisted of circular, longitudinal, and diagonal fibers over the trunk. Also distinct were the musculature of the gut and of the mouth and pharynx (circular, longitudinal, and radial). Dorsoventral fibers where restricted in this species to the head and tail regions. Circular muscle fibers in the body wall were often grouped into bands of up to four parallel strands. Surprisingly, diagonal fibers formed two distinct sets, one dorsal and one ventral. Certain diagonal muscle fibers entered the wall of the mouth and were continuous with some longitudinal muscles of the pharynx. Dorsoventral fibers in the rostrum occurred partly in regularly spaced pairs, a fact not known for free-living Plathelminthes. All muscle fibers appeared to be mononucleated. During postembryonic development, the number of circular muscle fibers can be estimated to increase by a factor of 3.5 and that of longitudinal muscles by a factor of 2. Apparently as many as 700–800 circular muscle cells must be added in the region of the gut alone during postembryonic development. Stem cells (neoblasts), identified by TEM in the caudalmost region of the gut, lie along the lateral nerve cords. In the same body region most perikarya of circular muscle cells occurred in a similar position. This suggests that the nucleus-containing part of the cell remains in the position where differentiation starts.     

G protein-dependent activation of smooth muscle eNOS via natriuretic peptide clearance receptor

In gastrointestinal smooth muscle, the neuropeptides vasoactiveintestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) induce relaxation by interacting withVIP₂/PACAP₃ receptors coupled via G_s toadenylyl cyclase and with distinct receptors coupled viaG_i1 and/orG_i2 to a smooth muscle endothelial nitric oxide synthase (eNOS). The present study identifies the receptoras the single-transmembrane natriuretic peptide clearance receptor(NPR-C). RT-PCR and Northern analysis demonstrated expression of thenatriuretic peptide receptors NPR-C and NPR-B but not NPR-A in rabbitgastric muscle cells. In binding studies using¹²⁵I-labeled atrial natriureticpeptide (¹²⁵I-ANP) and¹²⁵I-VIP as radioligands, VIP,ANP, and the selective NPR-C ligand cANP(4-23) bound with highaffinity to NPR-C. ANP, cANP-(4-23), and VIP initiated identicalsignaling cascades consisting ofCa²⁺ influx, activation of eNOSvia G_i1 andG_i2, stimulation of cGMP formation, and muscle relaxation. NOS activity and cGMP formation wereabolished (93 ± 3 to 96 ± 2% inhibition) by nifedipine,pertussis toxin, the NOS inhibitor,N^G-nitro-L-arginine,and the antagonists ANP-(1-11) and VIP-(10-28). NOS activitystimulated by all three ligands in muscle membranes was additivelyinhibited by G_i1 andG_i2 antibodies (82 ± 2 to 84 ± 1%). In reconstitution studies, VIP, cANP-(4-23), and guanosine 5'-O-(3-thiotriphosphate) stimulated NOS activity inmembranes of COS-1 cells cotransfected with NPR-C and eNOS. Theresults establish a unique mechanism for G protein-dependent activation of a constitutive NOS expressed in gastrointestinal smooth muscle involving interaction of the relaxant neuropeptides VIP and PACAP with a single-transmembrane natriuretic peptide receptor, NPR-C.

     

Inhibition of cyclooxygenase-2 and EP1 receptor antagonism reduces human colonic longitudinal muscle contractility in vitro

We investigated the contribution of cyclo-oxygenase enzyme inhibition and prostamide agonism on human colonic contractility in vitro. The effects of the non-specific COX inhibitor diclofenac were compared against selective COX-2 inhibition via nimesulide, the prostanoid EP₁ receptor antagonist SC19220 or the prostaglandin prodrug/prostamide receptor agonist bimatoprost, on potency of contraction to acetylcholine in human colonic circular and longitudinal muscle strips. Pre-treatment with either nimesulide (10^?5 M) or diclofenac (10^?6 M) caused a significant decrease in the potency of acetylcholine-evoked longitudinal muscle contraction, but did not inhibit acetylcholine-evoked circular muscle contraction. Pre-treatment with the EP₁ receptor antagonist SC19220 (10^?5 M) similarly decreased cholinergic potency in longitudinal muscle, without influence on circular muscle contraction. The prostamide agonist bimatoprost (10^?6 M) increased basal circular and longitudinal muscle tone, but did not alter cholinergic potency in either muscle layer. In conclusion, colonic longitudinal muscle contraction is augmented by COX-2 activity, most likely via PGE₂ acting at EP₁ receptors. While colonic contraction is tonically modulated by bimatoprost, it does not share the same functional properties attributed to other endogenous COX-2 metabolites on colonic contractile function.     

Mechanism of the exercise hyperkalemia: an alternate hypothesis

Wasserman, Karlman, William W. Stringer, Richard Casaburi,and Yong-Yu Zhang. Mechanism of the exercisehyperkalemia: an alternate hypothesis. J. Appl.Physiol. 83(2): 631-643, 1997.A progressivehyperkalemia is observed as exercise intensity increases. The currentmost popular hypothesis for the hyperkalemia is that theNa⁺-K⁺pump cannot keep pace with the K⁺efflux from muscle during the depolarization-repolarization process ofthe sarcolemmal membrane during muscle contraction. In this report, wepresent data that suggest an alternate hypothesis to those previouslydescribed. Because phosphocreatine (PCr) is a highly dissociated acidand creatine is neutral at cell pH, the concentration of nondiffusibleanions decreases, and an alkaline reaction takes place when PCrhydrolyzes. This creates a state of cation(K⁺) excess andH⁺ depletion in the cell. Toexamine the balance of K⁺ andH⁺ for exercising muscle duringthe early period of exercise when PCr changes most rapidly, catheterswere inserted into the brachial artery and femoral vein (FV) in fivehealthy subjects who performed two 6-min cycle ergometer exercise testsat 40 and 85% of peak oxygen uptake. FV blood was sampled every 5 sduring the first 2 min, then every 30 s for the remaining 4 min ofexercise and the first 3 min of recovery, and then less frequently forthe next 12 min. Arterial sampling was every 30 s during exercise andsimultaneous with FV sampling during recovery. ArterialK⁺ concentration([K⁺]) increase laggedFV [K⁺]increase. The hyperkalemia observed during early exerciseresults from K⁺ release fromskeletal muscle. FV[K⁺] increased by 5 sof the start of exercise and followed the rate ofH⁺ loss from the FV blood for thefirst 30 s of exercise. FV lactate andNa⁺ kinetics differed fromK⁺ kinetics during exercise andrecovery. As predicted from the PCr hydrolysis reaction, the exercisinglimb took up H⁺ and releasedK⁺ at the start of exercise (first30 s) at both exercise intensities, resulting in a FV metabolicalkalosis. K⁺ release wasessentially complete by 3 min, the time at which oxygen uptake (and,presumably, PCr) reached its asymptote. These findings lead us tohypothesize that the early K⁺release by the cell takes place withH⁺ exchange and that the majormechanism for the exercise hyperkalemia is the reduction innondiffusible intracellular anions in the myocyte as PCr hydrolyzes.

     

Decreased [3H]ouabain binding sites in skeletal muscle of rats with chronic heart failure

Pickar, Joel G., John P. Mattson, Steve Lloyd, and TimothyI. Musch. Decreased[³H]ouabainbinding sites in skeletal muscle of rats with chronic heart failure.J. Appl. Physiol. 83(1): 323-329, 1997.Abnormalities intrinsic to skeletal muscle are thought tocontribute to decrements in exercise capacity found in individualswith chronic heart failure (CHF).Na⁺-K⁺-adenosinetriphosphatase(the Na⁺ pump) is essential formaintaining muscle excitability and contractility. Therefore, weinvestigated the possibility that the number and affinity ofNa⁺ pumps in locomotor muscles ofrats with CHF are decreased. Myocardial infarction (MI) was induced in8 rats, and a sham operation was performed in 12 rats. The degree ofCHF was assessed ~180 days after surgery. Soleus and plantarismuscles were harvested, and Na⁺pumps were quantified by using a[³H]ouabain bindingassay. At the time of muscle harvest, MI and sham-operated rats weresimilar in age (458 ± 54 vs. 447 ± 34 days old, respectively).Compared with their sham-operated counterparts, MI rats had asignificant amount of heart failure, right ventricular-to-body weightratio was greater (48%), and the presence of pulmonary congestion wassuggested by an elevated lung-to-body weight ratio (29%). Leftventricular end-diastolic pressure was significantly increased in theMI rats (11 ± 1 mmHg) compared with the sham-operated controls (1 ± 1 mmHg). In addition, mean arterial blood pressure was lower inthe MI rats compared with their control counterparts. [³H]ouabain bindingsites were reduced 18% in soleus muscle (136 ± 12 vs. 175 ± 13 pmol/g wet wt, MI vs. sham, respectively) and 22% in plantaris muscle(119 ± 12 vs. 147 ± 8 pmol/g wet wt, MI vs. sham,respectively). The affinity of these[³H]ouabain bindingsites was similar for the two groups. The relationship between thereduction in Na⁺ pump number andthe reduced exercise capacity in individuals with CHF remains to bedetermined.

     

Cytoskeletal modulation of sodium current in human jejunal circular smooth muscle cells

ANa⁺ current is present in human jejunal circular smoothmuscle cells. The aim of the present study was to determine the role ofthe cytoskeleton in the regulation of the Na⁺ current.Whole cell currents were recorded by using standard patch-clamptechniques with Cs⁺ in the pipette to block K⁺currents. Cytochalasin D and gelsolin were used to disrupt the actincytoskeleton and phalloidin to stabilize it. Colchicine was used todisassemble the microtubule cytoskeleton (and intermediate filaments)and paclitaxel to stabilize it. Acrylamide was used to disrupt theintermediate filament cytoskeleton. Perfusion of the recording chamberat 10 ml/min increased peak Na⁺ current recorded fromjejunal smooth muscle cells by 27 ± 3%. Cytochalasin D andgelsolin abolished the perfusion-induced increase in Na⁺current, whereas incubation with phalloidin, colchicine, paclitaxel, oracrylamide had no effect. In conclusion, the Na⁺ currentexpressed in human jejunal circular smooth muscle cells appears to beregulated by the cytoskeleton. An intact actin cytoskeleton is requiredfor perfusion-induced activation of the Na⁺ current.

     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Muscle quality. II. Effects of strength training in 65-to 75-yr-old men and women

To determine theeffects of strength training (ST) on muscle quality (MQ,strength/muscle volume of the trained muscle group), 12 healthy oldermen (69 ± 3 yr, range 65-75 yr) and 11 healthy older women (68 ± 3 yr, range 65-73 yr) were studied before and after aunilateral leg ST program. After a warm-up set, four sets ofheavy-resistance knee extensor ST exercise were performed 3 days/wk for9 wk on the Keiser K-300 leg extension machine. The men exhibitedgreater absolute increases in the knee extension one-repetition maximum(1-RM) strength test (75 ± 2 and 94 ± 3 kg before andafter training, respectively) and in quadriceps muscle volume measuredby magnetic resonance imaging (1,753 ± 44 and 1,955 ± 43 cm³) than the women (42 ± 2 and 55 ± 3 kg for the 1-RM test and 1,125 ± 53 vs.1,261 ± 65 cm³ forquadriceps muscle volume before and after training, respectively, inwomen; both P < 0.05). However,percent increases were similar for men and women in the 1-RM test (27 and 29% for men and women, respectively), muscle volume (12% forboth), and MQ (14 and 16% for men and women, respectively).Significant increases in MQ were observed in both groups in the trainedleg (both P < 0.05) and in the 1-RMtest for the untrained leg (both P < 0.05), but no significant differences were observed between groups,suggesting neuromuscular adaptations in both gender groups. Thus,although older men appear to have a greater capacity for absolutestrength and muscle mass gains than older women in response to ST, the relative contribution of neuromuscular and hypertrophic factors to theincrease in strength appears to be similar between genders.

     

Effect of sprint cycle training on activities of antioxidant enzymes in human skeletal muscle

Hellsten, Ylva, Fred S. Apple, and Bertil Sjödin.Effect of sprint cycle training on activities of antioxidantenzymes in human skeletal muscle. J. Appl.Physiol. 81(4): 1484-1487, 1996.The effect ofintermittent sprint cycle training on the level of muscle antioxidantenzyme protection was investigated. Resting muscle biopsies, obtainedbefore and after 6 wk of training and 3, 24, and 72 h after the finalsession of an additional 1 wk of more frequent training, were analyzedfor activities of the antioxidant enzymes glutathione peroxidase (GPX),glutathione reductase (GR), and superoxide dismutase (SOD). Activitiesof several muscle metabolic enzymes were determined to assess the effectiveness of the training. After the first 6-wk training period, nochange in GPX, GR, or SOD was observed, but after the 7th week oftraining there was an increase in GPX from 120 ± 12 (SE) to 164 ± 24 µmol ^· min^{1 ·} gdry wt¹(P < 0.05) and in GR from 10.8 ± 0.8 to 16.8 ± 2.4 µmol ^· min^{1 ·} gdry wt¹(P < 0.05). There was no significantchange in SOD. Sprint cycle training induced a significant(P < 0.05) elevation in the activity of phosphofructokinase and creatine kinase, implying an enhanced anaerobic capacity in the trained muscle. The present studydemonstrates that intermittent sprint cycle training that induces anenhanced capacity for anaerobic energy generation also improves thelevel of antioxidant protection in the muscle.

     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

Trauma-induced changes of skeletal muscle membrane: decreased K+ and increased Na+ permeability

Hong, S. J., and C. C. Chang.Trauma-induced changes of skeletal muscle membrane: decreasedK⁺ and increasedNa⁺ permeability.J. Appl. Physiol. 83(4):1096-1103, 1997.Trauma of skeletal muscle causes membranedepolarization and reduces membrane resistance. The underlyingmechanisms were studied in isolated mouse phrenic nerve diaphragmssubject to sharp transections of muscle. Depolarization was most markedat the vicinity (~1 mm) of trauma, where the membrane potentialdropped rapidly from about 80 mV to zero and repolarized toabout 25 mV. At the end-plate region (located ~3 mm away fromthe cut end), the membrane gradually attained a plateau potentialaround 45 mV. The magnitude of depolarization was not reduced byinhibition of Na⁺,Ca²⁺, orCl channel, whereas theprogress of depolarization was delayed in low-Na⁺ medium. Activation of theK⁺ channel with lemakalim inducedsome hyperpolarization at damaged site but produced aglybenclamide-sensitive outward current and hyperpolarization ofend-plate region to the levels before trauma, as if there was nodiminution of transmembrane K⁺gradient in this area. Appropriate elevation of extracellular K⁺ to stimulateK⁺ conductance also hyperpolarizedthe end-plate region. The results suggest that depolarization atregions remote from trauma is related to decreasedK⁺ and increasedNa⁺ permeability. The cytoplasmacompartmentalization and permeability changes may protect muscle fiberfrom trauma.

     

Effects of Met-enkephalin on the mechanical activity and distribution of Met-enkephalin-like immunoreactivity in the cat large intestine.

The effects of Met-enkephalin on the spontaneous and electrically evoked activity were investigated in longitudinal and circular strips isolated from different regions of the large intestine, i.e., proximal colon, distal colon and rectum. Met-enkephalin induced dose-dependent contractile responses which were reversibly blocked by naloxone (10(-6) M). In all longitudinal strips and in the circular strips of the rectum, the effects of Met-enkephalin were prevented by TTX (10(-7) M), demonstrating their neurogenic nature. In the circular strips from the colon, Met-enkephalin induced contractile responses after TTX, proving the existence of smooth muscle opioid receptors. The comparison between the EC50 values of Met-enkephalin showed that the opioid receptors in the different regions have different sensitivity to Met-enkephalin, while the opioid receptors in the longitudinal and circular layers of the same region have equal affinity. Atropine (10(-6) M) and guanethidine (10(-6) M) did not alter significantly the EC50 values, showing that the neurogenic effects of Met-enkephalin on the spontaneous activity involve mainly nonadrenergic, noncholinergic (NANC) neurotransmitter mechanisms. When the preparations were stimulated electrically, Met-enkephalin (10(-9) M) suppressed the cholinergic components of the responses. Met-enkephalin-containing nerve fibers were found in the myenteric plexus of the three intestinal regions. In the colon, where direct smooth muscle effects were observed, fibers containing Met-enkephalin-like immunoreactivity were found to go deep into the circular layer, suggesting that they could supply Met-enkephalin input to the smooth muscle cells.     

Intracellular signaling specificity in response to uniaxial vs. multiaxial stretch: implications for mechanotransduction

Several lines of evidence suggest that muscle cells can distinguish between specific mechanical stimuli. To test this concept, we subjected C₂C₁₂ myotubes to cyclic uniaxial or multiaxial stretch. Both types of stretch induced an increase in extracellular signal-regulated kinase (ERK) and protein kinase B (PKB/Akt) phosphorylation, but only multiaxial stretch induced ribosomal S6 kinase (p70^S6k) phosphorylation. Further results demonstrated that the signaling events specific to multiaxial stretch (p70^S6k phosphorylation) were elicited by forces delivered through the elastic culture membrane and were not due to greater surface area deformations or localized regions of large tensile strain. Experiments performed using medium that was conditioned by multiaxial stretched myotubes indicated that a release of paracrine factors was not sufficient for the induction of signaling to p70^S6k. Furthermore, incubation with gadolinium(III) chloride (500 µM), genistein (250 µM), PD-98059 (250 µM), bisindolylmaleimide I (20 µM), or LY-294002 (100 µM ) did not block the multiaxial stretch-induced signaling to p70^S6k. However, disrupting the actin cytoskeleton with cytochalasin D did block the multiaxial signaling to p70^S6k, with no effect on signaling to PKB/Akt. These results demonstrate that specific types of mechanical stretch activate distinct signaling pathways, and we propose that this occurs through direct mechanosensory-mechanotransduction mechanisms and not through previously defined growth factor/receptor binding pathways. growth; hypertrophy; muscle; strain; tension     

Interactions of neurogenic responses of longitudinal and circular muscle in the guinea-pig ileum

The relationship between neurogenic responses of longitudinal and circular muscle was studied by measuring contractions and EMG or nonadrenergic, non-cholinergic (NANC) relaxations and NANC inhibitory junction potentials in different preparations of the guinea-pig ileum. NANC relaxation of longitudinal muscle was observed also without any preceding or concomitant circular muscle contraction ruling out the possibility that the latter might be the cause of the NANC relaxation. Circular muscle twitches or powerful contractions were absent if there was no preceding neurogenic or myogenic excitation of longitudinal muscle; in preparations with myenteric plexus-longitudinal muscle layers removed only small residual responses were seen although still under neurogenic influences. Thus excitation of longitudinal muscle seemed a prerequisite for synchronized and powerful contractions of circular muscle to occur. Cholinergic contraction and NANC relaxation of longitudinal muscle evoked by field stimulation were partly inhibited if the submucous plexus was also present suggesting the involvement of a more complex neuronal circuitry in these responses.     

Sodium/calcium exchange in amphibian skeletal muscle fibers and isolated transverse tubules

TheNa⁺/Ca²⁺ exchanger participates inCa²⁺ homeostasis in a variety of cells and has a key rolein cardiac muscle physiology. We studied in this work the exchanger ofamphibian skeletal muscle, using both isolated inside-out transversetubule vesicles and single muscle fibers. In vesicles, increasingextravesicular (intracellular) Na⁺ concentrationcooperatively stimulated Ca²⁺ efflux (reverse mode), withthe Hill number equal to 2.8. In contrast to the stimulation of thecardiac exchanger, increasing extravesicular (cytoplasmic)Ca²⁺ concentration ([Ca²⁺]) inhibited thisreverse activity with an IC₅₀ of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at90 mV. Photolysis of a cytoplasmic caged Ca²⁺ compoundactivated an inward current (forward mode) of 23 ± 10 nA(n = 3), with an average current density of 0.6 µA/µF. External Na⁺ withdrawal generated an outwardcurrent (reverse mode) with an average current density of 0.36 ± 0.17 µA/µF (n = 6) but produced a minimal increasein cytosolic [Ca²⁺]. These results suggest that, inskeletal muscle, the main function of the exchanger is to removeCa²⁺ from the cells after stimulation.

     

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

Spontaneous transient outward currents(STOCs) were recorded from smooth muscle cells of theguinea pig taenia coli using the whole cell patch-clamp technique.STOCs were resolved at potentials positive to 50 mV. Treatingcells with caffeine (1 mM) caused a burst of outward currentsfollowed by inhibition of STOCs. Replacing extracellularCa²⁺ with equimolarMn²⁺ caused STOCs to "rundown." Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM)inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs"remained in the presence of these drugs. Mini-STOCs were reduced byapamin (500 nM), an inhibitor of small-conductance Ca²⁺-activatedK⁺ channels (SK channels).Application of ATP or 2-methylthioadenosine 5'-triphosphate(2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATPpersisted in the presence of charybdotoxin but were blocked bycombination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did notincrease STOCs in the presence of pyridoxal phosphate6-azophenyl-2',4'-disulfonic acid, aP₂ receptor blocker. Similarly,pretreatment of cells with U-73122 (1 µM), an inhibitor ofphospholipase C (PLC), abolished the effects of 2-MeS-ATP. XestosponginC, an inositol 1,4,5-trisphosphate(IP₃) receptor blocker,attenuated STOCs, but these events were not affected by ryanodine. Thedata suggest that purinergic activation through P_2Y receptors results in localizedCa²⁺ release via PLC- andIP₃-dependent mechanisms. Releaseof Ca²⁺ is coupled to STOCs, whichare composed of currents mediated by large-conductanceCa²⁺-activatedK⁺ channels and SK channels. Thelatter are thought to mediate hyperpolarization and relaxationresponses of gastrointestinal muscles to inhibitory purinergic stimulation.

     

Elementary events of agonist-induced Ca2+ release in vascular endothelial cells

The subcellular spatial and temporal organization ofagonist-induced Ca²⁺ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca²⁺ concentration([Ca²⁺]_i)in peripheral cytoplasmic processes from where activation propagated asa[Ca²⁺]_iwave toward the central regions of the cell. The average propagation velocity of the[Ca²⁺]_iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca²⁺]_idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca²⁺]_ideclined monotonically, whereas in the central region[Ca²⁺]_idecreased in an oscillatory fashion. Propagating[Ca²⁺]_iwaves were preceded by small, highly localized[Ca²⁺]_itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa²⁺ release was 23 nM, and theunderlying flux of Ca²⁺ amountedto ~1-2 × 10¹⁸mol/s or ~0.3 pA, consistent with aCa²⁺ flux through a single orsmall number of endoplasmic reticulum Ca²⁺-release channels.

     

Phosphorylation of p70S6k correlates with increased skeletal muscle mass following resistance exercise

High-resistance exercise training results in an increase inmuscle wet mass and protein content. To begin to address the acute changes following a single bout of high-resistance exercise, a newmodel has been developed. Training rats twice a week for 6 wk resultedin 13.9 and 14.4% hypertrophy in the extensor digitorum longus (EDL)and tibialis anterior (TA) muscles, respectively. Polysome profilesafter high-resistance lengthening contractions suggest that the rate ofinitiation is increased. The activity of the 70-kDa S6 protein kinase(p70^S6k), a regulator oftranslation initiation, is also increased following high-resistancelengthening contractions (TA, 363 ± 29%; EDL, 353 ± 39%).Furthermore, the increase inp70^S6k activity 6 h after exercisecorrelates with the percent change in muscle mass after 6 wk oftraining (r = 0.998). The tightcorrelation between the activation ofp70^S6k and the long-term increasein muscle mass suggests thatp70^S6k phosphorylation may be agood marker for the phenotypic changes that characterize musclehypertrophy and may play a role in load-induced skeletal muscle growth.