D. F. Van Helden

Pacemaker potentials in lymphatic smooth muscle of the guinea‐pig mesentery.

1. Intracellular microelectrode recordings have been made from lymphatic smooth muscle (SM) to investigate pacemaker mechanisms underlying lymphatic pumping. 2. The SM of small lymphangions or that of short segments, cut from lymphangions of any length, behaved similarly. Both preparations exhibited spontaneous transient depolarizations (STDs) and exhibited simplified electrical characteristics approximating those of a spherical cell. 3. STDs were found to underlie activation of action potentials and hence constrictions. 4. The level of STD activity correlated to the pumping activity of lymphangions, the SM from more active chambers exhibiting increased STD activity. 5. Lymphatic SM exhibited STDs with properties similar to the STDs of mesenteric veins. STDs appeared to be of myogenic origin as they were present despite denervation or substantial destruction of the endothelium. 6. Noradrenaline enhanced the size and frequency of STDs. 7. STD activity was abolished by chelation of cytosolic Ca2+. 8. It is proposed that STDs provide a mechanism for pacemaking in the lymphatic SM studied here. Furthermore, it is postulated that STDs are the consequence of Ca(2+)‐dependent pulsatile release of an intracellular messenger, probably Ca2+ itself. This mechanism provides a novel means for pacemaking.

Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea‐pig gastric pylorus

1 Intracellular recordings made in single bundle strips of a visceral smooth muscle revealed rhythmic spontaneous membrane depolarizations termed slow waves (SWs). These exhibited ‘pacemaker’ and ‘regenerative’ components composed of summations of more elementary events termed spontaneous transient depolarizations (STDs). 2 STDs and SWs persisted in the presence of tetrodotoxin, nifedipine and ryanodine, and upon brief exposure to Ca2+‐free Cd2+‐containing solutions; they were enhanced by ACh and blocked by BAPTA AM, cyclopiazonic acid and caffeine. SWs were also inhibited in heparin‐loaded strips. 3 SWs were observed over a wide range of membrane potentials (e.g. ‐80 to ‐45 mV) with increased frequencies at more depolarized potentials. 4 Regular spontaneous SW activity in this preparation began after 1–3 h superfusion of the tissue with physiological saline following the dissection procedure. Membrane depolarization applied before the onset of this activity induced bursts of STD‐like events (termed the ‘initial’ response) which, when larger than threshold levels initiated regenerative responses. The combined initial‐regenerative waveform was termed the SW‐like action potential. 5 Voltage‐induced responses exhibited large variable latencies (typical range 0.3‐4 s), refractory periods of ≈11 s and a pharmacology that was indistinguishable from those of STDs and spontaneous SWs. 6 The data indicate that SWs arise through more elementary inositol 1,4,5‐trisphosphate (IP3) receptor‐induced Ca2+ release events which rhythmically synchronize to trigger regenerative Ca2+ release and induce inward current across the plasmalemma. The finding that action potentials, which were indistinguishable from SWs, could be evoked by depolarization suggests that membrane potential modulates IP3 production. Voltage feedback on intracellular IP3‐sensitive Ca2+ release is likely to have a major influence on the generation and propagation of SWs.

Endothelium‐dependent modulation of pacemaking in lymphatic vessels of the guinea‐pig mesentery.

1. Endothelial control of the rate of constrictions and the underlying pacemaker potentials has been studied in vitro in guinea‐pig mesenteric lymphatic vessels. 2. ACh stimulated 60% of intraluminally perfused vessels to slow or abolish lymphatic constrictions. This action was inhibited by atropine and was likely to be due to the release of endothelium‐derived nitric oxide (EDNO) as the effect was absent after endothelial lysis, mimicked by sodium nitroprusside (SNP), blocked by N omega‐nitro L‐arginine (NOLA) and partially inhibited by Methylene Blue (MB). 3. The remaining 40% of perfused vessels did not mechanically respond to ACh or SNP. In four of seven such vessels this appeared to be due to excessive perfusion‐associated release of EDNO, as incubation with NOLA restored the response to SNP. 4. Application of NOLA or MB in perfused vessels significantly increased constriction frequency, further indicating perfusion‐associated release of EDNO. 5. ACh induced a marked increase in endothelial [Ca2+]i of both mechanically responding and non‐responding vessels. This ACh‐induced increase could be repetitively induced when Ca2+ was present in the perfusate, but rapidly ran down when a Ca(2+)‐free EGTA perfusate was used. 6. Intracellular recordings from the smooth muscle of non‐perfused vessel segments demonstrated an ACh‐induced hyperpolarization and decrease in membrane resistance, changes which were prevented by atropine, NOLA, MB and endothelial lysis and mimicked by SNP. 7. ACh directly reduced the size of the underlying pacemaker potentials termed spontaneous transient depolarizations (STDs). 8. NOLA and MB enhanced STDs in non‐perfused vessel segments indicating an endogenous release of EDNO. 9. It is concluded that the lymphatic endothelium produces and releases EDNO endogenously, during perfusion or after stimulation with ACh, to decrease the efficacy of STDs to generate action potentials and resultant constrictions.

ENHANCED EXCITATORY JUNCTION POTENTIALS IN MESENTERIC ARTERIES FROM SPONTANEOUSLY HYPERTENSIVE RATS

Excitatory junction potentials (EJPs) were examined using intracellular recording techniques in mesenteric arteries isolated from 12- to 15-week-old spontaneously hypertensive (SHR), Wistar Kyoto (WKY) and Sprague Dawley (SD) rats. The amplitudes of EJPs evoked by single supramaximal stimuli were larger in arteries from SHRs (12.9±0.7 mV,n=16) than in arteries from either WKYs (5.2±0.5 mV,n=24) or SDs (8.6±0.8 mV,n=15). The time constant of decay of EJPs did not differ significantly, suggesting that the passive electrical properties of the vascular smooth muscle are similar in the three rat strains. Spontaneous EJPs recorded in tissues from SHRs and WKYs had similar amplitude frequency distributions, suggesting that the quantal size is also similar between strains. In some arteries from SHRs, EJPs evoked by single stimuli triggered muscle action potentials (MAPs). Visible constriction only occurred following a MAP. In tissues from all three strains, summation of EJPs triggered MAPs. As EJPs are generated by the sympathetic co-transmitter adenosine 5′-triphosphate (ATP), the findings of the present study indicate that purinergic transmission is enhanced in mesenteric arteries from SHRs, probably as a result of an increase in quantal release. A consequence is that when nerves are activated SHR arteries more readily undergo constriction that is dependent on voltage-activated Ca2+ influx.

Co-ordination of contractile activity in guinea-pig mesenteric lymphatics.

1. Intraluminally perfused lymphatic vessels from the mesentery of the guinea‐pig were examined in vitro to investigate their contractile activity and the co‐ordination of this activity between adjacent lymphangions. 2. Lymphangions constricted at fairly regular intervals and exhibited ‘refractory’ periods of up to 3 s during which constrictions did not occur. 3. The contractile activity of adjacent lymphangions was highly co‐ordinated. 4. The smooth muscle was found to be continuous between the adjacent lymphangions for the majority of valve regions examined morphologically (52 of 63 preparations). 5. Mechanical and electrical coupling between adjacent lymphangions was indicated, as some lymphangions underwent transient dilatations just prior to constriction, whereas direct electrophysiological measurements showed that the smooth muscle of most adjacent lymphangions was electrically coupled across the valve (15 out of 20 pairs of lymphangions). 6. It is concluded that perfused lymphangions of guinea‐pig mesenteric lymphatic vessels rhythmically constrict, with the contractile activity of adjacent lymphangions highly co‐ordinated. The findings also indicate that transmission of both mechanical and electrical signals between the adjacent lymphangions contribute to the co‐ordination of their contractile activity.

Spontaneous and noradrenaline‐induced transient depolarizations in the smooth muscle of guinea‐pig mesenteric vein.

1. Recordings of membrane current were made in the smooth muscle of short segments of mesenteric vein before or during stimulation with noradrenaline (NA). 2. Small veins (diameter less than 150 microns) when cut into short segments (of length less than 250 microns) had the passive electrical characteristics of short cables both before and during activation with NA. 3. Spontaneous transient depolarizations (STDs) or the underlying inward currents (STICs) were recorded in these preparations. STDs were of myogenic origin as they were not blocked by tetrodotoxin or antagonists to the alpha‐adrenoreceptor and persisted after either denervation or disruption of the endothelium. 4. STDs had time courses similar to the underlying currents and were generally slow compared to the membrane time constant of the short segments. 5. STDs and the underlying currents showed large variability in frequency and amplitude both within and between short segments. Currents were typically less than 0.3 nA, were characteristic in shape, had half‐durations normally in the range 0.1‐0.7 s and reversed at about ‐25 mV. 6. STDs persisted, but at markedly reduced frequencies, after exposure (3‐10 min) to a solution in which cobalt ions had been used to substitute for Ca2+. STDs were also substantially suppressed by exposure to low‐chloride solution. 7. Caffeine induced excitatory and inhibitory conductances. An initial component of the caffeine‐induced responses showed similar voltage dependence to STDs and was also suppressed by exposure to low‐chloride solution. 8. NA, through activation of alpha‐adrenoreceptors, caused a sustained depolarization or inward current (under voltage clamp) with considerable membrane potential or current noise often in the form of agonist‐induced spontaneous transient depolarizations (ASTDs) or currents (ASTICs). There were marked increases in amplitude and frequency of ASTDs with increase in NA concentrations. 9. ASTDs appeared to be generated within the smooth muscle as they were activated in preparations which had been denervated or in which the endothelium had been disrupted. 10. Except for the pathway of activation, ASTDs were indistinguishable from STDs having half‐durations in the same range (0.1‐2 s with the majority less than 0.7 s). The underlying currents again showed large variation in amplitude (typically less than 0.3 nA; maximum recorded 0.9 nA). They reversed at about ‐25 mV, could still be elicited in cobalt solution (but at reduced intensity for long exposures to this low‐Ca2+ solution) and were reduced by long term exposure to low‐chloride solution.(ABSTRACT TRUNCATED AT 400 WORDS)

PACEMAKER CURRENTS IN MOUSE LOCUS COERULEUS NEURONS

We have characterized the currents that flow during the interspike interval in mouse locus coeruleus (LC) neurons, by application of depolarizing ramps and pulses, and compared our results with information available for rats. A tetrodotoxin (TTX)-sensitive current was the only inward conductance active during the interspike interval; no TTX-insensitive Na(+) or oscillatory currents were detected. Ca(2+)-free and Ba(2+)-containing solutions failed to demonstrate a Ca(2+) current during the interspike interval, although a Ca(2+) current was activated at membrane potentials positive to -40 mV. A high- tetraethylammonium chloride (TEA) (15 mM) sensitive current accounted for almost all the K(+) conductance during the interspike interval. Ca(2+)-activated K(+), inward rectifier and low-TEA (10 muM) sensitive currents were not detected within the interspike interval. Comparison of these findings to those reported for neonatal rat LC neurons indicates that the pacemaker currents are similar, but not identical, in the two species with mice lacking a persistent Ca(2+) current during the interspike interval. The net pacemaking current determined by differentiating the interspike interval from averaged action potential recordings closely matched the net ramp-induced currents obtained either under voltage clamp or after reconstructing this current from pharmacologically isolated currents. In summary, our results suggest the interspike interval pacemaker mechanism in mouse LC neurons involves a combination of a TTX-sensitive Na(+) current and a high TEA-sensitive K(+) current. In contrast with rats, a persistent Ca(2+) current is not involved.

Multiple Modes of Ryanodine Receptor 2 Inhibition by Flecainide

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na+ and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca2+], [Mg2+], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca2+] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide.

Edge detection at multiple locations using a ‘radar’ tracking algorithm as exemplified in isolated guinea-pig lymphatic vessels

An algorithm based on the Kalman filter and used for tracking a target in a noisy environment has been applied to monitoring the position of moving boundaries as recorded by a video camera. The method, termed the Probabilistic Data Association Filter, is computationally efficient, allowing simultaneous monitoring at many positions along the boundary at video frame rates of 25 Hz. The present study uses the tracking procedure to investigate and correlate constrictions along lymphatic vessels. The procedure is well suited for real time tracking of visualised biological events including the simultaneous monitoring of constrictions in networks of blood vessels, the movement of cells or the trajectory of intracellular events monitored by intracellular fluorescent markers.

Oxytocin depolarizes mitochondria in isolated myometrial cells.

Oxytocin is known to play important roles in uterine contractions, mediated at least in part by increasing intracellular Ca2+ concentration ([Ca2+]i), through enhancing extracellular Ca2+ entry and Ca2+ release from the sarcoplasmic reticulum, processes that are intimately linked with mitochondria. This study examined the effects of oxytocin on mitochondrial function. This was achieved by measuring the ratiometric JC‐1 fluorescence signal in isolated myometrial cells, which provides a relative measure of the mitochondrial membrane potential (ψ  m), and also by loading the cells with Oregon Green BAPTA‐AM to examine changes in [Ca2+]i. Oxytocin (1 nm) depolarized the ψ  m to 73.8 ± 3.7% of the control value (P < 0.05; perfused for 11 min) and also caused a transient increase in [Ca2+]i. The depolarization of mitochondrial membrane potential was effectively reversed by 2‐aminoethoxydiphenyl borate, nifedipine, Ca2+‐free solution or oligomycin, with the ratiometric JC‐1 fluorescence signal becoming no different from the control value in all cases (i.e. P > 0.05). The reduction in ψ  m is likely to occur at least in part through the oxytocin‐induced increase in [Ca2+]i, causing enhanced mitochondrial uptake of Ca2+ and resultant dissipation of the mitochondrial electrochemical gradient. ATP synthase is also stimulated, which would further contribute to a decrease in ψ  m.