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p21-activated kinases (PAKs) are highly conserved serine/threonine protein kinases, which are divided into two groups: group-I (PAKs1–3) and group-II (PAKs4–6). In various tissues, Group-II PAKs play important roles in cytoskeletal dynamics and cell growth as well as neoplastic development/progression. However, little is known about Group-II PAK’s role in a number of physiological events, including their ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth factors (GFs). We used rat pancreatic acini to explore the ability of GI hormones/neurotransmitters/GFs to activate Group-II-PAKs and the signaling cascades involved. Only PAK4 was detected in pancreatic acini. PAK4 was activated by endothelin, secretagogues-stimulating phospholipase C (bombesin, CCK-8, and carbachol), by pancreatic GFs (insulin, insulin-like growth factor 1, hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor), and by postreceptor stimulants (12-O-tetradecanoylphobol-13-acetate and A23187 ). CCK-8 activation of PAK4 required both high- and low-affinity CCK1-receptor state activation. It was reduced by PKC-, Src-, p44/42-, or p38-inhibition but not with phosphatidylinositol 3-kinase-inhibitors and only minimally by thapsigargin. A protein kinase D (PKD)-inhibitor completely inhibited CCK-8-stimulated PKD-activation; however, stimulated PAK4 phosphorylation was only inhibited by 60%, demonstrating that it is both PKD-dependent and PKD-independent. PF-3758309 and LCH-7749944, inhibitors of PAK4, decreased CCK-8-stimulated PAK4 activation but not PAK2 activation. Each inhibited ERK1/2 activation and amylase release induced by CCK-8 or bombesin. These results show that PAK4 has an important role in modulating signal cascades activated by a number of GI hormones/neurotransmitters/GFs that have been shown to mediate both physiological/pathological responses in acinar cells. Therefore, in addition to the extensive studies on PAK4 in pancreatic cancer, PAK4 should also be considered an important signaling molecule for pancreatic acinar physiological responses and, in the future, should be investigated for a possible role in pancreatic acinar pathophysiological responses, such as in pancreatitis.NEW & NOTEWORTHY This study demonstrates that the only Group-II p21-activated kinase (PAK) in rat pancreatic acinar cells is PAK4, and thus differs from islets/pancreatic cancer. Both gastrointestinal hormones/neurotransmitters stimulating PLC and pancreatic growth factors activate PAK4. With cholecystokinin (CCK), activation is PKC-dependent/-independent, requires both CCK1-R affinity states, Src, p42/44, and p38 activation. PAK4 activation is required for CCK-mediated p42/44 activation/amylase release. These results show PAK4 plays an important role in mediating CCK physiological signal cascades and suggest it may be a target in pancreatic acinar diseases besides cancer.

Pancreatic α-cells exhibit oscillations in cytosolic Ca2+(Ca2+c), which control pulsatile glucagon (GCG) secretion. However, the mechanisms that modulate α-cell Ca2+coscillations have not been elucidated. As β-cell Ca2+coscillations are regulated in part by Ca2+-activated K+(Kslow) currents, this work investigated the role of Kslowin α-cell Ca2+handling and GCG secretion. α-Cells displayed Kslowcurrents that were dependent on Ca2+influx through L- and P/Q-type voltage-dependent Ca2+channels (VDCCs) as well as Ca2+released from endoplasmic reticulum stores. α-Cell Kslowwas decreased by small-conductance Ca2+-activated K+(SK) channel inhibitors apamin and UCL 1684, large-conductance Ca2+-activated K+(BK) channel inhibitor iberiotoxin (IbTx), and intermediate-conductance Ca2+-activated K+(IK) channel inhibitor TRAM 34. Moreover, partial inhibition of α-cell Kslowwith apamin depolarized membrane potential ( Vm) (3.8 ± 0.7 mV) and reduced action potential (AP) amplitude (10.4 ± 1.9 mV). Although apamin transiently increased Ca2+influx into α-cells at low glucose (42.9 ± 10.6%), sustained SK (38.5 ± 10.4%) or BK channel inhibition (31.0 ± 11.7%) decreased α-cell Ca2+influx. Total α-cell Ca2+cwas similarly reduced (28.3 ± 11.1%) following prolonged treatment with high glucose, but it was not decreased further by SK or BK channel inhibition. Consistent with reduced α-cell Ca2+cfollowing prolonged Kslowinhibition, apamin decreased GCG secretion from mouse (20.4 ± 4.2%) and human (27.7 ± 13.1%) islets at low glucose. These data demonstrate that Kslowactivation provides a hyperpolarizing influence on α-cell Vmthat sustains Ca2+entry during hypoglycemic conditions, presumably by preventing voltage-dependent inactivation of P/Q-type VDCCs. Thus, when α-cell Ca2+cis elevated during secretagogue stimulation, Kslowactivation helps to preserve GCG secretion.

The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound ( R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4 H-benzo[ f]imidazo[1,5- a][1,4] diazepine-3-carboxylate (SH-053-2′F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca2+]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2′F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca2+]i, store-operated Ca2+ entry, and methacholine-induced Ca2+ oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm.

Triggered activity in cardiac muscle and intracellular Ca2+have been linked in the past. However, today not only are there a number of cellular proteins that show clear Ca2+dependence but also there are a number of arrhythmias whose mechanism appears to be linked to Ca2+-dependent processes. Thus we present a systematic review of the mechanisms of Ca2+transport (forward excitation-contraction coupling) in the ventricular cell as well as what is known for other cardiac cell types. Second, we review the molecular nature of the proteins that are involved in this process as well as the functional consequences of both normal and abnormal Ca2+cycling (e.g., Ca2+waves). Finally, we review what we understand to be the role of Ca2+cycling in various forms of arrhythmias, that is, those associated with inherited mutations and those that are acquired and resulting from reentrant excitation and/or abnormal impulse generation (e.g., triggered activity). Further solving the nature of these intricate and dynamic interactions promises to be an important area of research for a better recognition and understanding of the nature of Ca2+and arrhythmias. Our solutions will provide a more complete understanding of the molecular basis for the targeted control of cellular calcium in the treatment and prevention of such.

Diminished insulin (Ins) sensitivity is a characteristic feature of various pathological conditions such as the cardiometabolic syndrome, Type 2 diabetes, and hypertension. Persons with essential hypertension are more prone than normotensive persons to develop diabetes, and this propensity may reflect decreased ability of Ins to promote relaxation and glucose transport in vascular and skeletal muscle tissue, respectively. There are increasing data suggesting that ANG II acting through its ANG type 1 receptor inhibits the actions of Ins in vascular and skeletal muscle tissue, in part, by interfering with Ins signally through phosphatidylinositol 3-kinase (PI3K) and its downstream protein kinase B (Akt) signaling pathways. This inhibitory action of ANG II is mediated, in part, through stimulation of RhoA activity and oxidative stress. Activated RhoA and increased reactive oxygen species inhibition of PI3K/Akt signaling results in decreased endothelial cell production of nitric oxide, increased myosin light chain activation with vasoconstriction, and reduced skeletal muscle glucose transport.

Endothelin is a potent vasoconstricting agent released by vascular endothelial cells. Endothelin affects multiple organ systems and occurs at high levels with heart disease. Changes in cellular calcium levels and Ca2+ sensitization of myofilaments mediate the endothelin pathway. The ability to modulate endothelin levels may lead to many effective therapies.

Maintenance of cell calcium homeostasis and transepithelial transport of this cation require its extrusion from the cell against a steep electrochemical gradient. Because it has been proposed that a membrane Ca-ATPase activated by micromolar concentrations of Ca2+ prevailing in the cell participates in these processes, we attempted in this study to determine whether such an enzyme is present in the rabbit nephron. A magnesium-dependent ATPase, maximally activated by Ca2+ (Ca-Mg-ATPase) concentrations between 1.1 and 2.3 microM (apparent Km = 0.3-0.4 microM), was found in all segments of the nephron. Ca-Mg-ATPase (pmol.mm-1.h-1) was highest in the distal convoluted tubule (243) and cortical collecting tubule (208), intermediate in the proximal convoluted tubule (140) and medullary thick ascending limb of Henle's loop (135), and lower in the pars recta (97), cortical thick ascending limb (50), and medullary collecting tubule (51). The enzyme was insensitive to ouabain and vanadate, but was inhibited by ruthenium red in a dose-dependent manner (Ki congruent to 2.10(-6) M). Sodium azide, an inhibitor of mitochondrial ATPase, did not affect Ca-Mg-ATPase, suggesting that the enzyme was located in the plasma membrane. The Ca-Mg-ATPase activity measured in most segments of the rabbit nephron in this study appears sufficient to account in theory for the active component of the unidirectional (lumen-to-bath) calcium flux found in the corresponding region of the nephron with in vitro single tubule microperfusion techniques.

In primary hypertension an enhanced phospholipase C responsiveness appears to be one of the primary events involved in the impaired cell calcium homeostasis that finally results in increased periphreal resistance and tension.