Eliana Scemes

Evidence for secretory pathway localization of a voltage-dependent anion channel isoform

Voltage-dependent anion channels (VDACs) are pore-forming proteins (porins) that form the major pathway for movement of adenine nucleotides through the outer mitochondrial membrane. Electrophysiological studies indicate that VDAC-like channel activity is also prevalent in the cell membranes of many mammalian cells. However, the multitopological localization of porins outside the mitochondrion has remained an extremely controversial issue. Herein, we show that usage of two alternative first exons of the murine VDAC-1 gene leads to expression of two porins differing within their N termini. One porin (plasmalemmal VDAC-1) harboring a hydrophobic leader peptide is primarily targeted through the Golgi apparatus to the cell membrane. In contrast, the second isoform lacking the N-terminal leader (mitochondrial VDAC-1) is translocated more efficiently into the outer mitochondrial membrane. Thus, our data provide unique genetic evidence in favor of a multitopological localization of a mitochondrial porin.

Volume changes in cardiac ventricles from Aplysia brasiliana upon exposure to hyposmotic shock

We investigated the possible role of ion channels and transporters in cell volume control using Aplysia brasiliana ventricular tissues exposed to a 26% hyposmotic shock, by assessing changes in wet weight, intracellular water and ionic contents. Thirty minutes after the shock, the wet weight of isolated ventricles increase about 20% above control levels and then attain near original weight within 60 min after the shock. At the time when the wet weight returned to control values, intracellular water and KCl contents are decreased by 22 and 20%, respectively. The K(+) channel blockers, 4-AP and TEA, but not the cotransport blockers, hydrochlorothiazide and furosemide, greatly affect the magnitude of wet weight gain and the time course of weight recovery, indicating that KCl loss occur through conductive pathways. Intracellular recordings performed on ventricular myocytes during exposure to the osmotic shock showed an immediate membrane hyperpolarization and blockade of spontaneous electrical activity; diastolic membrane potential recover over time and spontaneous action potentials are completely restored 60 min after the hyposmotic shock. Because significant weight loss is observed during the exposure of ventricular tissues to 26% hypo-ionic, but isosmotic saline, it is suggested that ventricular volume restoration is accomplished by two distinct but simultaneously occurring processes: a volume-dependent and a volume-independent mechanism. Because wet weight restoration is completely prevented by exposing ventricular tissue to a Ca(2+)-free hyposmotic solution, we postulate that both processes involved in A. brasiliana ventricular weight restoration are Ca(2+)-dependent mechanisms.

Ionic requirements for PCH-induced pigment aggregation in the freshwater shrimp, Macrobrachium potiuna, erythrophores☆

Abstract The effects of either cation removal or ionic channel blockade were determined on the dose-response curve (DRC) to PCH (pigment-concentrating hormone) in Macrobrachium potiuna erythrophores. In sodium-, potassium- and calcium-free salines, the pigment-aggregating responses to PCH were depressed; in the former condition, maximal aggregation was not achieved and the slope of the regression curve determined from the DRC was significantly different from control. Tetrodotoxin, verapamil or tetraethylammonium (TEA) treatments also diminished the pigment-aggregating responses to PCH, and the slopes of the regression curves were different from control in the presence of 10 −6 M verapamil or 10 −6 M TEA. Interestingly, the DRC determined in the absence of both sodium and calcium ions was not significantly different from control. When verapamil was applied in sodium-free conditions, maximal aggregation was prevented. The erythrophore resting membrane potential ranged from −62 mV to −78 mV and did not vary during PCH-induced pigment aggregation as compared to the control. Our results suggest that transient modifications of potassium equilibrium potential may interfere with PCH signal transduction, revealing a more relevant role of potassium in the process, and that a sodium influx and an intracellular calcium mobilization are necessary to maintain a cytosolic balance between the ions for normality of PCH-induced responses.

Rethinking the Role of Cholinergic Neurotransmission in the Cnidaria

After Otto Loewi’s confirmation of the concept of chemical synaptic transmission, a major emphasis in the field of comparative physiology of neuromuscular systems concerned the question of which invertebrates employ adrenergic or cholinergic transmission. This problem was of particular interest to zoologists attempting to establish phylogenetic relationships among invertebrates. Thus, Bacq and Pantin began a series of studies around 1935 to verify the presence of adrenaline, acetylcholine (ACh) and cholinesterase in invertebrates ranging from the Coelenterata to the Tunicata. Following their finding of a widespread sensitivity of animals to the application of ACh, adrenaline and related substances, these authors proposed that chemical transmission occcurred in most animals, but that the problem of neurotransmission in the coelenterates had been solved by employing chemical mechanisms other than those described for the vertebrate neuromuscular junction.

Behavioral modifications of Liriope tetraphylla (Chamisso and Eysenhardt) (Cnidaria, Hydrozoa, Trachymedusae) induced by hyposmotic conditions

The osmoregulatory capability and swimming behavior of Liriope tetraphylla (Chamisso and Eysenhardt) were analyzed under hyposmotic conditions. These hydromedusae are shown to be osmoconforming animals that are not able to maintain constant osmotic and ionic concentrations of their body fluids when exposed to anisosmotic media. Swimming behavior (as measured by the pulsation rate and the swimming index) is transiently affected by osmotic shock, being restored 30 min after exposure. However, even when swimming activity was restored, two different swimming patterns were observed depending on the hypotonic period. Thirty minutes exposure led to an increase of the quiescent period with no changes on the swimming burst characteristics while five hours exposure to hypotonicity shortened both the quiescent period and burst durations. Such behavioral modifications were related to the changes in body fluid ionic composition (mainly to extracellular K+ levels) that occur during the osmotic adjustment process. The fact that swimming activity was impaired during hypoionic shocks and this condition caused morphological changes in the animals suggests that these early metazoans are endowed with mechanisms to compensate for ionic gradient changes induced by hyposmotic, but not for the ones caused by hypoionic condition.

Absence of cholinesterase activity in body wall homogenates from the sea anemone Bunodosoma caissarum Corrêa

Abstract 1. 1. Attempts to detect cholinesterase activity in body wall homogenates of the sea anemone Bunodosoma caissarum were made using gasometric and colorimetric techniques. 2. 2. No cholinesterase activity was found in the homogenates using either of these techniques. 3. 3. Control experiments for cholinesterase activity were performed using crude homogenates from the polychaete Eurythoe complanata and the marine fish Bathygobius soporator. 4. 4. An evolutionary line of acetylcholine-meditated transmission at the junction level is discussed.

Cholinergic mechanism in Liriope tetraphylla (Cnidaria, Hydrozoa)

Crude whole body homogenates of Liriope tetraphylla exhibit a cholinesterase particularly active on acetylthiocholine but not on butyrylthiocholine. The acetylthiocholine hydrolysis is completely blocked by neostigmine. The Michaelis-Menten constant for acetylthiocholine is 0.14 mM. The pharmacological analysis of the responses to the choline esters nicotine and atropine suggests the involvement in Liriope tetraphylla of a cholinergic mechanism in the pointing reflex. Butyrylcholine, nicotine and atropine (but not muscarinic agonists) caused the contraction of the subumbrellar radial muscles. The effects of atropine were dose-dependent and were depressed in competition with muscarinic agonists. MgCl2 interfered with the action of atropine. The results were explained by suggesting the existence, at least at the neuromuscular junction, of excitatory (nicotinic) and inhibitory (muscarinic) pre-synaptic receptors modulating the release of the (unknown) transmitter acting post-synaptically.

Pharmacology of the radial neuromuscular system of Liriope tetraphylla (Hydrozoa, Trachymedusae)

Abstract 1. 1. In an attempt to analyse the cholinergic pre-synaptic modulatory hypothesis (Scemes and Mendes, 1986), the effects of magnesium and calcium ions and of cholinergic agonists and antagonists on the behavior of Liriope tetraphylla were studied. 2. 2. The medusae were submitted to the action of atropine in the presence of magnesium and different concentrations of calcium. 3. 3. The enhancement of the responses to atropine caused by acetylcholine and nicotine agree with the idea of the presence of nicotinic excitatory and muscarinic inhibitory receptors modulating the release of a still unknown neurotransmitter at the neuromuscular junction of the radial pathway.