Santhosh M. Baby

Anesthetics and control of breathing

An important side effect of general anesthetics is respiratory depression. Anesthetics have multiple membrane targets of which ionotropic receptors such as gamma-aminobutyric acid-A (GABA(A)), glycine, N-methyl-D-aspartate and nicotinic acetylcholinergic (nACh) receptors are important members. GABA, glutamate and ACh are crucial neurotransmitters in the respiratory neuronal network, and the ability of anesthetics to modulate their release and interact with their receptors implies complex effects on respiration. Metabotropic receptors and intracellular proteins are other important targets for anesthetics suggesting complex effects on intracellular signaling pathways. Here we briefly overview the effects of general anesthetics on protein targets as far as these are relevant for respiratory control. Subsequently, we describe some methods with which the overall effect of anesthetics on the control of breathing can be measured, as well as some promising in vivo approaches to study their synaptic effects. Finally, we summarize the most important respiratory effects of volatile anesthetics in humans and animals and those of some intravenous anesthetics in animals.

Activation of HIF-1α mRNA by hypoxia and iron chelator in isolated rat carotid body

Abstract The hypoxia inducible factor-1α (HIF-1α) protein level is increased by hypoxia and iron chelator (ciclopirox olamine) in isolated rat carotid body (CB) and glomus cells. Reverse transcription and polymerase chain reaction (RT-PCR) are performed to test whether this increase is caused, at least in part, by increased HIF-1α gene transcription. HIF-1α mRNA levels dose-dependently increased and decreased in the rat CBs incubated for 1 h in a medium saturated with O 2 levels that were varied around nominally normoxic level of 21% in the 0–95% range. The iron chelator, ciclopirox olamine (5 μM), stimulated HIF-1α mRNA production under normoxic condition. Thus, in the CB, the main systemic O 2 -sensing organ, HIF-1α transcription is regulated by O 2 supply around the normoxic level; this may contribute to cellular and organismal adaptations to chronic changes in ambient O 2 .

Effects of iron-chelators on ion-channels and HIF-1α in the carotid body

Abstract Acute hypoxia instantaneously increases the chemosensory discharge from the carotid body, increasing ventilation mostly by inhibiting the oxygen sensitive ion channels and exciting the mitochondrial functions in the glomus cells. On the other hand, Fe 2+ -chelation mimics hypoxia by inhibiting the prolyl hydroxylases and the degradation of HIF-1α in non-excitable cells. Whether Fe 2+ -chelation can inhibit the ion channels giving rise to the sensory responses in excitable cells was the question. We characterized the responses to Fe 2+ -chelators on excitable glomus cells of the rat, and found that they instantaneously blocked the ion-channels, exciting the chemosensory discharge, and later causing a gradual accumulation of HIF-1α. Although initiated by the same stimuli, the two effects (on ion channels and cytosolic HIF-1α) possibly occurred by two different mechanisms.

Ventilatory Chemosensory Drive Is Blunted in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO2 and pH and high PaCO2 in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure.

Differential Expression of Utrophin-A and -B Promoters in the Central Nervous System (CNS) of Normal and Dystrophic mdx Mice

Utrophin (Utrn) is the autosomal homolog of dystrophin, the Duchene Muscular Dystrophy (DMD) locus product and of therapeutic interest, as its overexpression can compensate dystrophins absence. Utrn is transcribed by Utrn‐A and ‐B promoters with mRNAs differing at their 5′ ends. However, previous central nervous system (CNS) studies used C‐terminal antibodies recognizing both isoforms. As this distinction may impact upregulation strategies, we generated Utrn‐A and ‐B promoter‐specific antibodies, Taqman Polymerase chain reaction (PCR)‐based absolute copy number assays, and luciferase‐reporter constructs to study CNS of normal and dystrophic mdx mice. Differential expression of Utrn‐A and ‐B was noted in microdissected and capillary‐enriched fractions. At the protein level, Utrn‐B was predominantly expressed in vasculature and ependymal lining, whereas Utrn‐A was expressed in neurons, astrocytes, choroid plexus and pia mater. mRNA quantification demonstrated matching patterns of differential expression; however, transcription–translation mismatch was noted for Utrn‐B in caudal brain regions. Utrn‐A and Utrn‐B proteins were significantly upregulated in olfactory bulb and cerebellum of mdx brain. Differential promoter activity, mRNA and protein expressions were studied in cultured C2C12, bEnd3, neurons and astrocytes. Promoter activity ranking for Utrn‐A and ‐B was neurons > astrocytes >  C2C12 > bEnd3 and bEnd3 > astrocytes >  neurons > C2C12, respectively. Our results identify promoter usage patterns for therapeutic targeting and define promoter‐specific differential distribution of Utrn isoforms in normal and dystrophic CNS.

ATP causes glomus cell [Ca2+]c increase without corresponding increases in CSN activity

The hypothesis that an increase in intracellular calcium [Ca(2+)](c) in carotid body (CB) glomus cells will cause enhanced afferent carotid sinus nerve (CSN) activities was tested in the rat CB in-vitro with the use of extracellular ATP. ATP caused a dose dependent [Ca(2+)](c) increase in identified glomus cells. A major part of total [Ca(2+)](c) increase (2/3) was due to the [Ca(2+)] influx. The rest of [Ca(2+)](c) increase (1/3) was due to the release of [Ca(2+)] from the endoplasmic reticulum (ER) [Ca(2+)] stores, and it was inhibited by the pretreatment of cells with cyclopiazonic acid (CPA), an intracellular Ca(2+)-ATPase blocker. Suramin, a purinergic P(2) receptor membrane blocker, blocked [Ca(2+)] influx due to ATP in the presence of extracellular [Ca(2+)]. Perfusion with 5 and 10 microM ATP stimulated CSN activities in both normoxia (Nx) and hypoxia (Hx). Above that level, 100 microM ATP induced slight initial stimulation in CSN activities which were subsided subsequently in Nx and partly diminished in Hx, while 500 microM ATP completely inhibited CSN activities in Nx and Hx after a slight initial stimulation. Electrophysiological measurements of the glomus cell membrane potential in the presence of ATP (100 microM) during Nx indicated cellular enhanced outward K(+) current and hyperpolarization, suggesting potential mechanism for the inhibition of CSN activities. Thus, ATP dependent linear increases in [Ca(2+)](c) did not give rise to a corresponding increase in CSN activities, contravening the normally expected increase in CSN activities following [Ca(2+)](c) rise.

Heregulin-induced epigenetic regulation of the utrophin-A promoter

Utrophin is the autosomal homolog of dystrophin, the product of the Duchennes muscular dystrophy (DMD) locus. Utrophin is of therapeutic interest since its over‐expression can compensate dystrophins absence. Utrophin is enriched at neuromuscular junctions due to heregulin‐mediated utrophin‐A promoter activation. We demonstrate that heregulin activated MSK1/2 and phosphorylated histone H3 at serine 10 in cultured C2C12 muscle cells, in an ERK‐dependent manner. MSK1/2 inhibition suppressed heregulin‐mediated utrophin‐A activation. MSK1 over‐expression potentiated heregulin‐mediated utrophin‐A activation and chromatin remodeling at the utrophin‐A promoter. These results identify MSK1/2 as key effectors modulating utrophin‐A expression as well as identify novel targets for DMD therapy.

Role of central and peripheral opiate receptors in the effects of fentanyl on analgesia, ventilation and arterial blood-gas chemistry in conscious rats

This study determined the effects of the peripherally restricted μ-opiate receptor (μ-OR) antagonist, naloxone methiodide (NLXmi) on fentanyl (25μg/kg, i.v.)-induced changes in (1) analgesia, (2) arterial blood gas chemistry (ABG) and alveolar-arterial gradient (A-a gradient), and (3) ventilatory parameters, in conscious rats. The fentanyl-induced increase in analgesia was minimally affected by a 1.5mg/kg of NLXmi but was attenuated by a 5.0mg/kg dose. Fentanyl decreased arterial blood pH, pO2 and sO2 and increased pCO2 and A-a gradient. These responses were markedly diminished in NLXmi (1.5mg/kg)-pretreated rats. Fentanyl caused ventilatory depression (e.g., decreases in tidal volume and peak inspiratory flow). Pretreatment with NLXmi (1.5mg/kg, i.v.) antagonized the fentanyl decrease in tidal volume but minimally affected the other responses. These findings suggest that (1) the analgesia and ventilatory depression caused by fentanyl involve peripheral μ-ORs and (2) NLXmi prevents the fentanyl effects on ABG by blocking the negative actions of the opioid on tidal volume and A-a gradient.

l-Cysteine ethyl ester reverses the deleterious effects of morphine on, arterial blood–gas chemistry in tracheotomized rats

This study determined whether the membrane-permeable ventilatory stimulant, L-cysteine ethylester (L-CYSee), reversed the deleterious actions of morphine on arterial blood-gas chemistry in isoflurane-anesthetized rats. Morphine (2 mg/kg, i.v.) elicited sustained decreases in arterial blood pH, pO₂ and sO₂, and increases in pCO₂ (all responses indicative of hypoventilation) and alveolar-arterial gradient (indicative of ventilation-perfusion mismatch). Injections of L-CYSee (100 μmol/kg, i.v.) reversed the effects of morphine in tracheotomized rats but were minimally active in non-tracheotomized rats. L-cysteine or L-serine ethylester (100 μmol/kg, i.v.) were without effect. It is evident that L-CYSee can reverse the negative effects of morphine on arterial blood-gas chemistry and alveolar-arterial gradient but that this positive activity is negated by increases in upper-airway resistance. Since L-cysteine and L-serine ethylester were ineffective, it is evident that cell penetrability and the sulfur moiety of L-CYSee are essential for activity. Due to its ready penetrability into the lungs, chest wall muscle and brain, the effects of L-CYSee on morphine-induced changes in arterial blood-gas chemistry are likely to involve both central and peripheral sites of action.

Ca2+ responses to hypoxia are mediated by IP3-R on Ca2+ store depletion.

Inositol 1,4, 5-triphosphate (IP3) is generated from the hydrolysis of phosphatidylinositol 4, 5-triphosphate (PIP2) which is a component of plasma membrane. IP3 acts as a messenger to link with receptors IP3-Rs which are located on intracellular Ca2+ stores, such as the endoplasmic reticulum (ER) (Belousov et al., 1995; Berridge, 1993). The ER also often shares the same domain of the mitochondria which have a low affinity, high-capacity for Ca2+ uptake mechanism, and Ca2+ concentrations influence mitochondrial metabolism (Duchen, 2000). Thus, IP3-Rs are an ideal candidate for Ca2+- related cellular functions, Rizzuto et al. (1993) have shown that [Ca2+]m increases rapidly and transiently upon stimulation with agonists coupled to IP3- R generation. This leads to high [Ca2+]i close to IP3-R and sensed by mitochondria. Conversely, metabolites generated by energy production may influence IP3-mediated Ca2+ dynamics. Depletion of cellular energy resources leads to the accumulation of cytoplasmic reduced NADH (Veech et al., 1970). Hypoxia decreases mitochondrial respiration by inhibiting the terminal step in the electron transport chains, provoking a rapid rise in intracellular Ca2+ (Biscoe and Duchen, 1990; Kaplin et al., 1996; McCormack et al., 1990). Hypoxia also increases cytoplasmic NADH levels as a result of enhanced glycolysis (Yager et al., 1991). Also, NADH selectively stimulates the release of Ca2+ mediated by IP3-R (Ferris and Snyder, 1992).