Development, validation, and application of a fast, simple, and robust SPE-based LC-MS/MS method for quantification of angiotensin I-converting enzyme inhibiting tripeptides Val-Pro-Pro, Ile-Pro-Pro, and Leu-Pro-Pro in yoghurt and other fermented dairy products

Dairy products are an important part of a nutritionally balanced diet as their constituents can affect the human state of health. By inhibiting the angiotensin I-converting enzyme, the tripeptides Val-Pro-Pro, Ile-Pro-Pro, and Leu-Pro-Pro can lower blood pressure. As these peptides are produced during fermentation, they are found in various dairy products like cheese, yoghurt, etc., but except for cheese only little is known about their content. To investigate how other dairy products contribute to a supply of these antihypertensive peptides, we developed and validated a fast and sensitive assay for quantification of the three tripeptides with LC-MS/MS combined with a simple protocol for extraction and SPE-purification from yoghurt, curd, or other products. Finally, the entire method was successfully applied to survey peptide concentrations in samples from local dairies and thus expands our awareness on the content of antihypertensive peptides in our food. (C) 2019 Elsevier Ltd. All rights reserved.Peer reviewe

Characterization of the molecular and electrophysiological properties of the T-type calcium channel in human myometrium

Rises in intracellular calcium are essential for contraction of human myometrial smooth muscle (HMSM) and hence parturition. The T-type calcium channel may play a role in this process. The aim was to investigate the role of the T-Type calcium channel in HMSM by characterizing mRNA expression, protein localization, electrophysiological properties and function of the channel subunits Cav3.1(α1G), Cav3.2(α1H), and Cav3.3(α1I). QRT-PCR, immuno-histochemistry, electrophysiology and invitro contractility were performed on human myometrial samples from term, preterm, labour and not in labour. QRT-PCR analysis of Cav3.1, Cav3.2 and Cav3.3 demonstrated expression of Cav3.1 and Cav3.2 with no significant change (P>0.05) associated with gestation or labour status. Immuno-histochemistry localized Cav3.1 to myometrial and vascular smooth muscle cells whilst Cav3.2 localized to vascular endothelial cells and invading leukocytes. Voltage clamp studies demonstrated a T-type current in 55% of cells. Nickel block of T-type current was voltage sensitive (IC50 of 118.57±68.9 μM at - 30mV). Activation and inactivation curves of ICa currents in cells expressing T-type channels overlapped demonstrating steady state window currents at the resting membrane potential of myometrium at term. Current clamp analysis demonstrated that hyperpolarizing pulses to a membrane potential greater than -80mV elicited rebound calcium spikes that were blocked reversibly by 100μM nickel. Contractility studies demonstrated a reversible decrease in contraction frequency during application of 100μM nickel (P<0.05). We conclude that the primary T-type subunit expressed in some MSMCs is Cav3.1. We found that application of 100 μM nickel to spontaneously contracting human myometrium reversibly slows contraction frequency

Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node

The sympathetic nervous system increases heart rate by activating β adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current (If) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. If is produced by hyperpolarization-activated cyclic nucleotide–sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. β adrenergic regulation of If in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of β adrenergic agonists to shift the voltage dependence of If in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of If. Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate

Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization

Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms. The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research

Evidence both L-type and non-L-type voltage-dependent calcium channels contribute to cerebral artery vasospasm following loss of NO in the rat

We recently found block of NO synthase in rat middle cerebral artery caused spasm, associated with depolarizing oscillations in membrane potential (Em) similar in form but faster in frequency (circa 1 Hz) to vasomotion. T-type voltage-gated Ca2+ channels contribute to cerebral myogenic tone and vasomotion, so we investigated the significance of T-type and other ion channels for membrane potential oscillations underlying arterial spasm. Smooth muscle cell membrane potential (Em) and tension were measured simultaneously in rat middle cerebral artery. NO synthase blockade caused temporally coupled depolarizing oscillations in cerebrovascular Em with associated vasoconstriction. Both events were accentuated by block of smooth muscle BKCa. Block of T-type channels or inhibition of Na+/K+-ATPase abolished the oscillations in Em and reduced vasoconstriction. Oscillations in Em were either attenuated or accentuated by reducing [Ca2+]o or block of KV, respectively. TRAM-34 attenuated oscillations in both Em and tone, apparently independent of effects against KCa3.1. Thus, rapid depolarizing oscillations in Em and tone observed after endothelial function has been disrupted reflect input from T-type calcium channels in addition to L-type channels, while other depolarizing currents appear to be unimportant. These data suggest that combined block of T and L-type channels may represent an effective approach to reverse cerebral vasospasm

The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity

HCN4 subunit expression in fast-spiking interneurons of the rat spinal cord and hippocampus

Hyperpolarisation-activated (Ih) currents are considered important for dendritic integration, synaptic transmission, setting membrane potential and rhythmic action potential (AP) discharge in neurons of the central nervous system. Hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels underlie these currents and are composed of homo- and hetero-tetramers of HCN channel subunits (HCN1–4), which confer distinct biophysical properties on the channel. Despite understanding the structure–function relationships of HCN channels with different subunit stoichiometry, our knowledge of their expression in defined neuronal populations remains limited. Recently, we have shownthat HCN subunit expression is a feature of a specific population of dorsal horn interneurons that exhibit high-frequency AP discharge. Here we expand on this observation and use neuroanatomical markers to first identify well-characterised neuronal populations in the lumbar spinal cord and hippocampus and subsequently determine whether HCN4 expression correlates with high-frequency AP discharge in these populations. In the spinal cord, HCN4 is expressed in several putative inhibitory interneuron populations including parvalbumin (PV)-expressing islet cells (84.1%; SD: ±2.87), in addition to all putative Renshaw cells and Ia inhibitory interneurons. Similarly, virtually all PVexpressing cells in the hippocampal CA1 subfield (93.5%;±3.40) and the dentate gyrus (90.9%; ±6.38) also express HCN4. This HCN4 expression profile in inhibitory interneurons mirrors both the prevalence of Ih sub-threshold currents and high-frequency AP discharge. Our findings indicate that HCN4 subunits are expressed in several populations of spinal and hippocampal interneurons, which are known to express both Ih sub-threshold currents and exhibit high-frequency AP discharge. As HCN channel function plays a critical role in pain perception, learning and memory,and sleep as well as the pathogenesis of several neurologicaldiseases, these findings provide important insights into the identity and neurochemical status of cells that could underlie such conditions

An in vivo role for Rho kinase activation in the tumour vascular disrupting activity of combretastatin A-4 3-O-phosphate

Background and Purpose Combretastatin A-4 3-O-phosphate (CA4P) is in clinical trial as a tumour vascular disrupting agent (VDA) but the cause of blood flow disruption is unclear. We tested the hypothesis that activation of Rho/Rho kinase (ROCK) is fundamental to the effects of this drug in vivo. Experimental Approach Mouse models of human colorectal carcinoma (SW1222 and LS174T) were used. Effects of the ROCK inhibitor, Y27632, alone or in combination with CA4P, on ROCK activity, vascular function, necrosis and immune cell infiltration in solid tumours were determined. Mean arterial BP (MABP) was measured to monitor systemic interactions and the vasodilator, hydralazine, was used to control for the hypotensive effects of Y27632. Key Results Y27632 caused a rapid drop in blood flow in SW1222 tumours, with recovery by around 3 h, which was paralleled by MABP changes. Y27632 pretreatment reduced CA4P-induced ROCK activation and partially blocked CA4P-induced tumour vascular effects, in both tumour types. Y27632 also partially inhibited CA4P-induced tumour necrosis and was associated with reduced immune cell infiltration in SW1222 tumours. Hydralazine caused a similar hypotensive effect as Y27632 but had no protective effect against CA4P treatment. Conclusions and Implications These results demonstrate that ROCK activity is critical for full manifestation of the vascular activity of CA4P in vivo, providing the evidence for pharmacological intervention to enhance the anti-tumour efficacy of CA4P and related VDAs

STIM2 regulates AMPA receptor trafficking and plasticity at hippocampal synapses

STIM2 is an integral membrane protein of the endoplasmic reticulum (ER) that regulates the activity of plasma membrane (PM) channels at ER-PM contact sites. Recent studies show that STIM2 promotes spine maturation and surface expression of the AMPA receptor (AMPAR) subunit GluA1, hinting at a probable role in synaptic plasticity. Here, we used a Stim2 cKO mouse to explore the function of STIM2 in Long-Term Potentiation (LTP) and Depression (LTD), two widely-studied models of synaptic plasticity implicated in information storage. We found that STIM2 is required for the stable expression of both LTP and LTD at CA3-CA1 hippocampal synapses. Altered plasticity in Stim2 cKO mice is associated with subtle alterations in the shape and density of dendritic spines in CA1 neurons. Further, surface delivery of GluA1 in response to LTP-inducing chemical manipulations was markedly reduced in excitatory neurons derived from Stim2 cKO mice. GluA1 endocytosis following chemically-induced LTD was also impaired in Stim2 cKO neurons. We conclude that STIM2 facilitates synaptic delivery and removal of AMPARs and regulates activity-dependent changes in synaptic strength through a unique mode of communication between the ER and the synapse

Dynamic analysis of Ca2+ level during bovine oocytes maturation and early embryonic development

Mammalian oocyte maturation and early embryo development processes are Ca2+-dependent. In this study, we used confocal microscopy to investigate the distribution pattern of Ca2+ and its dynamic changes in the processes of bovine oocytes maturation, in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) embryo development. During the germinal vesicle (GV) and GV breakdown stage, Ca2+ was distributed in the cortical ooplasm and throughout the oocytes from the MI to MII stage. In IVF embryos, Ca2+ was distributed in the cortical ooplasm before the formation of the pronucleus. In 4-8 cell embryos and morulas, Ca2+ was present throughout the blastomere. In PA embryos, Ca2+ was distributed throughout the blastomere at 48 h, similar to in the 4-cell and 8-cell phase and the morula. At 6 h after activation, there was almost no distribution of Ca2+ in the SCNT embryos. However, Ca2+ was distributed in the donor nucleus at 10 h and it was distributed throughout the blastomere in the 2-8 cell embryos. In this study, Ca2+ showed significant fluctuations with regularity of IVF and SCNT groups, but PA did not. Systematic investigation of the Ca2+ location and distribution changes during oocyte maturation and early embryo development processes should facilitate a better understanding of the mechanisms involved in oocyte maturation, reconstructed embryo activation and development, ultimately improving the reconstructed embryo development rate