Tissue-specific mitochondrial toxicity of cigarette smoke concentrate: consequence to oxidative phosphorylation.

Cigarette smoke exposure is a well-known risk factor for developing numerous chronic health conditions, including pulmonary disease and cardiometabolic disorders. However, the cellular mechanisms mediating the toxicity of cigarette smoke in extrapulmonary tissues are still poorly understood. Therefore, the purpose of this study was to characterize the acute dose-dependent toxicity of cigarette smoke on mitochondrial metabolism by determining the susceptibility and sensitivity of mitochondrial respiration from murine skeletal (gastrocnemius and soleus) and cardiac muscles, as well as the aorta to cigarette smoke concentrate (CSC). In all tissues, exposure to CSC inhibited tissue-specific respiration capacity, measured by high-resolution respirometry, according to a biphasic pattern. With a break point of 451 ± 235 µg/mL, the aorta was the least susceptible to CSC-induced mitochondrial respiration inhibition compared with the gastrocnemius (151 ± 109 µg/mL; P = 0.008, d = 2.3), soleus (211 ± 107 µg/mL; P = 0.112; d = 1.7), and heart (94 ± 51 µg/mL; P < 0.001; d = 2.6) suggesting an intrinsic resistance of the vascular smooth muscle mitochondria to cigarette smoke toxicity. In contrast, the cardiac muscle was the most susceptible and sensitive to the effects of CSC, demonstrating the greatest decline in tissue-specific respiration with increasing CSC concentration (P < 0.001, except the soleus). However, when normalized to citrate synthase activity to account for differences in mitochondrial content, cardiac fibers' sensitivity to cigarette smoke inhibition was no longer significantly different from both fast-twitch gastrocnemius and slow-twitch soleus muscle fibers, thus suggesting similar mitochondrial phenotypes. Collectively, these findings established the acute dose-dependent toxicity of cigarette smoke on oxidative phosphorylation in permeabilized tissues involved in the development of smoke-related cardiometabolic diseases.NEW & NOTEWORTHY Despite numerous investigations into the mechanisms underlying cigarette smoke-induced mitochondrial dysfunction, no studies have investigated the tissue-specific mitochondrial toxicity to cigarette smoke. We demonstrate that, while aorta is least sensitive and susceptible to cigarette smoke-induced toxicity, the degree of cigarette smoke-induced toxicity in striated muscle depends on the tissue-specific mitochondrial content. We conclude that while the mitochondrial content influences cigarette smoke-induced toxicity in striated muscles, aorta is intrinsically protected against cigarette smoke-induced mitochondrial toxicity.

Phase-Locking Requires Efficient Ca2+ Extrusion at the Auditory Hair Cell Ribbon Synapse.

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca2+ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca2+ imaging to examine Ca2+ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca2+ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca2+ ATPase (PMCA), the mitochondrial Ca2+ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca2+ ATPase (SERCA), but not the Na+/Ca2+ exchanger (NCX). Furthermore, we found that both the basal Ca2+ level and the Ca2+ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca2+ buffer, multiple Ca2+ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.SIGNIFICANCE STATEMENT Hair cell synapses can transmit sound-driven signals precisely in the kHz range. However, previous studies of Ca2+ handling in auditory hair cells have often been conducted in immature hair cells, with elevated extracellular Ca2+ concentration, or through steady-state stimulation that may not be physiologically relevant. Here we examine Ca2+ clearance from hair cell synaptic ribbons in a fully mature preparation at physiological concentration of external Ca2+ and at physiological temperature. By stimulating hair cells with sinusoidal voltage commands that mimic pure sound tones, we recapitulated the phase-locking of hair cell exocytosis with an in vitro approach. This allowed us to reveal the Ca2+ extrusion mechanisms that are required for phase-locking at auditory hair cell ribbon synapses.

Activation of the (pro)renin receptor in the paraventricular nucleus increases sympathetic outflow in anesthetized rats.

Previous studies have indicated that hyperactivity of brain prorenin receptors (PRR) is implicated in neurogenic hypertension. However, the role of brain PRR in regulating arterial blood pressure (ABP) is not well understood. Here, we test the hypothesis that PRR activation in the hypothalamic paraventricular nucleus (PVN) contributes to increased sympathetic nerve activity (SNA). In anaesthetized adult Sprague-Dawley (SD) rats, bilateral PVN microinjection of human prorenin (2 pmol/side) significantly increased splanchnic SNA (SSNA; 71 ± 15%, n = 7). Preinjection of either prorenin handle region peptide, the PRR binding blocker (PRRB), or tiron (2 nmol/side), the scavenger of reactive oxygen species (ROS), significantly attenuated the increase in SSNA (PRRB: 32 ± 5% vs. control, n = 6; tiron: 8 ± 10% vs. control, n = 5; P < 0.05) evoked by prorenin injection. We further investigated the effects of PRR activation on ROS production as well as downstream gene expression using cultured hypothalamus neurons from newborn SD rats. Incubation of brain neurons with human prorenin (100 nM) dramatically enhanced ROS production and induced a time-dependent increase in mRNA levels of inducible nitric oxide synthase (iNOS), NAPDH oxidase 2 subunit cybb, and FOS-like antigen 1 (fosl1), a marker for neuronal activation and a component of transcription factor activator protein-1 (AP-1). The maximum mRNA increase in these genes occurred 6 h following incubation (iNOS: 201-fold; cybb: 2 -fold; Ffosl1: 11-fold). The increases in iNOS and cybb mRNA were not attenuated by the AT1 receptor antagonist losartan but abolished by the AP-1 blocker curcumin. Our results suggest that PVN PRR activation induces sympathoexcitation possibly through stimulation of an ANG II-independent, ROS-AP-1-iNOS signaling pathway.

P2X7 receptors in neurohypophysial terminals: evidence for their role in arginine-vasopressin secretion.

Arginine-vasopressin (AVP) plays a major role in maintaining cardiovascular function and related pathologies. The mechanism involved in its release into the circulation is complex and highly regulated. Recent work has implicated the purinergic receptor, P2X7R, in a role for catecholamine-enhanced AVP release in the rat hypothalamic-neurohypophysial (NH) system. However, the site of P2X7R action in this endocrine system, and whether or not it directly mediates release in secretory neurons have not been determined. We hypothesized that the P2X7R is expressed and mediates AVP release in NH terminals. P2X7R function was first examined by patch-clamp recordings in isolated NH terminals. Results revealed that subpopulations of isolated terminals displayed either high ATP-sensitivity or low ATP-sensitivity, the latter of which was characteristic of the rat P2X7R. Additional recordings showed that terminals showing sensitivity to the P2X7R-selective agonist, BzATP, were further inhibited by P2X7R selective antagonists, AZ10606120 and brilliant blue-G. In confocal micrographs from tissue sections and isolated terminals of the NH P2X7R-immunoreactivity was found to be localized in plasma membranes. Lastly, the role of P2X7R on AVP release was tested. Our results showed that BzATP evoked sustained AVP release in NH terminals, which was inhibited by AZ10606120. Taken together, our data lead us to conclude that the P2X7R is expressed in NH terminals and corroborates its role in AVP secretion.

Modulation/physiology of calcium channel sub-types in neurosecretory terminals.

The hypothalamic-neurohypophysial system (HNS) controls diuresis and parturition through the release of arginine-vasopressin (AVP) and oxytocin (OT). These neuropeptides are chiefly synthesized in hypothalamic magnocellular somata in the supraoptic and paraventricular nuclei and are released into the blood stream from terminals in the neurohypophysis. These HNS neurons develop specific electrical activity (bursts) in response to various physiological stimuli. The release of AVP and OT at the level of neurohypophysis is directly linked not only to their different burst patterns, but is also regulated by the activity of a number of voltage-dependent channels present in the HNS nerve terminals and by feedback modulators. We found that there is a different complement of voltage-gated Ca(2+) channels (VGCC) in the two types of HNS terminals: L, N, and Q in vasopressinergic terminals vs. L, N, and R in oxytocinergic terminals. These channels, however, do not have sufficiently distinct properties to explain the differences in release efficacy of the specific burst patterns. However, feedback by both opioids and ATP specifically modulate different types of VGCC and hence the amount of AVP and/or OT being released. Opioid receptors have been identified in both AVP and OT terminals. In OT terminals, µ-receptor agonists inhibit all VGCC (particularly R-type), whereas, they induce a limited block of L-, and P/Q-type channels, coupled to an unusual potentiation of the N-type Ca(2+) current in the AVP terminals. In contrast, the N-type Ca(2+) current can be inhibited by adenosine via A(1) receptors leading to the decreased release of both AVP and OT. Furthermore, ATP evokes an inactivating Ca(2+)/Na(+)-current in HNS terminals able to potentiate AVP release through the activation of P2X2, P2X3, P2X4 and P2X7 receptors. In OT terminals, however, only the latter receptor type is probably present. We conclude by proposing a model that can explain how purinergic and/or opioid feedback modulation during bursts can mediate differences in the control of neurohypophysial AVP vs. OT release.

Involvement of the brain (pro)renin receptor in cardiovascular homeostasis.

RATIONALE: Despite overwhelming evidence of the importance of brain renin-angiotensin system (RAS), the very existence of intrinsic brain RAS remains controversial. OBJECTIVE: To investigate the hypothesis that the brain (pro)renin receptor (PRR) is physiologically important in the brain RAS regulation and cardiovascular functions. METHODS AND RESULTS: PRR is broadly distributed within neurons of cardiovascular-relevant brain regions. The physiological functions of PRR were studied in the supraoptic nucleus (SON) because this brain region showed greater levels of PRR mRNA in the spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto (WKY) rats. Adeno-associated virus (AAV)-mediated overexpression of human PRR in the SON of normal rats resulted in increases in plasma and urine vasopressin, and decreases in H(2)O intake and urine output without any effects on mean arterial pressure and heart rate. Knockdown of endogenous PRR by AAV-short hairpin RNA in the SON of SHRs attenuated age-dependent increases in mean arterial pressure and caused a decrease in heart rate and plasma vasopressin. Incubation of neuronal cells in culture with human prorenin and angiotensinogen resulted in increased generation of angiotensin I and II. Furthermore, renin treatment increased phosphorylation of extracellular signal-regulated kinase ½ in neurons from both WKY rats and SHRs; however, the stimulation was 50% greater in the SHR. CONCLUSIONS: The study demonstrates that brain PRR is functional and plays a role in the neural control of cardiovascular functions. This may help resolve a long-held controversy concerning the existence of intrinsic and functional brain RAS.

A current view of brain renin-angiotensin system: Is the (pro)renin receptor the missing link?

The renin-angiotensin system (RAS) plays a central role in the brain to regulate blood pressure (BP). This role includes the modulation of sympathetic nerve activity (SNA) that regulates vascular tone; the regulation of secretion of neurohormones that have a critical role in electrolyte as well as fluid homeostasis; and by influencing behavioral processes to increase salt and water intake. Based on decades of research it is clear that angiotensin II (Ang II), the major bioactive product of the RAS, mediates these actions largely via its Ang II type 1 receptor (AT1R), located within hypothalamic and brainstem control centers. However, the mechanisms of brain RAS function have been questioned, due in large part to low expression levels of the rate limiting enzyme renin within the central nervous system. Tissue localized RAS has been observed in heart, kidney tubules and vascular cells. Studies have also given rise to the hypothesis for localized RAS function within the brain, so that Ang II can act in a paracrine manner to influence neuronal activity. The recently discovered (pro)renin receptor (PRR) may be key in this mechanism as it serves to sequester renin and prorenin for localized RAS activity. Thus, the PRR can potentially mitigate the low levels of renin expression in the brain to propagate Ang II action. In this review we examine the regulation, expression and functional properties of the various RAS components in the brain with particular focus on the different roles that PRR may have in BP regulation and hypertension.

Phosphate-activated glutaminase-containing neurons in the rat paraventricular nucleus express angiotensin type 1 receptors.

The centrally mediated cardiovascular regulatory actions of angiotensin II in normal and hypertensive rats include angiotensin II type 1 receptor (AT1R)-mediated actions at the paraventricular nucleus (PVN) of the hypothalamus. Because the PVN consists of multiple neuronal populations, it is important to understand which neuronal types in the PVN are influenced by angiotensin II. Here we have developed a viral vector (Adeno-associated vector 2 [AAV2]-PAG-eGFP [PAG; phosphate-activated glutaminase promoter]) to drive expression of green fluorescent protein (GFP) primarily within glutamate neurons. At 10 to 14 days after bilateral microinjection (200 nL per side; 1.2 x10(12) genome copies) of AAV2-PAG-eGFP into adult Sprague-Dawley rat PVN, animals were euthanized and brains removed and used for isolation and culture of PVN neurons. Fluorescence microscopy and immunostaining using neuron and PAG-specific antibodies revealed the presence of GFP-containing glutamatergic neurons in these PVN cultures. Whole-cell patch-clamp recordings demonstrated that angiotensin II (100 nmol/L) produced a 16% decrease in delayed rectifier potassium current in approximately 50% of the GFP-containing neurons, an effect that was abolished by the AT1R antagonist losartan (1 mumol/L). Consistently, 9 of 28 GFP/PAG-expressing neurons contained AT1R mRNA, as indicated by single-cell RT-PCR. Furthermore, specific GFP/PAG-positive neurons in the PVN that project to the rostral ventrolateral medulla of the brain stem express immunoreactive AT1R. In conclusion, we have demonstrated the presence of functional AT1R on PAG-positive (largely glutamate) neurons within rat PVN, certain of which project to the rostral ventrolateral medulla.

M2, M3, and M4 muscarinic receptors are expressed in the guinea pig gallbladder.

AIM: The identity of muscarinic acetylcholine receptors (mAchR) involved in cholinergic-mediated contraction of the guinea pig gallbladder has been a matter of debate. Different groups have suggested the involvement of M(1), M(2), M(3), or M(4) receptor subtypes in the contraction of this tissue. The objective of this study was to identify the mAchR subtypes expressed in the guinea pig gallbladder by RT-PCR. METHODS: Total RNA prepared from frozen guinea pig gallbladder tissue was amplified by using specific primers for the M(1)-M(4) receptor subtypes. RESULTS: M(2), M(3), and M(4) transcripts were detected in the following rank order: M(4) > M(2) > M(3). We were unable to demonstrate the expression of the M(1) receptor subtype in this tissue. CONCLUSIONS: Our results are in agreement with our previous binding and functional data.

Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects.

Homeobox transcription factor Nkx2-5, highly expressed in heart, is a critical factor during early embryonic cardiac development. In this study, using tamoxifen-inducible Nkx2-5 knockout mice, we demonstrate the role of Nkx2-5 in conduction and contraction in neonates within 4 days after perinatal tamoxifen injection. Conduction defect was accompanied by reduction in ventricular expression of the cardiac voltage-gated Na+ channel pore-forming alpha-subunit (Na(v)1.5-alpha), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca2+ for contraction (conduction-contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca2+ is released from sarcoplasmic reticulum, was substantially reduced in Nkx2-5 knockout mice. These results indicate that Nkx2-5 function is critical not only during cardiac development but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction.

Characterization of a functional (pro)renin receptor in rat brain neurons.

(Pro)renin receptor (PRR), the newest member of the renin-angiotensin system (RAS), is turning out to be an important player in the regulation of the cardiovascular system. It plays a pivotal role in activation of the local RAS and stimulates signalling pathways involved in proliferative and hypertrophic mechanisms. However, the role of PRR in the brain remains unknown. Thus, our objective in this study was to determine whether a functional PRR is present in neurons within the brain. Neuronal co-cultures from the hypothalamus and brainstem areas of neonatal rat brain express PRR mRNA. Immunoreactivity for PRR was primarily localized on the neuronal cell soma and in discrete areas in the neurites. Treatment of neurons with renin, in the presence of 2 microm losartan, caused a time- and dose-dependent stimulation of phosphorylation of extracellular signal related kinase ERK1 (p44) and ERK2 (p42) isoforms of mitogen-activated protein kinase. Optimal stimulation of fourfold was observed within 2 min with 20 nm renin. Electrophysiological recordings showed that treatment of the neurons with renin, in the presence of 2 microm losartan, resulted in a steady and stable decrease in action potential frequency. A 46% decrease in action potential frequency was observed within 5 min of treatment and was attenuated by co-incubation with a PRR blocking peptide. These observations demonstrate that the PRR is present in neurons within the brain and that its activation by renin initiates the MAP kinase signalling pathway and inhibition of neuronal activity.

Mechanisms of M(1) muscarinic receptor-mediated up-regulation of neuronal nitric oxide synthase in N1E-115 neuroblastoma cells.

The neuronal form of nitric oxide synthase (nNOS) was generally assumed to be constitutively expressed at a constant level. However, it is now becoming recognized that its expression can be modulated by a number of physiological and pathophysiological conditions. Previously, we reported that nNOS expression is up-regulated after prolonged muscarinic M(1) receptor stimulation. In this work, we report that muscarinic receptor activation signals the up-regulation of nNOS via multiple pathways in N1E-115 mouse neuroblastoma cells. These include protein kinase C (PKC) activation, cytosolic calcium mobilization and NO production. Further characterization showed that the half-life of nNOS is slightly, but significantly, increased in agonist-pretreated cells compared with vehicle-treated control cells. Based on these data, it appears that the level of nNOS expression is modulated in a complex manner by a number of mechanisms that include, but might not be limited to, those described here.

AMPA receptor synaptic targeting regulated by stargazin interactions with the Golgi-resident PDZ protein nPIST.

Regulation of AMPA receptors (AMPARs) at synapses plays a critical role in alterations of synaptic strength in the brain. Stargazin, an AMPAR-interacting protein, is critical for clustering and regulation of synaptic AMPARs. Stargazin interacts with AMPARs via its extracellular domain and with PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] proteins via its C-terminal PDZ-binding motif, and these interactions are necessary for stargazin and AMPAR synaptic targeting. By studying the expression of stargazin mutant constructs in cultured hippocampal neurons, we identified a novel domain corresponding to residues 243-283 within the cytoplasmic C terminus of stargazin that is also required for stargazin and AMPAR synaptic clustering. To identify proteins that interact with this stargazin synaptic clustering domain, we performed a yeast two-hybrid assay and found that this stargazin domain binds to nPIST (neuronal isoform of protein-interacting specifically with TC10), a Golgi-enriched protein implicated in trafficking of transmembrane proteins. Using in situ hybridization, immunohistochemistry, coimmunoprecipitation studies, and biochemical fractionation, we found that stargazin and nPIST colocalize and interact in the brain. Finally, by studying AMPAR clustering in transfected hippocampal neurons, we found that overexpression of nPIST enhances AMPAR synaptic clustering, whereas transfection of a dominant-negative nPIST construct attenuates AMPAR synaptic clustering. These studies identify a novel stargazin domain necessary for synaptic clustering of AMPARs and suggest that nPIST and stargazin interactions play a critical role in AMPAR trafficking to the synapse.

Beta-catenin signaling is required for neural differentiation of embryonic stem cells.

Culture of embryonic stem (ES) cells at high density inhibits both beta-catenin signaling and neural differentiation. ES cell density does not influence beta-catenin expression, but a greater proportion of beta-catenin is targeted for degradation in high-density cultures. Moreover, in high-density cultures, beta-catenin is preferentially localized to the membrane further reducing beta-catenin signaling. Increasing beta-catenin signaling by treatment with Wnt3a-conditioned medium, by overexpression of beta-catenin, or by overexpression of a dominant-negative form of E-cadherin promotes neurogenesis. Furthermore, beta-catenin signaling is sufficient to induce neurogenesis in high-density cultures even in the absence of retinoic acid (RA), although RA potentiates the effects of beta-catenin. By contrast, RA does not induce neurogenesis in high-density cultures in the absence of beta-catenin signaling. Truncation of the armadillo domain of beta-catenin, but not the C terminus or the N terminus, eliminates its proneural effects. The proneural effects of beta-catenin reflect enhanced lineage commitment rather than proliferation of neural progenitor cells. Neurons induced by beta-catenin overexpression either alone or in association with RA express the caudal neuronal marker Hoxc4. However, RA treatment inhibits the beta-catenin-mediated generation of tyrosine hydroxylase-positive neurons, suggesting that not all of the effects of RA are dependent upon beta-catenin signaling. These observations suggest that beta-catenin signaling promotes neural lineage commitment by ES cells, and that beta-catenin signaling may be a necessary co-factor for RA-mediated neuronal differentiation. Further, enhancement of beta-catenin signaling with RA treatment significantly increases the numbers of neurons generated from ES cells, thus suggesting a method for obtaining large numbers of neural species for possible use in for ES cell transplantation.

Endogenous expression of nNOS protein in several neuronal cell lines.

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma x glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells.