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Vacuum sealing drainage (VSD) is frequently used in abdominal surgeries. However, relevant guidelines are rare. Chinese Trauma Surgeon Association organized a committee composed of 28 experts across China in July 2017, aiming to provide an evidence-based recommendation for the application of VSD in abdominal surgeries.Eleven questions regarding the use of VSD in abdominal surgeries were addressed: (1) which type of materials should be respectively chosen for the intraperitoneal cavity, retroperitoneal cavity and superficial incisions? (2) Can VSD be preventively used for a high-risk abdominal incision with primary suture? (3) Can VSD be used in severely contaminated/infected abdominal surgical sites? (4) Can VSD be used for temporary abdominal cavity closure under some special conditions such as severe abdominal trauma, infection, liver transplantation and intra-abdominal volume increment in abdominal compartment syndrome? (5) Can VSD be used in abdominal organ inflammation, injury, or postoperative drainage? (6) Can VSD be used in the treatment of intestinal fistula and pancreatic fistula? (7) Can VSD be used in the treatment of intra-abdominal and extra-peritoneal abscess? (8) Can VSD be used in the treatment of abdominal wall wounds, wound cavity, and defects? (9) Does VSD increase the risk of bleeding? (10) Does VSD increase the risk of intestinal wall injury? (11) Does VSD increase the risk of peritoneal adhesion?Focusing on these questions, evidence-based recommendations were given accordingly. VSD was strongly recommended regarding the questions 2–4. Weak recommendations were made regarding questions 1 and 5–11. Proper use of VSD in abdominal surgeries can lower the risk of infection in abdominal incisions with primary suture, treat severely contaminated/infected surgical sites and facilitate temporary abdominal cavity closure. Keywords: Guideline, Vacuum sealing drainage, Abdominal surgery

We previously reported that short-term (2 h) plating of cat atrial myocytes on the extracellular matrix protein, laminin (LMN) decreases adenylate cyclase activity and β1-adrenergic receptor (β1-AR) stimulation of L-type Ca2+ current (ICa,L). The present study sought to determine whether LMN-mediated down-regulation of β1 signalling is due to down-regulation of adenylate cyclase and to gain insight into the signalling mechanisms responsible. β1-AR stimulation was achieved by 0.01 μm isoproterenol (isoprenaline) plus 0.1 μm ICI 118551, a selective β2-AR antagonist. Atrial myocytes were plated for at least 2 h on uncoated cover-slips (−LMN) or cover-slips coated with LMN (+LMN). As previously reported, β1-AR stimulation of ICa,L was significantly smaller in +LMN compared to −LMN atrial myocytes. In −LMN myocytes, 10 μm LY294002 (LY), a specific inhibitor of PI-(3)K, had no effect on β1-AR stimulation of ICa,L. In +LMN myocytes, however, LY significantly increased β1-AR stimulation of ICa,L. Western blots revealed that compared with −LMN myocytes, +LMN myocytes showed a significant increase in Akt phosphorylation at Ser-473, which was prevented by LY. In another approach, +LMN myocytes were infected (multiplicity of infection (MOI), 100; 24 h) with replication-defective adenoviruses (Adv) expressing dominant-negative inhibitors of focal adhesion kinase (FAK) (Adv-FRNK or Adv-Y397F-FAK) or Akt (Adv-dnAkt). Compared with control cells infected with Adv-β-galactosidase, cells infected with Adv-FRNK, Adv-Y397F-FAK or Adv-dnAkt each exhibited a significantly greater β1-AR stimulation of ICa,L. In −LMN myocytes LY had no effect on forskolin (FSK)-stimulated ICa,L. However, in +LMN myocytes LY significantly increased FSK-stimulated ICa,L. Similar results were obtained in +LMN atrial myocytes infected with Adv-FRNK. We conclude that LMN binding to β1-integrin receptors acts via FAK/PI-(3)K/Akt to inhibit adenylate cyclase activity and thereby down-regulates β1-AR-mediated stimulation of ICa,L. These findings provide new insight into the cellular mechanisms by which the extracellular matrix can modulate atrial β-AR signalling.

In this study we sought to determine whether contractile activity has a role as a signalling mechanism in the activation of intracellular nitric oxide (NOi) production induced by electrical stimulation of cat ventricular myocytes. Field stimulation (FS) of single ventricular myocytes elicited frequency-dependent increases in NOi that were blocked by the calmodulin (CaM) inhibitor 10 μm W-7 and partially inhibited by the phosphatidylinositol 3′-kinase (PI-(3)K) inhibitor 10 μm LY294002. Increasing extracellular [Ca2+] caused a concentration-dependent increase in FS-induced NOi that was partially inhibited by LY294002. The negative inotropic agents BDM (5 mm) or blebbistatin (10 μm) decreased cell shortening and NOi production without concomitant changes in L-type Ca2+ current (ICa,L) or [Ca2+]i transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 μm) increased cell shortening and NOi production without concomitant changes in ICa,L or [Ca2+]i transients. FS-induced NOi production was decreased in myocytes infected (100 multiplicity of viral infection (MOI); 24 h) with a replication-deficient adenovirus expressing a dominant-negative mutant of protein kinase B (Akt) compared with cells infected with a control adenovirus expressing β-galactosidase. FS-induced NOi was partially inhibited by either endothelial (eNOS) or neuronal nitric oxide synthase (nNOS) inhibitors and completely blocked by simultaneous exposure to both. FS-induced [Ca2+]i transients were increased by the nNOS inhibitor nNOS-I (0.24 μm), decreased by the eNOS inhibitor L-NIO (1 μm) and unchanged by exposure to both inhibitors. We conclude that in cat ventricular myocytes, FS-induced NOi production requires both Ca2+-dependent CaM signalling and Ca2+-independent PI-(3)K–Akt signalling activated by contractile activity. FS activates NOi production from both eNOS and nNOS, and each source of NOi exerts opposing effects on [Ca2+]i transient amplitude. These findings are important for understanding the regulation of NOi signalling in the normal and mechanically failing heart.

The present study determined the effects of acetylcholine (ACh) on the L-type Ca(2+) current (I(Ca,L)) stimulated by beta(1)- or beta(2)-adrenergic receptor (AR) agonists in cat atrial myocytes. When isoproterenol (ISO; 0.1 microM) plus the beta(2)-AR antagonist ICI 118,551 (ISO-beta(1)-AR stimulation) or 0.1 microM fenoterol, a beta(2)-AR agonist (FEN-beta(2)-AR stimulation) increased I(Ca,L), ACh (1 microM) inhibited I(Ca,L) by -60 +/- 4 and -63 +/- 6 %, respectively. When ISO plus the beta(1)-AR antagonist atenolol (ISO-beta(2)-AR stimulation) or 1 microM zinterol (ZIN-beta(2)-AR stimulation) increased I(Ca,L), ACh-induced inhibition of I(Ca,L) was significantly smaller, at -21 +/- 3 and -24 +/- 3 %, respectively. L-N(5)-(1-iminoethyl)ornithine (L-NIO, 10 microM), an inhibitor of nitric oxide (NO) synthase, enhanced ACh-induced inhibition of I(Ca,L) when stimulated by ZIN-beta(2)-ARs, but not when stimulated by ISO-beta(1)-ARs or FEN-beta(2)-ARs. Haemoglobin (50 microM), a NO scavenger, also enhanced ACh-induced inhibition when I(Ca,L) was stimulated by ZIN-beta(2)-ARs, but not when stimulated by FEN-beta(2)-ARs. ACh-induced inhibition of I(Ca,L) stimulated by ZIN-beta(2)-ARs was not affected by 10 microM 1H-[1,2,4] oxadiazolo[4,3-a] quinoxaline-1-one (ODQ) a guanylate cyclase inhibitor, but was significantly enhanced by 500 microM reduced glutathione or 100 microM dithiothreitol, agents that act as sinks for S-nitrosylation. ACh-induced inhibition was smaller when I(Ca,L) was stimulated by spermine/NO, a NO donor, than by milrinone, a phosphodiesterase type III inhibitor. ISO (ISO-beta(1)/beta(2)-AR stimulation) increased I(Ca,L) and even though ISO releases NO, ACh prominently inhibited I(Ca,L). This inhibitory effect of ACh was enhanced by L-NIO. Stimulation of ZIN-beta(2)-ARs increased intracellular NO, whereas ISO-beta(1)-ARs or FEN-beta(2)-ARs failed to increase intracellular NO. These results indicate that in atrial myocytes, NO released by selective beta(2)-AR stimulation prevents ACh-induced inhibition of I(Ca,L) stimulated by beta(2)-ARs. NO acts via a cGMP-independent, S-nitrosylation mechanism. Although FEN acts via beta(2)-ARs, it fails to stimulate G(i)-/NO signalling and preferentially stimulates G(s)-/adenylate cyclase signalling, similar to beta(1)-ARs. These findings indicate that NO signalling modulates muscarinic receptor inhibition of atrial function stimulated by beta(2)-ARs.

1. Perforated patch recordings were used to determine how plating atrial cells on laminin alters beta-adrenergic receptor (beta-AR) regulation of L-type Ca2+ current (ICa,L). 2. Isoproterenol (isoprenaline; ISO; 0.01 microM), a non-selective beta-AR agonist, elicited a greater stimulation of ICa,L in cells plated on laminin (+79 +/- 16 %; n = 17) than on glass (+33 +/- 5 %; n = 23). Also, desensitization to ISO was greater in cells on laminin (-16 +/- 2 %) than on glass (-3 +/- 1 %). Atenolol (0.1 microM), a selective beta1-AR antagonist, inhibited the effects of ISO in cells on glass but not laminin. Conversely, 0.1 microM ICI 118,551, a selective beta2-AR antagonist, inhibited the effects of ISO in cells on laminin but not glass. With beta2-ARs blocked, ISO-induced stimulation of ICa,L was greater in cells on glass than laminin. 3. Zinterol (0.01-0.1 microM), a selective beta2-AR agonist, elicited a greater stimulation of ICa,L in cells on laminin than on glass. The effects of zinterol were blocked by ICI 118,551. 4. ISO-induced stimulation of ICa,L was greater in cells plated on an alphabeta1-integrin antibody than on glass. Also, addition of 20 microM cytochalasin D to cells on laminin prevented the enhanced effects of ISO typically elicited in cells on laminin alone. 5. We conclude that laminin binding to alphabeta1-integrins, in conjunction with the actin cytoskeleton, reduces beta1-AR and enhances beta2-AR signalling which regulates ICa,L. This novel mechanism may contribute to remodelling of beta-AR signalling in the failing heart.

A perforated patch recording method was used to determine how plating cells on laminin (20 microg ml(-1); >2 h) alters cholinergic regulation of L-type Ca(2+) current (I(Ca,L)) in atrial myocytes. Acetylcholine (ACh; 1 microm)-induced inhibition of basal I(Ca,L) was not different between cells on glass and laminin. However, stimulation of I(Ca,L) elicited by ACh withdrawal was significantly smaller in cells on laminin (10 +/- 2 %) than on glass (48 +/- 5 %) (P < 0.001). Stimulation of I(Ca,L) induced by either spermine-NO (200 microm), milrinone (10 microm), IBMX (100 microm) or forskolin (1 microm) was significantly smaller in cells plated on laminin than on glass. However, stimulation of I(Ca,L) by 100 microm 8-CPT-cAMP or intracellular dialysis with 50 microM cAMP was not different between cells plated on laminin or glass. Basal, forskolin- and IBMX-stimulated cAMP content was significantly smaller in cells plated on laminin than on glass. Stimulation of I(Ca,L) by ACh withdrawal was significantly smaller in cells plated on an alpha beta 1-integrin antibody (10 +/- 4 %) than on glass (3 +/- 6 %; P < 0.001). In cells on laminin, prior exposure to 100 microg ml-1 YIGSR, a laminin receptor-binding peptide, restored ACh-induced stimulation of I(Ca,L) (58 +/- 14 %)laminin alone (7 +/- 2 %; P < 0. 05). Addition of 20 microm cytochalasin D or 1 microM latrunculin A, agents that prevent actin polymerization, to cells on laminin restored ACh-induced stimulation of I(Ca,L). We conclude that laminin binding to beta 1 integrins acts in association with the actin-based cytoskeleton to attenuate adenylate cyclase activity. As a result, laminin inhibits NO-mediated stimulation of I(Ca,L) elicited by ACh withdrawal. Laminin-integrin signalling may be relevant to changes in autonomic regulation that occur during cardiac development and/or disease.

Electromechanical alternans was characterized in single cat atrial and ventricular myocytes by simultaneous measurements of action potentials, membrane current, cell shortening and changes in intracellular Ca2+ concentration ([Ca2+]i). Using laser scanning confocal fluorescence microscopy, alternans of electrically evoked [Ca2+]i transients revealed marked differences between atrial and ventricular myocytes. In ventricular myocytes, electrically evoked [Ca2+]i transients during alternans were spatially homogeneous. In atrial cells Ca2+ release started at subsarcolemmal peripheral regions and subsequently spread toward the centre of the myocyte. In contrast to ventricular myocytes, in atrial cells propagation of Ca2+ release from the sarcoplasmic reticulum (SR) during the small-amplitude [Ca2+]i transient was incomplete, leading to failures of excitation-contraction (EC) coupling in central regions of the cell. The mechanism underlying alternans was explored by evaluating the trigger signal for SR Ca2+ release (voltage-gated L-type Ca2+ current, ICa,L) and SR Ca2+ load during alternans. Voltage-clamp experiments revealed that peak ICa,L was not affected during alternans when measured simultaneously with changes of cell shortening. The SR Ca2+ content, evaluated by application of caffeine pulses, was identical following the small-amplitude and the large-amplitude [Ca2+]i transient. These results suggest that the primary mechanism responsible for cardiac alternans does not reside in the trigger signal for Ca2+ release and SR Ca2+ load. beta-Adrenergic stimulation with isoproterenol (isoprenaline) reversed electromechanical alternans, suggesting that under conditions of positive cardiac inotropy and enhanced efficiency of EC coupling alternans is less likely to occur. The occurrence of electromechanical alternans could be elicited by impairment of glycolysis. Inhibition of glycolytic flux by application of pyruvate, iodoacetate or beta-hydroxybutyrate induced electromechanical and [Ca2+]i transient alternans in both atrial and ventricular myocytes. The data support the conclusion that in cardiac myocytes alternans is the result of periodic alterations in the gain of EC coupling, i. e. the efficacy of a given trigger signal to release Ca2+ from the SR. It is suggested that the efficiency of EC coupling is locally controlled in the microenvironment of the SR Ca2+ release sites by mechanisms utilizing ATP, produced by glycolytic enzymes closely associated with the release channel.

Abstract Cholinergic inhibition of atrial contraction is typically followed by a rebound positive inotropic response. In the present study, we used a nystatin–perforated patch whole-cell recording method to determine whether acetylcholine (ACh) elicits a rebound stimulation of L-type Ca 2+ current (I Ca,L ) in cat atrial myocytes. ACh (1 μmol/L) decreased basal I Ca,L (−19±2%). Within ≈30 s of returning to ACh-free solution, basal I Ca,L exhibited a rebound increase above the control level (+61±7%) that returned to the control level within 4 to 5 minutes. ACh elicited concomitant changes in cell shortening, ie, a decrease followed by a rebound increase. The EC 50 and maximal response of ACh-induced inhibition and rebound stimulation of I Ca,L were 1.9×10 −9 mol/L and −30%, respectively, and 2.9×10 −8 mol/L and +64%, respectively. All effects of ACh on I Ca,L were blocked by prior exposure to 1 μmol/L atropine or 100 μmol/L AFDX116 and unaffected by 0.2 μmol/L pirenzepine or 1 μmol/L propranolol. In the presence of ACh, exposure to atropine elicited stimulation of I Ca,L . ACh-induced inhibition and rebound stimulation of current were independent of external Ca 2+ . Rebound stimulation of I Ca,L was associated with a negative shift in the voltage dependence of I Ca,L activation. Inhibition of protein kinase A by 50 μmol/L Rp-cAMPs decreased basal I Ca,L by 36±1% and abolished the rebound stimulation of I Ca,L . Forskolin (0.01 μmol/L) or isoproterenol (0.01 μmol/L) had no effect on basal I Ca,L , but each accentuated the rebound increase in I Ca,L . When adenylate cyclase was maximally stimulated with 1 μmol/L isoproterenol plus 2 μmol/L forskolin, ACh decreased I Ca,L but failed to elicit rebound stimulation of I Ca,L . Milrinone (10 μmol/L) increased basal I Ca,L by 70±7% and significantly attenuated the rebound stimulation of I Ca,L . Exposure to 1 mmol/L 8-bromo-cGMP elicited a small decrease in basal I Ca,L , attenuated ACh-induced inhibition, and enhanced the rebound stimulation of I Ca,L . Incubation in pertussis toxin prevented all ACh-induced changes in I Ca,L . Inhibition of nitric oxide synthase by 100 μmol/L N G -monomethyl- l -arginine (L-NMMA) decreased basal I Ca,L by −20±5%, prevented ACh-induced inhibition, and markedly attenuated the rebound stimulation of I Ca,L . We conclude that in cat atrial myocytes ACh acts via M 2 muscarinic receptors and pertussis toxin–sensitive G protein to inhibit basal I Ca,L and that on withdrawal ACh elicits a rebound stimulation of I Ca,L . Rebound stimulation of I Ca,L is mediated via cAMP-dependent protein kinase A enhanced by ACh-induced inhibition of phosphodiesterase. This mechanism contributes directly to the positive inotropic response that follows cholinergic inhibition of atrial contraction.

A nystatin-perforated patch whole cell recording method was used to study the effects of acetylcholine (ACh) on ACh-induced K+ currents in atrial myocytes isolated from cat hearts. The general protocol involved an initial 4-min exposure to ACh (ACh1), followed by a 4-min washout in ACh-free Tyrode solution and then a second 4-min ACh exposure (ACh2). Voltage ramps (40 mV/s) between -130 and +30 mV were used to assess changes in total membrane conductance. ACh2 (10 microM) induced an increase in K+ conductance that was significantly larger than that induced by ACh1 (10 microM) at voltages both negative and positive to the reversal potential. The potentiated current induced by ACh2 reversed at about -80 mV and inwardly rectified at voltages positive to the reversal potential. External Ba2+ (5 mM) or tetraethylammonium (10 mM) abolished all ACh2-induced increases in membrane conductance. The sensitivity to K+ channel blockers, reversal potential, and the rectifying properties indicate that the current potentiated by ACh2 is a K+ current. Atropine (1 microM) blocked all effects of ACh on K+ currents. Potentiation of K+ current by ACh2 required 1) ACh1 concentrations > or = 1 microM, 2) ACh1 duration > or = 2 min, and 3) recovery interval > or = 2 min. We conclude that an initial exposure to ACh potentiates subsequent ACh-induced increases in K+ current. ACh-induced potentiation depends on the concentration and duration of the initial ACh exposure and the recovery interval between consecutive ACh exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NO i ) in cat atrial myocytes. Field stimulation (1 Hz) of cells or exposure of quiescent cells to ACh (1 to 10 μmol/L) had no effect on NO i . However, in field-stimulated cells, ACh exposure increased NO i , and ACh withdrawal elicited an additional, prominent increase in NO i production. During ACh exposure, addition of 1 μmol/L atropine increased NO i production similar to ACh withdrawal. ACh-induced increases in NO i were reduced by prior exposure to 1 mmol/L extracellular Ca 2+ ([Ca 2+ ] o ) and prevented by 0.5 mmol/L [Ca 2+ ] o , 1 μmol/L verapamil, 1 μmol/L atropine, 10 μmol/L L- N 5 -(1-iminoethyl)ornithine, 10 μmol/L W-7, or incubating cells in pertussis toxin or 10 μmol/L LY294002 (inhibits phosphatidylinositol 3-kinase). Switching to 0.5 mmol/L [Ca 2+ ] o during ACh withdrawal prevented the additional increase in NO i . ACh exposure increased phosphorylation (Ser 473 ) of protein kinase B (Akt), and this effect was blocked by LY294002 and unaffected in low (0.5 mmol/L) [Ca 2+ ] o . Confocal microscopy revealed that ACh exposure increased NO i at local subsarcolemmal sites, and ACh withdrawal additionally increased NO i by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-β-cyclodextrin abolished ACh-induced NO i production. We conclude that in cat atrial myocytes, ACh stimulates NO i release from local subsarcolemmal sites. ACh-induced increases in NO i requires both muscarinic receptor–mediated G i protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca 2+ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NO i elicited by ACh withdrawal result from the recovery of Ca 2+ influx after ACh inhibition. NO signaling elicited by ACh withdrawal stimulates rapid recovery from cholinergic atrial inhibition.

The purpose of this study is to determine the effects of brief rapid pacing (RP; ∼200–240 beats/min for ∼5 min) on contractile function in ventricular myocytes. RP was followed by a sustained inhibition of peak systolic cell shortening (−44 ± 4%) that was not due to changes in diastolic cell length, membrane voltage, or L-type Ca2+current ( ICa,L). During RP, baseline and peak intracellular Ca2+concentration ([Ca2+]i) increased markedly. After RP, Ca2+transients were similar to control. The effects of RP on cell shortening were not prevented by 1 μM calpain inhibitor I, 25 μMl- N5-(1-iminoethyl)-orthinthine, or 100 μM NG-monomethyl-l-arginine. However, RP-induced inhibition of cell shortening was prevented by lowering extracellular [Ca2+] (0.5 mM) during RP or exposure to chelerythrine (2–4 μM), a protein kinase C (PKC) inhibitor, or LY379196 (30 nM), a selective inhibitor of PKC-β. Exposure to phorbol ester (200 nM phorbol 12-myristate 13-acetate) inhibited cell shortening (−46 ± 7%). Western blots indicated that cat myocytes express PKC-α, -δ, and -ε as well as PKC-β. These findings suggest that brief RP of ventricular myocytes depresses contractility at the myofilament level via Ca2+/PKC-dependent signaling. These findings may provide insight into the mechanisms of contractile dysfunction that follow paroxysmal tachyarrhythmias.

Background Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca 2+ influx via L-type Ca 2+ current and stimulate Ca 2+ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca 2+ . Overload of cellular Ca 2+ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca 2+ -induced delayed afterdepolarizations (DADs) in atrial myocytes. Methods and Results A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca 2+ ([Ca 2+ ] i ), respectively. Withdrawal of ACh (1 μmol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca 2+ current ( I Ca,L ) (+45%) and Na/Ca exchange current ( I NaCa ) (+16%) and the appearance of transient inward current ( I ti ) and spontaneous [Ca 2+ ] i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 μmol/L) or H-89 (2 μmol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 μmol/L) abolished I NaCa and the appearance of I ti without decreasing the rebound stimulation of I Ca,L elicited by withdrawal of ACh. Conclusions Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca 2+ influx via I Ca,L and uptake of SR Ca 2+ . As a result, cellular Ca 2+ overload causes enhanced SR Ca 2+ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.

In atrial myocytes, an initial exposure to acetylcholine (ACh1) exerts a short-term conditioning effect such that a second ACh exposure (ACh2) activates ATP-sensitive K+ current (IK,ATP). The purpose of the present study was to determine the mechanism underlying the short-term conditioning induced by ACh that results in subsequent ACh-induced activation of IK.ATP. Cat atrial myocytes were studied using a nystatin-perforated patch whole cell recording method. Changes in L-type Ca2+ current (ICa,L) amplitude were used as an index of relative changes in cyclic AMP (cAMP). The results show that when atrial myocytes are treated with two consecutive exposures to 10 microM ACh separated by a recovery interval, ACh2 activates a larger increase in potassium conductance (gK+) than ACh1. The additional ACh2-induced increase in gK+ is selectively blocked by 10 microM glibenclamide, identifying the current as IK,ATP. Moreover, ICa,L activated immediately after the withdrawal of ACh1 exhibited a transient increase in amplitude above control (+ 76%), consistent with rebound stimulation of cAMP. Rp-cAMPs (50 microM), a selective antagonist of cAMP-dependent protein kinase A, blocked the rebound stimulation of ICa,L and abolished ACh2-induced activation of IK,ATP. Thapsigargin (5 microM), an inhibitor of Ca2+ ATPase in the sarcoplasmic reticulum (SR), abolished ACh2-induced activation of IK,ATP without decreasing rebound stimulation of ICa,L. Rebound stimulation of ICa,L and ACh2-induced activation of IK,ATP both varied as a function of ACh1 duration. We conclude that withdrawal of an initial ACh exposure elicits a rebound cAMP-mediated stimulation of SR Ca2+ uptake. This mechanism induces a short-term conditioning in atrial myocytes such that a subsequent ACh exposure activates IK,ATP. The present results demonstrate novel cholinergic signaling mechanisms in the regulation of IK,ATP.

Vagal nerve-induced inhibition of the heartbeat is followed by a postvagal increase in heart rate above control levels, postvagal tachycardia. In the present study, we used a perforated-patch/whole-cell recording method to determine the role of L-type Ca 2+ current (I Ca,L ) and the hyperpolarization-activated inward current (I f ) in the positive chronotropic response elicited by withdrawal of acetylcholine (ACh). Experiments were performed on sinoatrial node (SAN) and latent atrial pacemaker (LAP) cells isolated from cat right atrium. Withdrawal of a 2-minute exposure to 1 μmol/L ACh elicited a rebound stimulation of I Ca,L in both SAN (33±4%) and LAP (50±6%) cells above control. Similarly, withdrawal of ACh (1 μmol/L) elicited a rebound stimulation of I f in both SAN (21±4%) and LAP (20±6%) cells. During the rebound stimulation of I Ca,L , peak amplitude was increased throughout the voltage range, and the voltage dependence of activation was shifted to more negative voltages. Action potential recordings from both SAN and LAP cells showed that following ACh-induced inhibition, withdrawal of ACh elicited a concomitant rebound increase in action potential amplitude (+21±2% and +21±3%, respectively) and decrease in pacemaker cycle length (30±5% and 44±5%, respectively) compared with control. H-89 (2 μmol/L), an inhibitor of cAMP-dependent protein kinase A, abolished the rebound increase of I Ca,L , I f , action potential amplitude, and decrease in pacemaker cycle length elicited by withdrawal of ACh. In the presence of 2 mmol/L cesium, a blocker of I f , the rebound decrease in pacemaker cycle length elicited by withdrawal of ACh was unchanged. We conclude that in SAN and LAP cells, withdrawal of ACh elicits a positive chronotropic response primarily through a cAMP-mediated rebound stimulation of I Ca,L . These findings are the first demonstration of an intrinsic cellular mechanism that may contribute directly to the nonadrenergic component of postvagal tachycardia.

Abstract Our previous work on atrial myocytes suggested that the effect of acetylcholine (ACh) to increase K + conductance can be potentiated by prior loading of the sarcoplasmic reticulum (SR) with Ca 2+ . The present study, therefore, sought to determine whether prior exposure to isoproterenol (ISO) potentiates ACh-induced increases in K + conductance and the underlying mechanisms. A nystatin–perforated patch whole-cell configuration was used to record from cat atrial myocytes. Voltage-clamp ramps (40 mV/s) were used to assess total membrane conductance. The experimental protocol consisted of two consecutive 30-second ACh exposures (ACh 1 and ACh 2 ) separated by a 6-minute recovery period in ACh-free solution. In general, experimental interventions, such as exposure to ISO, were imposed during the period between ACh 1 and ACh 2 to determine their effects on the response to ACh 2 in relation to ACh 1 . Under control conditions, K + conductances induced by ACh 1 and ACh 2 were not different from one another with or without activation of L-type Ca 2+ current (I Ca,L ) during the recovery period. When 1 μmol/L ISO plus I Ca,L activation was imposed during the recovery period, ACh 2 induced a significantly larger increase in K + conductance than ACh 1 . The ACh 2 -induced K + current potentiated by ISO was time independent and selectively blocked by 10 μmol/L glibenclamide and therefore identified as ATP-sensitive K + current (I K,ATP ). The effect of ISO to induce ACh 2 -activated I K,ATP was mimicked by 1 μmol/L forskolin or 200 μmol/L 8-(4-chlorophenylthio)-cAMP, but not by 0.5 μmol/L BAY K 8644, and was selectively abolished by (1) 5 μmol/L thapsigargin or 1 μmol/L ryanodine, agents that prevent accumulation of SR Ca 2+ , (2) inhibition of L-type Ca 2+ current (I Ca,L ) by 1 μmol/L nisoldipine or zero external Ca 2+ , (3) 50 μmol/L Rp-cAMPs, an inhibitor of cAMP-dependent protein kinase A, or (4) 2 μmol/L propranolol. Atropine (1 μmol/L) abolished all ACh-induced currents. Moreover, ACh 2 -activated I K,ATP was selectively blocked by 0.2 μmol/L pirenzepine, an M 1 muscarinic receptor antagonist, or 0.1 μmol/L calphostin C, a selective inhibitor of protein kinase C. AFDX116 (100 μmol/L), an M 2 muscarinic receptor antagonist, blocked the conventional ACh-activated K + current and revealed ACh 2 -activated I K,ATP . These results indicate that prior exposure to ISO potentiates ACh-induced increases in K + current via ACh-activated I K,ATP . ISO acts via β-adrenergic receptors and cAMP to enhance both Ca 2+ influx via I Ca,L and SR Ca 2+ uptake. By loading SR Ca 2+ , ISO facilitates ACh-induced stimulation of the M 1 receptor/phosphoinositol signaling pathway to activate I K,ATP . These findings suggest that prior β-adrenergic stimulation accentuates subsequent cholinergic inhibition of atrial function via ACh-induced activation of ATP-sensitive K + channels.

The preceding paper [Y. G. Wang and S. L. Lipsius, Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H1313–H1321, 1995.] showed that when an atrial myocyte is treated with two consecutive exposures to acetylcholine (ACh) separated by a recovery interval, the second ACh exposure elicits a larger increase in K+ conductance than the first ACh exposure. In the present study a nystatin-perforated patch whole cell recording method was used to determine the mechanisms underlying the potentiating effect of ACh on ACh-induced K+ currents and the nature of the potentiated K+ current. The K+ current potentiated by the second ACh exposure was selectively abolished by 1) M1 muscarinic receptor block by 0.1 microM pirenzepine, 2) depletion of sarcoplasmic reticulum (SR) Ca2+ stores by 1 microM ryanodine or 10 mM caffeine, 3) intracellular dialysis with 10 mM ethylene glycolbis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 4) omitting external Ca2+, 5) 50% external Na+, 6) inhibition of protein kinase C by 0.01 microM staurosporine or 0.1 microM calphostin C, or 7) inhibition of ATP-sensitive K+ channels with 10 microM glibenclamide (Glib). AFDX-116 (100 microM), an M2 muscarinic receptor antagonist, selectively abolished the conventional ACh-activated K+ current and revealed an ACh-activated Glib-sensitive K+ current. In addition, with K+ conductances blocked and zero external Ca2+, 10 microM ACh induced a small nonselective inward current carried by Na+.(ABSTRACT TRUNCATED AT 250 WORDS)