Mohammad S. Imtiaz

Ca2+ phase waves: a basis for cellular pacemaking and long‐range synchronicity in the guinea‐pig gastric pylorus

Ca2+ imaging and multiple microelectrode recording procedures were used to investigate a slow wave‐like electrical rhythmicity in single bundle strips from the circular muscle layer of the guinea‐pig gastric pylorus. The ‘slow waves’ (SWs) consisted of a pacemaker and regenerative component, with both potentials composed of more elementary events variously termed spontaneous transient depolarizations (STDs) or unitary potentials. STDs and SW pacemaker and regenerative potentials exhibited associated local and distributed Ca2+ transients, respectively. Ca2+ transients were often larger in cellular regions that exhibited higher basal Ca2+ indicator‐associated fluorescence, typical of regions likely to contain intramuscular interstitial cells of Cajal (ICCIM). The emergence of rhythmicity arose through entrainment of STDs resulting in pacemaker Ca2+ transients and potentials, events that exhibited considerable spatial synchronicity. Application of ACh to strips exhibiting weak rhythmicity caused marked enhancement of SW synchronicity. SWs and underlying Ca2+ increases exhibited very high ‘apparent conduction velocities’ (‘CVs’) orders of magnitude greater than for sequentially conducting Ca2+ waves. Central interruption of either intercellular connectivity or inositol 1,4,5‐trisphosphate receptor (IP3R)‐mediated store Ca2+ release in strips caused SWs at the two ends to run independently of each other, consistent with a coupled oscillator‐based mechanism. Central inhibition of stores required much wider regions of blockade than inhibition of connectivity indicating that stores were voltage‐coupled. Simulations, made using a conventional store array model but now including depolarization coupled to IP3R‐mediated Ca2+ release, predicted the experimental findings. The linkage between membrane voltage and Ca2+ release provides a means for stores to interact as strongly coupled oscillators, resulting in the emergence of Ca2+ phase waves and associated pacemaker potentials. This distributed pacemaker triggers regenerative Ca2+ release and resultant SWs.

Generation and propagation of gastric slow waves

1. Mechanisms underlying the generation and propagation of gastrointestinal slow wave depolarizations have long been controversial. The present review aims to collate present knowledge on this subject with specific reference to slow waves in gastric smooth muscle.

Spontaneous transient depolarizations in lymphatic vessels of the guinea pig mesentery: pharmacology and implication for spontaneous contractility

Guinea pig mesenteric lymphatic vessels exhibit rhythmic constrictions induced by action potential (AP)-like spikes and initiated by entrainment of spontaneous transient depolarizations (STDs). To characterize STDs and the signaling mechanisms responsible for their occurrence, we used intracellular microelectrodes, Ca2+ imaging, and pharmacological agents. In our investigation of the role of intracellular Ca2+ released from Ca2+ stores, we observed that intracellular Ca2+ transients accompanied some STDs, although there were many exceptions where Ca2+ transients occurred without accompanying STDs. STD frequency and amplitude were markedly affected by activators/inhibitors of inositol 1,4,5-trisphosphate receptors (IP3Rs) but not by treatments known to alter Ca2+ release via ryanodine receptors. A role for Ca2+-activated Cl(-) (Cl(Ca)) channels was indicated, as STDs were dependent on the Cl(-) but not Na+ concentration of the superfusing solution and were inhibited by the Cl(Ca) channel blockers niflumic acid (NFA), anthracene 9-carboxylic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid but not by the volume-regulated Cl(-) blocker DIDS. Increases in STD frequency and amplitude induced by agonist stimulation were also inhibited by NFA. Nifedipine, the hyperpolarization-activated inward current blocker ZD-7288, and the nonselective cation/store-operated channel blockers SKF-96365, Gd3+, and Ni2+ had no or marginal effects on STD activity. However, nifedipine, 2-aminoethoxydiphenyl borate, NFA, SKF-96365, Gd3+, and Ni2+ altered the occurrence of spontaneous APs. Our findings support a role for Ca2+ release through IP3Rs and a resultant opening of Cl(Ca) channels in STD generation and confirm the importance of these events in the initiation of lymphatic spontaneous APs and subsequent contractions. The abolition of spontaneous APs by blockers of other excitatory ion channels suggests a contribution of these conductances to lymphatic pacemaking.

A Theoretical Model of Slow Wave Regulation Using Voltage-Dependent Synthesis of Inositol 1,4,5-Trisphosphate

A qualitative mathematical model is presented that examines membrane potential feedback on synthesis of inositol 1,4,5-trisphosphate (IP(3)), and its role in generation and modulation of slow waves. Previous experimental studies indicate that slow waves show voltage dependence, and this is likely to result through membrane potential modulation of IP(3). It is proposed that the observed response of the tissue to current pulse, pulse train, and maintained current injection can be explained by changes in IP(3), modulated through a voltage-IP(3) feedback loop. Differences underlying the tissue responses to current injections of opposite polarities are shown to be due to the sequence of events following such currents. Results from this model are consistent with experimental findings and provide further understanding of these experimental observations. Specifically, we find that membrane potential can induce, abolish, and modulate slow wave frequency by altering the excitability of the tissue through the voltage-IP(3) feedback loop.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca2+ release. Although dantrolene inhibits Ca2+ release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca2+ release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca2+] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca2+ release in the absence of CaM, but reduced Ca2+ wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

Synchronization of Ca2+ oscillations: a coupled oscillator‐based mechanism in smooth muscle

Entrained oscillations in Ca2+ underlie many biological pacemaking phenomena. In this article, we review a long‐range signaling mechanism in smooth muscle that results in global outcomes of local interactions. Our results are derived from studies of the following: (a) slow‐wave depolarizations that underlie rhythmic contractions of gastric smooth muscle; and (b) membrane depolarizations that drive rhythmic contractions of lymphatic smooth muscle. The main feature of this signaling mechanism is a coupled oscillator‐based synchronization of Ca2+ oscillations across cells that drives membrane potential changes and causes coordinated contractions. The key elements of this mechanism are as follows: (a) the Ca2+ release–refill cycle of endoplasmic reticulum Ca2+ stores; (b) Ca2+‐dependent modulation of membrane currents; (c) voltage‐dependent modulation of Ca2+ store release; and (d) cell–cell coupling through gap junctions or other mechanisms. In this mechanism, Ca2+ stores alter the frequency of adjacent stores through voltage‐dependent modulation of store release. This electrochemical coupling is many orders of magnitude stronger than the coupling through diffusion of Ca2+ or inositol 1,4,5‐trisphosphate, and thus provides an effective means of long‐range signaling.

Intracranial pressure elevation reduces flow through collateral vessels and the penetrating arterioles they supply. A possible explanation for ‘collateral failure' and infarct expansion after ischemic stroke

Recent human imaging studies indicate that reduced blood flow through pial collateral vessels (‘collateral failure’) is associated with late infarct expansion despite stable arterial occlusion. The cause for ‘collateral failure’ is unknown. We recently showed that intracranial pressure (ICP) rises dramatically but transiently 24 hours after even minor experimental stroke. We hypothesized that ICP elevation would reduce collateral blood flow. First, we investigated the regulation of flow through collateral vessels and the penetrating arterioles arising from them during stroke reperfusion. Wistar rats were subjected to intraluminal middle cerebral artery (MCA) occlusion (MCAo). Individual pial collateral and associated penetrating arteriole blood flow was quantified using fluorescent microspheres. Baseline bidirectional flow changed to MCA-directed flow and increased by 4450% immediately after MCAo. Collateral diameter changed minimally. Second, we determined the effect of ICP elevation on collateral and watershed penetrating arteriole flow. Intracranial pressure was artificially raised in stepwise increments during MCAo. The ICP increase was strongly correlated with collateral and penetrating arteriole flow reductions. Changes in collateral flow post-stroke appear to be primarily driven by the pressure drop across the collateral vessel, not vessel diameter. The ICP elevation reduces cerebral perfusion pressure and collateral flow, and is the possible explanation for ‘collateral failure’ in stroke-in-progression.

ß-Adrenergic Stimulation Increases RyR2 Activity via Intracellular Ca2+ and Mg2+ Regulation

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca2+ and Mg2+ and the role of these changes in SR Ca2+ release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca2+] <1 µM, ß-adrenergic stimulation increased luminal Ca2+ activation of single RyR channels, decreased luminal Mg2+ inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg2+. At cytoplasmic [Ca2+] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg2+ and Ca2+ inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca2+ activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca2+ binding to the luminal Ca2+ site and decreasing its affinity for luminal Mg2+ and 2) decreasing affinity of the low-affinity Ca2+/Mg2+ cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.

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.

In silico assessment of kinetics and state dependent binding properties of drugs causing acquired LQTS

The Kv11.1 or hERG potassium channel is responsible for one of the major repolarising currents (IKr) in cardiac myocytes. Drug binding to hERG can result in reduction in IKr, action potential prolongation, acquired long QT syndrome and fatal cardiac arrhythmias. The current guidelines for pre-clinical assessment of drugs in development is based on the measurement of the drug concentration that causes 50% current block, i.e., IC50. However, drugs with the same apparent IC50 may have very different kinetics of binding and unbinding, as well as different affinities for the open and inactivated states of Kv11.1. Therefore, IC50 measurements may not reflect the true risk of drug induced arrhythmias. Here we have used an in silico approach to test the hypothesis that drug binding kinetics and differences in state-dependent affinity will influence the extent of cardiac action potential prolongation independent of apparent IC50 values. We found, in general that drugs with faster overall kinetics and drugs with higher affinity for the open state relative to the inactivated state cause more action potential prolongation. These characteristics of drug-hERG interaction are likely to be more arrhythmogenic but cannot be predicted by IC50 measurement alone. Our results suggest that the pre-clinical assessment of Kv11.1-drug interactions should include descriptions of the kinetics and state dependence of drug binding. Further, incorporation of this information into sophisticated in silico models should be able to better predict arrhythmia risk and therefore more accurately assess safety of new drugs in development.