Mitigation of Cardiovascular Disease and Toxicity through NRF2 Signalling.

Cardiovascular toxicity and diseases are phenomena that have a vastly detrimental impact on morbidity and mortality. The pathophysiology driving the development of these conditions is multifactorial but commonly includes the perturbance of reactive oxygen species (ROS) signalling, iron homeostasis and mitochondrial bioenergetics. The transcription factor nuclear factor erythroid 2 (NFE2)-related factor 2 (NRF2), a master regulator of cytoprotective responses, drives the expression of genes that provide resistance to oxidative, electrophilic and xenobiotic stresses. Recent research has suggested that stimulation of the NRF2 signalling pathway can alleviate cardiotoxicity and hallmarks of cardiovascular disease progression. However, dysregulation of NRF2 dynamic responses can be severely impacted by ageing processes and off-target toxicity from clinical medicines including anthracycline chemotherapeutics, rendering cells of the cardiovascular system susceptible to toxicity and subsequent tissue dysfunction. This review addresses the current understanding of NRF2 mechanisms under homeostatic and cardiovascular pathophysiological conditions within the context of wider implications for this diverse transcription factor.

Kir6.2-D323 and SUR2A-Q1336: an intersubunit interaction pairing for allosteric information transfer in the KATP channel complex.

ATP-sensitive potassium (KATP) channels are widely expressed and play key roles in many tissues by coupling metabolic state to membrane excitability. The SUR subunits confer drug and enhanced nucleotide sensitivity to the pore-forming Kir6 subunit, and so information transfer between the subunits must occur. In our previous study, we identified an electrostatic interaction between Kir6 and SUR2 subunits that was key for allosteric information transfer between the regulatory and pore-forming subunit. In this study, we demonstrate a second putative interaction between Kir6.2-D323 and SUR2A-Q1336 using patch clamp electrophysiological recording, where charge swap mutation of the residues on either side of the potential interaction compromise normal channel function. The Kir6.2-D323K mutation gave rise to a constitutively active, glibenclamide and ATP-insensitive KATP complex, further confirming the importance of information transfer between the Kir6 and SUR2 subunits. Sensitivity to modulators was restored when Kir6.2-D323K was co-expressed with a reciprocal charge swap mutant, SUR-Q1336E. Importantly, equivalent interactions have been identified in both Kir6.1 and Kir6.2 suggesting this is a second important interaction between Kir6 and the proximal C terminus of SUR.

A novel form of glycolytic metabolism-dependent cardioprotection revealed by PKCα and ß inhibition.

KEY POINTS: Acute hyperglycaemia at the time of a heart attack worsens the outcome for the patient. Acute hyperglycaemia is not limited to diabetic patients and can be due to a stress response in non-diabetics. This study suggests that the damaging cardiac effects of hyperglycaemia can be reversed by selective PKC inhibition. If PKCα/ß isoforms are inhibited, then high glucose itself becomes protective against ischaemic damage. Selective PKC inhibition may therefore be a useful therapeutic tool to limit the damage that can occur during a heart attack by stress-induced hyperglycaemia. ABSTRACT: Hyperglycaemia has a powerful association with adverse prognosis for patients with acute coronary syndromes (ACS). Previous work shows that high glucose prevents ischaemic preconditioning and causes electrical and mechanical disruption via protein kinase C α/ß (PKCα/ß) activation. The present study aimed to: (i) determine whether the adverse clinical association of hyperglycaemia in ACS can be replicated in preclinical cellular models of ACS and (ii) determine the importance of PKCα/ß activation to the deleterious effect of glucose. Freshly isolated rat, guinea pig or rabbit cardiomyocytes were exposed to simulated ischaemia after incubation in the presence of normal (5 mm) or high (20 mm) glucose in the absence or presence of small molecule or tat-peptide-linked PKCαß inhibitors. In each of the four conditions, the following hallmarks of cardioprotection were recorded using electrophysiology or fluorescence imaging: cardiomyocyte contraction and survival, action potential stability and time to failure, intracellular calcium and ATP, mitochondrial depolarization, ischaemia-sensitive leak current, and time to Kir 6.2 opening. High glucose alone resulted in decreased cardiomyocyte contraction and survival; however, it also imparted cardioprotection in the presence of PKCα/ß inhibitors. This cardioprotective phenotype displayed improvements in all of the measured parameters and decreased myocardium damage during whole heart coronary ligation experiments. High glucose is deleterious to cellular and whole-heart models of simulated ischaemia, in keeping with the clinical association of hyperglycaemia with an adverse outcome in ACS. PKCαß inhibition revealed high glucose to show a cardioprotective phenotype in this setting. The results of the present study suggest the potential for the therapeutic application of PKCαß inhibition in ACS associated with hyperglycaemia.

Small-Molecule G Protein-Coupled Receptor Kinase Inhibitors Attenuate G Protein-Coupled Receptor Kinase 2-Mediated Desensitization of Vasoconstrictor-Induced Arterial Contractions.

Vasoconstrictor-driven G protein-coupled receptor (GPCR)/phospholipase C (PLC) signaling increases intracellular Ca2+ concentration to mediate arterial contraction. To counteract vasoconstrictor-induced contraction, GPCR/PLC signaling can be desensitized by G protein-coupled receptor kinases (GRKs), with GRK2 playing a predominant role in isolated arterial smooth muscle cells. In this study, we use an array of GRK2 inhibitors to assess their effects on the desensitization of UTP and angiotensin II (AngII)-mediated arterial contractions. The effects of GRK2 inhibitors on the desensitization of UTP- or AngII-stimulated mesenteric third-order arterial contractions, and PLC activity in isolated mesenteric smooth muscle cells (MSMC), were determined using wire myography and Ca2+ imaging, respectively. Applying a stimulation protocol to cause receptor desensitization resulted in reductions in UTP- and AngII-stimulated arterial contractions. Preincubation with the GRK2 inhibitor paroxetine almost completely prevented desensitization of UTP- and attenuated desensitization of AngII-stimulated arterial contractions. In contrast, fluoxetine was ineffective. Preincubation with alternative GRK2 inhibitors (Takeda compound 101 or CCG224063) also attenuated the desensitization of UTP-mediated arterial contractile responses. In isolated MSMC, paroxetine, Takeda compound 101, and CCG224063 also attenuated the desensitization of UTP- and AngII-stimulated increases in Ca2+, whereas fluoxetine did not. In human uterine smooth muscle cells, paroxetine reversed GRK2-mediated histamine H1 receptor desensitization, but not GRK6-mediated oxytocin receptor desensitization. Utilizing various small-molecule GRK2 inhibitors, we confirm that GRK2 plays a central role in regulating vasoconstrictor-mediated arterial tone, highlighting a potentially novel strategy for blood pressure regulation through targeting GRK2 function.

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release.

Aberrant Zn(2+) homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn(2+) modulates cardiac ryanodine receptor gating and Ca(2+) dynamics in isolated cardiomyocytes. We reveal that Zn(2+) is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn(2+) concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca(2+). At concentrations of free Zn(2+) >1 nm, Zn(2+) is the main activating ligand, and the dependence on Ca(2+) is removed. Zn(2+) is therefore a higher affinity activator of RyR2 than Ca(2+). Millimolar levels of free Zn(2+) were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn(2+) modulates both the frequency and amplitude of Ca(2+) waves in a concentration-dependent manner and that physiological levels of Zn(2+) elicit Ca(2+) release in the absence of activating levels of cytosolic Ca(2+). This highlights a new role for intracellular Zn(2+) in shaping Ca(2+) dynamics in cardiomyocytes through modulation of RyR2 gating.

Early opening of sarcolemmal ATP-sensitive potassium channels is not a key step in PKC-mediated cardioprotection.

ATP-sensitive potassium (KATP) channels are abundantly expressed in the myocardium. Although a definitive role for the channel remains elusive they have been implicated in the phenomenon of cardioprotection, but the precise mechanism is unclear. We set out to test the hypothesis that the channel protects by opening early during ischemia to shorten action potential duration and reduce electrical excitability thus sparing intracellular ATP. This could reduce reperfusion injury by improving calcium homeostasis. Using a combination of contractile function analysis, calcium fluorescence imaging and patch clamp electrophysiology in cardiomyocytes isolated from adult male Wistar rats, we demonstrated that the opening of sarcolemmal KATP channels was markedly delayed after cardioprotective treatments: ischemic preconditioning, adenosine and PMA. This was due to the preservation of intracellular ATP for longer during simulated ischemia therefore maintaining sarcolemmal KATP channels in the closed state for longer. As the simulated ischemia progressed, KATP channels opened to cause contractile, calcium transient and action potential failure; however there was no indication of any channel activity early during simulated ischemia to impart an energy sparing hyperpolarization or action potential shortening. We present compelling evidence to demonstrate that an early opening of sarcolemmal KATP channels during simulated ischemia is not part of the protective mechanism imparted by ischemic preconditioning or other PKC-dependent cardioprotective stimuli. On the contrary, channel opening was actually delayed. We conclude that sarcolemmal KATP channel opening is a consequence of ATP depletion, not a primary mechanism of ATP preservation in these cells.

Defining the roles of arrestin2 and arrestin3 in vasoconstrictor receptor desensitization in hypertension.

Prolonged vasoconstrictor-stimulated phospholipase C activity can induce arterial constriction, hypertension, and smooth muscle hypertrophy/hyperplasia. Arrestin proteins are recruited by agonist-occupied G protein-coupled receptors to terminate signaling and counteract changes in vascular tone. Here we determine whether the development of hypertension affects arrestin expression in resistance arteries and how such changes alter arterial contractile signaling and function. Arrestin2/3 expression was increased in mesenteric arteries of 12-wk-old spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) controls, while no differences in arrestin expression were observed between 6-wk-old SHR and WKY animals. In mesenteric artery myography experiments, high extracellular K(+)-stimulated contractions were increased in both 6- and 12-wk-old SHR animals. Concentration-response experiments for uridine 5'-triphosphate (UTP) acting through P2Y receptors displayed a leftward shift in 12-wk, but not 6-wk-old animals. Desensitization of UTP-stimulated vessel contractions was increased in 12-wk-old (but not 6-wk-old) SHR animals. Dual IP3/Ca(2+) imaging in mesenteric arterial cells showed that desensitization of UTP and endothelin-1 (ET1) responses was enhanced in 12-wk-old (but not 6-wk-old) SHR compared with WKY rats. siRNA-mediated depletion of arrestin2 for UTP and arrestin3 for ET1, reversed the desensitization of PLC signaling. In conclusion, arrestin2 and 3 expression is elevated in resistance arteries during the emergence of the early hypertensive phenotype, which underlies an enhanced ability to desensitize vasoconstrictor signaling and vessel contraction. Such regulatory changes may act to compensate for increased vasoconstrictor-induced vessel contraction.

Sulfonylurea receptors regulate the channel pore in ATP-sensitive potassium channels via an intersubunit salt bridge.

ATP-sensitive potassium channels play key roles in many tissues by coupling metabolic status to membrane potential. In contrast with other potassium channels, the pore-forming Kir6 subunits must co-assemble in hetero-octameric complexes with ATP-binding cassette (ABC) family sulfonylurea receptor (SUR) subunits to facilitate cell surface expression. Binding of nucleotides and drugs to SUR regulates channel gating but how these responses are communicated within the complex has remained elusive to date. We have now identified an electrostatic interaction, forming part of a functional interface between the cytoplasmic nucleotide-binding domain-2 of SUR2 subunits and the distal C-terminus of Kir6 polypeptides that determines channel response to nucleotide, potassium channel opener and antagonist. Mutation of participating residues disrupted physical interaction and regulation of expressed channels, properties that were restored in paired charge-swap mutants. Equivalent interactions were identified in Kir6.1- and Kir6.2-containing channels suggesting a conserved mechanism of allosteric regulation.

PKC-mediated toxicity of elevated glucose concentration on cardiomyocyte function.

While it is well established that mortality risk after myocardial infarction (MI) increases in proportion to blood glucose concentration at the time of admission, it is unclear whether there is a direct, causal relationship. We investigated potential mechanisms by which increased blood glucose may exert cardiotoxicity. Using a Wistar rat or guinea-pig isolated cardiomyocyte model, we investigated the effects on cardiomyocyte function and electrical stability of alterations in extracellular glucose concentration. Contractile function studies using electric field stimulation (EFS), patch-clamp recording, and Ca2+ imaging were used to determine the effects of increased extracellular glucose concentration on cardiomyocyte function. Increasing glucose from 5 to 20 mM caused prolongation of the action potential and increased both basal Ca2+ and variability of the Ca2+ transient amplitude. Elevated extracellular glucose concentration also attenuated the protection afforded by ischemic preconditioning (IPC), as assessed using a simulated ischemia and reperfusion model. Inhibition of PKCα and ß, using Gö6976 or specific inhibitor peptides, attenuated the detrimental effects of glucose and restored the cardioprotected phenotype to IPC cells. Increased glucose concentration did not attenuate the cardioprotective role of PKCε, but rather activation of PKCα and ß masked its beneficial effect. Elevated extracellular glucose concentration exerts acute cardiotoxicity mediated via PKCα and ß. Inhibition of these PKC isoenzymes abolishes the cardiotoxic effects and restores IPC-mediated cardioprotection. These data support a direct link between hyperglycemia and adverse outcome after MI. Cardiac-specific PKCα and ß inhibition may be of clinical benefit in this setting.

Identification and characterisation of functional Kir6.1-containing ATP-sensitive potassium channels in the cardiac ventricular sarcolemmal membrane.

BACKGROUND AND PURPOSE: The canonical Kir6.2/SUR2A ventricular KATP channel is highly ATP-sensitive and remains closed under normal physiological conditions. These channels activate only when prolonged metabolic compromise causes significant ATP depletion and then shortens the action potential to reduce contractile activity. Pharmacological activation of KATP channels is cardioprotective, but physiologically, it is difficult to understand how these channels protect the heart if they only open under extreme metabolic stress. The presence of a second KATP channel population could help explain this. Here, we characterise the biophysical and pharmacological behaviours of a constitutively active Kir6.1-containing KATP channel in ventricular cardiomyocytes. EXPERIMENTAL APPROACH: Patch-clamp recordings from rat ventricular myocytes in combination with well-defined pharmacological modulators was used to characterise these newly identified K+ channels. Action potential recording, calcium (Fluo-4) fluorescence measurements and video edge detection of contractile function were used to assess functional consequences of channel modulation. KEY RESULTS: Our data show a ventricular K+ conductance whose biophysical characteristics and response to pharmacological modulation were consistent with Kir6.1-containing channels. These Kir6.1-containing channels lack the ATP-sensitivity of the canonical channels and are constitutively active. CONCLUSION AND IMPLICATIONS: We conclude there are two functionally distinct populations of ventricular KATP channels: constitutively active Kir6.1-containing channels that play an important role in fine-tuning the action potential and Kir6.2/SUR2A channels that activate with prolonged ischaemia to impart late-stage protection against catastrophic ATP depletion. Further research is required to determine whether Kir6.1 is an overlooked target in Comprehensive in vitro Proarrhythmia Assay (CiPA) cardiac safety screens.

Kir6.2 limits Ca(2+) overload and mitochondrial oscillations of ventricular myocytes in response to metabolic stress.

ATP-sensitive K(+) (KATP) channels are abundant membrane proteins in cardiac myocytes that are directly gated by intracellular ATP and form a signaling complex with metabolic enzymes, such as creatine kinase. KATP channels are known to be essential for adaption to cardiac stress, such as ischemia; however, how all the molecular components of the stress response interact is not fully understood. We examined the effects of decreasing the KATP current density on Ca(2+) and mitochondrial homeostasis and ischemic preconditioning. Acute knockdown of the pore-forming subunit, Kir6.2, was achieved using adenoviral delivery of short hairpin RNA targeted to Kir6.2. The acute nature of the knockdown of Kir6.2 accurately shows the effects of Kir6.2 depletion without any compensatory effects that may arise in transgenic studies. We also investigated the effect of reducing the KATP current while maintaining KATP channel protein in the sarcolemmal membrane using a nonconducting Kir6.2 construct. Only 50% KATP current remained after Kir6.2 knockdown, yet there were profound effects on myocyte responses to metabolic stress. Kir6.2 was essential for cardiac myocyte Ca(2+) homeostasis under both baseline conditions before any metabolic stress and after metabolic stress. Expression of nonconducting Kir6.2 also resulted in increased Ca(2+) overload, showing the importance of K(+) conductance in the protective response. Both ischemic preconditioning and protection during ischemia were lost when Kir6.2 was knocked down. KATP current density was also important for the mitochondrial membrane potential at rest and prevented mitochondrial membrane potential oscillations during oxidative stress. KATP channel density is important for adaption to metabolic stress.

Slowly activating voltage-gated potassium current potentiation by ML277 is a novel cardioprotective intervention.

Cardiovascular disease is thought to account for nearly a third of deaths worldwide, with ischemic heart disease, including acute coronary syndromes such as myocardial infarction, accounting for 1.7 million deaths per year. There is a clear need for interventions to impart cardioprotection against ischemia. Here, we show that the slowly activating voltage-gated potassium current (IKs) potentiator ML277 imparts cardioprotection against ischemia in cellular and whole-heart models by modulating the action potential duration. In three different metabolic inhibition and reperfusion models, an increased contractile recovery and cell survival was observed with ML277, indicative of protection. Finally, ML277 reduced infarct size in an ex vivo Langendorff coronary ligation model, including if only applied on reperfusion. In conclusion, potentiation of the IKs with ML277 imparted a cardioprotection that was equivalent to the protection reported previously by ischemic preconditioning. These data suggest that IKs potentiation may be therapeutically useful in acute coronary syndromes.

Selective protein kinase C inhibition switches time-dependent glucose cardiotoxicity to cardioprotection.

Hyperglycaemia at the time of myocardial infarction has an adverse effect on prognosis irrespective of a prior diagnosis of diabetes, suggesting glucose is the damaging factor. In ex vivo models of ischaemia, we demonstrated that deleterious effects of acutely elevated glucose are PKCα/ß-dependent, and providing PKCα/ß are inhibited, elevated glucose confers cardioprotection. Short pre-treatments with high glucose were used to investigate time-dependent glucose cardiotoxicity, with PKCα/ß inhibition investigated as a potential mechanism to reverse the toxicity. Freshly isolated non-diabetic rat cardiomyocytes were exposed to elevated glucose to investigate the time-dependence toxic effects. High glucose challenge for >7.5 min was cardiotoxic, proarrhythmic and lead to contractile failure, whilst cardiomyocytes exposed to metabolic inhibition following 5-min high glucose, displayed a time-dependent protection lasting ∼15 min. This protection was further enhanced with PKCα/ß inhibition. Cardioprotection was measured as a delay in contractile failure and KATP channel activation, improved contractile and Ca2+ transient recovery and increased cell survival. Finally, the effects of pre-ischaemic treatment with high glucose in a whole-heart coronary ligation protocol, where protection was evident with PKCα/ß inhibition. Selective PKCα/ß inhibition enhances protection suggesting glycaemic control with PKC inhibition as a potential cardioprotective therapeutics in myocardial infarction and elective cardiac surgery.

The integrin ligand SVEP1 regulates GPCR-mediated vasoconstriction via integrins α9ß1 and α4ß1.

BACKGROUND AND PURPOSE: Vascular tone is regulated by the relative contractile state of vascular smooth muscle cells (VSMCs). Several integrins directly modulate VSMC contraction by regulating calcium influx through L-type voltage-gated Ca2+ channels (VGCCs). Genetic variants in ITGA9, which encodes the α9 subunit of integrin α9ß1, and SVEP1, a ligand for integrin α9ß1, associate with elevated blood pressure; however, neither SVEP1 nor integrin α9ß1 has reported roles in vasoregulation. We determined whether SVEP1 and integrin α9ß1 can regulate VSMC contraction. EXPERIMENTAL APPROACH: SVEP1 and integrin binding were confirmed by immunoprecipitation and cell binding assays. Human induced pluripotent stem cell-derived VSMCs were used in in vitro [Ca2+ ]i studies, and aortas from a Svep1+/- knockout mouse model were used in wire myography to measure vessel contraction. KEY RESULTS: We confirmed the ligation of SVEP1 to integrin α9ß1 and additionally found SVEP1 to directly bind to integrin α4ß1. Inhibition of SVEP1, integrin α4ß1 or α9ß1 significantly enhanced [Ca2+ ]i levels in isolated VSMCs to Gαq/11 -vasoconstrictors. This response was confirmed in whole vessels where a greater contraction to U46619 was seen in vessels from Svep1+/- mice compared to littermate controls or when integrin α4ß1 or α9ß1 was inhibited. Inhibition studies suggested that this effect was mediated via VGCCs, PKC and Rho A/Rho kinase dependent mechanisms. CONCLUSIONS AND IMPLICATIONS: Our studies reveal a novel role for SVEP1 and the integrins α4ß1 and α9ß1 in reducing VSMC contractility. This could provide an explanation for the genetic associations with blood pressure risk at the SVEP1 and ITGA9 loci.

Deep-Channel uses deep neural networks to detect single-molecule events from patch-clamp data.

Single-molecule research techniques such as patch-clamp electrophysiology deliver unique biological insight by capturing the movement of individual proteins in real time, unobscured by whole-cell ensemble averaging. The critical first step in analysis is event detection, so called "idealisation", where noisy raw data are turned into discrete records of protein movement. To date there have been practical limitations in patch-clamp data idealisation; high quality idealisation is typically laborious and becomes infeasible and subjective with complex biological data containing many distinct native single-ion channel proteins gating simultaneously. Here, we show a deep learning model based on convolutional neural networks and long short-term memory architecture can automatically idealise complex single molecule activity more accurately and faster than traditional methods. There are no parameters to set; baseline, channel amplitude or numbers of channels for example. We believe this approach could revolutionise the unsupervised automatic detection of single-molecule transition events in the future.

IP(3)R-mediated Ca(2+) release is modulated by anandamide in isolated cardiac nuclei.

Cannabinoids (CBs) are known to alter coronary vascular tone and cardiac performance. They also exhibit cardioprotective properties, particularly in their ability to limit the damage produced by ischaemia reperfusion injury. The mechanisms underlying these effects are unknown. Here we investigate the intracellular localisation of CB receptors in the heart and examine whether they may modulate localised nuclear Ca(2+) release. In isolated cardiac nuclear preparations, expression of both the inositol 1,4,5-trisphosphate receptor type 2 (IP(3)R) and CB receptors (CB(1)R and CB(2)R) was demonstrated by immunoblotting. Both receptors localised to the nucleus and purity of the nuclear preparations was confirmed by co-expression of the nuclear marker protein nucleolin but absence of cytoplasmic actin. To measure effects of IP(3)R and CBR agonists on nuclear Ca(2+) release, isolated nuclei were loaded with Fluo5N-AM. This dye accumulates in the nuclear envelope. Isolated nuclei responded to IP(3) with rapid and transient Ca(2+) release from the nuclear envelope. Anandamide inhibited this IP(3)-mediated release. Preincubation of nuclear preparations with either the CB(1)R antagonist (AM251) or the CB(2)R antagonist (AM630) reversed anandamide-mediated inhibition to 80% and 60% of control values respectively. When nuclei were pre-treated with both CBR antagonists, anandamide-mediated inhibition of IP(3)-induced Ca(2+) release was completely reversed. These results are the first to demonstrate the existence of cardiac nuclear CB receptors. They are also the first to show that anandamide can negatively modulate IP(3)-mediated nuclear Ca(2+) release. As such, this provides evidence for a novel key mechanism underlying the action of CBs and CBRs in the heart.

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria.

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.

Detachment of surface membrane invagination systems by cationic amphiphilic drugs.

Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulin inhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs also caused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of human platelets. Anionic amphiphiles, U73343 (a less electrophilic U73122 analogue) and a range of kinase inhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cell membrane where they bind to anionic lipids, especially PI(4,5)P2. We therefore propose that surface detachment of membrane invaginations results from an ability of CADs to interfere with PI(4,5)P2 interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a large class of pharmaceutical compounds.