Central chemoreception in the region of the ventral respiratory group in the rat

Nattie, Eugene E., and Aihua Li. Centralchemoreception in the region of the ventral respiratory group in therat. J. Appl. Physiol. 81(5):1987-1995, 1996.We injected acetazolamide (AZ; 5 × 10⁶ M, 1 nl) into theregion of the ventral respiratory group (VRG) of anesthetized paralyzedventilated rats. Control injections (mock cerebrospinal fluid,n = 6, or the inactive AZ analogue 2-acetylamino-1,3,4-thiadiazole-5-sulfon-t-butylamide,n = 6) did not increase the integratedphrenic neurogram [phrenic nerve amplitude (PNA)]. The AZinjections produced a focal region of tissue acidosis with a radius < 300-400 µm and are used as a probe for sites of centralchemosensitivity. Injection location is determined by anatomicanalysis. Of 22 VRG injections of AZ, 14 increased the amplitude of thePNA over 15-90 min; 8 had no effect. In 17 cases, we measuredmedullary tissue pH at the injection center and/or at a distantsite and reaffirmed the size of the acidotic region produced by suchsmall AZ injections. Of injections with pH electrodes within300-400 µm of the injection center, all responders showed anacid pH; three nonresponders showed an acid pH, and one an alkaline pH.In a subgroup of five rats, at VRG sites with known respiratory effectsidentified by prior glutamate injection (10 nl, 100 mM), all subsequentAZ injections produced a PNA response. Simultaneous measurement of PNAand tissue pH responses at the injection center of eight rats did notshow a uniform correlation in time; initially, both changed with asimilar time course, but PNA recovered more quickly. We conclude that1) the region of the VRG containssites of ventilatory chemoreception,2) ineffective AZ injections doproduce a tissue acidosis but at sites with minimal impact onbreathing, and 3) tissuepH does not uniquely represent the chemoreceptor stimulus.

     

Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue

The role of amiloride-sensitive Na+ channels (ASSCs) in the transduction of salty taste stimuli in rat fungiform taste buds has been well established. Evidence for the involvement of ASSCs in salt transduction in circumvallate and foliate taste buds is, at best, contradictory. In an attempt to resolve this apparent controversy, we have begun to look for functional ASSCs in taste buds isolated from fungiform, foliate, and circumvallate papillae of male Sprague-Dawley rats. By use of a combination of whole-cell and nystatin-perforated patch-clamp recording, cells within the taste bud that exhibited voltage-dependent currents, reflective of taste receptor cells (TRCs), were subsequently tested for amiloride sensitivity. TRCs were held at - 70 mV, and steady-state current and input resistance were monitored during superfusion of Na(+)-free saline and salines containing amiloride (0.1 microM to 1 mM). Greater than 90% of all TRCs from each of the papillae responded to Na+ replacement with a decrease in current and an increase in input resistance, reflective of a reduction in electrogenic Na+ movement into the cell. ASSCs were found in two thirds of fungiform and in one third of foliate TRCs, whereas none of the circumvallate TRCs was amiloride sensitive. These findings indicate that the mechanism for Na+ influx differs among taste bud types. All amiloride-sensitive currents had apparent inhibition constants in the submicromolar range. These results agree with afferent nerve recordings and raise the possibility that the extensive labeling of the ASSC protein and mRNA in the circumvallate papillae may reflect a pool of nonfunctional channels or a pool of channels that lacks sensitivity to amiloride.     

Grasping for calcium binding sites in sodium channels with an EF hand

Amino acid sequences near the carboxy terminal end of the Electrophorus electricus electric organ and rat brain sodium channel polypeptides were discovered to be putative EF hand calcium binding sites. This conclusion was made using the following criteria: the Tufty-Kretsinger and Szebenyi-Moffat EF hand tests, a computer generated analysis, the revised guidelines of Chou & Fasman, and sequence comparisons with other published EF hand calcium binding regions. These results suggest that the sodium channel may be a calcium binding protein.     

Cyclic nucleotide-gated cation channels mediate sodium and calcium influx in rat colon

We found mRNA for the three isoforms ofthe cyclic nucleotide-gated nonselective cation channel expressed inthe mucosal layer of the rat intestine from the duodenum to the colonand in intestinal epithelial cell lines in culture. Because thesechannels are permeable to sodium and calcium and are stimulated by cGMPor cAMP, we measured 8-bromo-cGMP-stimulated sodium-mediatedshort-circuit current (I_sc) inproximal and distal colon and unidirectional⁴⁵Ca²⁺fluxes in proximal colon to determine whether these channels couldmediate transepithelial sodium and calcium absorption across the colon.Sodium-mediatedI_sc, stimulatedby 8-bromo-cGMP, were inhibited by dichlorobenzamil andl-cis-diltiazem, blockers of cyclicnucleotide-gated cation channels, suggesting that these ion channelscan mediate transepithelial sodium absorption. Sodium-mediated I_sc and nettransepithelial⁴⁵Ca²⁺absorption were stimulated by heat-stable toxin fromEscherichia coli that increases cGMP.Addition of l-cis-diltiazem inhibited the enhanced transepithelial absorption of both ions. These results suggest that cyclic nucleotide-gated cation channels simultaneously increase net sodium and calcium absorption in the colon of the rat.

     

Histrionicotoxins: Effects on binding of radioligands for sodium,potassium, and calcium channels in brain membranes

A series of eight histrionicotoxins and two synthetic analogs inhibit binding of [³H]batrachotoxinin B to sites on voltage dependent sodium channels in brain membranes. Perhydrohistrionicotoxin (IC₅₀ 0.33 M) and octahydrohistrionicotoxin (IC₅₀ 1.2 M) are comparable in activities to potent local anesthetics. Histrionicotoxin (IC₅₀ 17 M) and the other histrionicotoxins are much less potent. The histrionicotoxins also inhibit binding of [³H]phencyclidine to putative potassium channels in brain membranes. Histrionicotoxin (IC₅₀ 15 M) and the other histrionicotoxins are much more potent than perhydrohistrionicotoxin (IC₅₀ 200 M), but are at least 200-fold less potent than phencyclidine. The histrionicotoxins enhance binding of [³H]nitrendipine to sites on calcium channels in brain membranes, with the exception of perhydrohistrionicotoxin, which inhibits binding. Structure activity relationships at these channel sites and at the sites for noncompetitive blockers on the nicotinic acetylcholine receptor channel (AChR) complex differ. The histrionicotoxins are more potent at the sites on the AChR complex than at sites on other channels with the exception of perhydrohistrionicotoxin, which has comparable potency at the AChR complex and sodium channels.     

Fine gating properties of channels responsible for persistent sodium current generation in entorhinal cortex neurons

The gating properties of channels responsible for the generation of persistent Na(+) current (I(NaP)) in entorhinal cortex layer II principal neurons were investigated by performing cell-attached, patch-clamp experiments in acutely isolated cells. Voltage-gated Na(+)-channel activity was routinely elicited by applying 500-ms depolarizing test pulses positive to -60 mV from a holding potential of -100 mV. The channel activity underlying I(NaP) consisted of prolonged and frequently delayed bursts during which repetitive openings were separated by short closings. The mean duration of openings within bursts was strongly voltage dependent, and increased by e times per every approximately 12 mV of depolarization. On the other hand, intraburst closed times showed no major voltage dependence. The mean duration of burst events was also relatively voltage insensitive. The analysis of burst-duration frequency distribution returned two major, relatively voltage-independent time constants of approximately 28 and approximately 190 ms. The probability of burst openings to occur also appeared largely voltage independent. Because of the above "persistent" Na(+)-channel properties, the voltage dependence of the conductance underlying whole-cell I(NaP) turned out to be largely the consequence of the pronounced voltage dependence of intraburst open times. On the other hand, some kinetic properties of the macroscopic I(NaP), and in particular the fast and intermediate I(NaP)-decay components observed during step depolarizations, were found to largely reflect mean burst duration of the underlying channel openings. A further I(NaP) decay process, namely slow inactivation, was paralleled instead by a progressive increase of interburst closed times during the application of long-lasting (i.e., 20 s) depolarizing pulses. In addition, long-lasting depolarizations also promoted a channel gating modality characterized by shorter burst durations than normally seen using 500-ms test pulses, with a predominant burst-duration time constant of approximately 5-6 ms. The above data, therefore, provide a detailed picture of the single-channel bases of I(NaP) voltage-dependent and kinetic properties in entorhinal cortex layer II neurons.     

Kinetics of the sodium current through EDTA-modified calcium channels of the molluscan neuron somatic membrane

The kinetics of the slow current carried by sodium ions through potential-dependent calcium channels after addition of EDTA to calcium-free external solution was investigated in experiments by the intracellular dialysis method on isolatedHelix pomatia neurons. The activation kinetics of this current was similar to that of the calcium current and could be described by the use of the square of the activation variable m in Hodgkin-Huxley equations. The decay (inactivation) kinetics of the induced sodium current during prolonged depolarization is biexponential in character. It is suggested that decay of the sodium currents takes place as a result of two independent processes: potential-dependent inactivation with a time constant τ_h～1 sec, taking place as far as a certain steady-state level h_∞, and a decrease in current connected with Na⁺ accumulation inside the cell during passage of the current and a consequent change in the sodium electrochemical potential (τ_c～10 sec). It is concluded that modification of the calcium channels, so that they acquire the ability to conduct sodium, has no significant effect on the gating mechanisms responsible for opening and closing of the channels.     

Molecular properties of sodium and calcium channels

Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium during electrical signals in cell membranes. Their principal subunits are members of a gene family and can function as voltage-gated ion channels by themselves. They are expressed in association with one or more auxiliary subunits which increase functional expression and modify the functional properties of the principal subunits. Structural elements which are required for voltage-dependent activation, selective ion conductance, and inactivation have been identified, and their mechanisms of action are being explored through mutagenesis, expression in heterologous cells, and functional analysis. These experiments reveal that these two channels are built on a common structural theme with variations appropriate for functional specialization of each channel type.     

G proteins and calcium channels in myocardium.

Calcium mobilization has been demonstrated to possess functional importance in myocardial excitation-contraction as well as cellular metabolism. So far, much progress has been made to explore the possibility of involvement of guanine nucleotide binding regulatory protein in the opening and closing of calcium channels as well as intracellular second messenger (cyclic adenosine monophosphate and inositol triphosphate) -mediated calcium mobilization, although such work is still in its preliminary stage, the results have proved highly interesting and significant.     

Evidence for functional sodium and calcium ion channels in the membrane of cultured cardiomyocytes of the adult rat

Characteristics are reported for electrical activity of adult rat cardiomyocytes in long-term primary culture. Cells in vitro for 12 to 28 days have mean membrane potential of -53 mV, are electrically excitable, and some are spontaneously contractile. The action potential of these cells has a slow rate of depolarization and is abolished by methoxyverapamil (D-600) but not by tetrodotoxin (TTX). When cells are hyperpolarized by passage of an inward current, spontaneous action potentials cease and action potentials evoked by depolarizing pulses are then TTX sensitive. Fetal bovine serum is a constituent of the culture medium. Its temporary removal causes spontaneous contractility to cease but the cells remain electrically excitable.     

Distribution of epithelial sodium channels and mineralocorticoid receptors in cardiovascular regulatory centers in rat brain

Epithelial sodium channels (ENaC) are important for regulating sodium transport across epithelia. Functional studies indicate that neural mechanisms acting through mineralocorticoid receptors (MR) and sodium channels (presumably ENaC) are crucial to the development of sympathoexcitation and hypertension in experimental models of salt-sensitive hypertension. However, expression and localization of the ENaC in cardiovascular regulatory centers of the brain have not yet been studied. RT-PCR and immunohistochemistry were performed to study ENaC and MR expression at the mRNA and protein levels, respectively. Both mRNA and protein for alpha-, beta-, and gamma-ENaC subunits and MR were found to be expressed in the rat brain. All three ENaC subunits and MR were present in the supraoptic nucleus, magnocellular paraventricular nucleus, hippocampus, choroid plexus, ependyma, and brain blood vessels, suggesting the presence of multimeric channels and possible regulation by mineralocorticoids. In most cortical areas, thalamus, amygdala, and suprachiasmatic nucleus, notable expression of gamma-ENaC was undetectable, whereas alpha- and beta-ENaC were abundantly expressed pointing to the possibility of a heterogeneous population of channels. The findings suggest that stoichiometrically different populations of ENaC may be present in both epithelial and neural components in the brain, which may contribute to regulation of cerebrospinal fluid and interstitial Na+ concentration as well as neuronal excitation.     

Frequency-related blocking of sodium channels in the membrane of isolated rat cardiomyocytes by the calcium antagonist verapamil

Use-dependent blocking of sodium current in the membrane of single rat cardiomyocytes by verapamil (in the concentration range of 5-50 mumol/l) has been observed. The data obtained suggest that verapamil binding with sodium channels which are in the open or inactivated state underlies suppression of sodium current.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Block of current through single calcium channels by Fe, Co, and Ni. Location of the transition metal binding site in the pore

The blocking actions of Fe2+, Co2+, and Ni2+ on unitary currents carried by Ba2+ through single dihydropyridine-sensitive Ca2+ channels were recorded from cell-attached patches on myotubes from the mouse C2 cell line. Adding millimolar concentrations of blocker to patch electrodes containing 110 mM BaCl2 produced discrete excursions to the closed channel level. The kinetics of blocking and unblocking were well described with a simple model of open channel block. Hyperpolarization speeded the exit of all of the blockers from the channel, as expected if the blocking site resides within the pore. The block by Ni2+ differs from that produced by Fe2+ and Co2+ because Ni2+ enters the channel approximately 20 times more slowly and exits approximately 50 times more slowly. Ni2+ also differs from the other transition metals because at millimolar concentrations it reduces the amplitude of the unitary current in a concentration-dependent manner. The results are consistent with the idea that the rate-limiting step for ion entry into the channel is water loss at its inner coordination sphere; unblocking, on the other hand, cannot be explained in terms of simple coulombic interactions arising from differences in ion size.     

Regulation of sodium and calcium channels by signaling complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.     

Distribution and function of voltage-gated sodium channels in the nervous system

Voltage-gated sodium channels (VGSCs) are the basic ion channels for neuronal excitability, which are crucial for the resting potential and the generation and propagation of action potentials in neurons. To date, at least nine distinct sodium channel isoforms have been detected in the nervous system. Recent studies have identified that voltage-gated sodium channels not only play an essential role in the normal electrophysiological activities of neurons but also have a close relationship with neurological diseases. In this study, the latest research findings regarding the structure, type, distribution, and function of VGSCs in the nervous system and their relationship to neurological diseases, such as epilepsy, neuropathic pain, brain tumors, neural trauma, and multiple sclerosis, are reviewed in detail.     

The characteristics of the sodium currents across EGTA-modified calcium channels in the membrane of cultured rat cardiomyocytes]

In isolated, cultured neonatal rat ventricular myocytes sodium currents through calcium channels induced by lowering of extracellular calcium concentration 100 nmol/l have been investigated by whole-cell patch clamp technique. Such Na(+)-carried currents are modulated by classic Ca2+ agonists and antagonists. The potential-dependent characteristics of Na+ current are shifted at 20 mV in hyperpolarizing direction as compared to initial Ca(2+)-carried current. The inactivation decay of Na+ current through Ca2+ channels has the monoexponential behaviour. The possible action of extracellular Ca2+ lowering on Ca2+ channel selective filter and gating mechanisms is suggested.     

Hepatic retinol metabolism. Distribution of retinoids, enzymes, and binding proteins in isolated rat liver cells

The main retinoids and some binding proteins and enzymes involved in retinol metabolism have been quantified in different types of rat liver cells. Hepatic perisinusoidal stellate cells contained 28-34 nmol of retinoids/10(6) cells, and parenchymal liver cells contained 0.5-0.8 nmol of retinoids/10(6) cells, suggesting that as much as 80% of more of total liver retinoids might be stored in stellate cells with the rest stored in parenchymal cells. Isolated endothelial cells and Kupffer cells contained very low levels of retinoids. More than 98% of the retinoids recovered in stellate cells were retinyl esters. Isolated parenchymal and stellate cell preparations both contained considerable retinyl palmitate hydrolase and acyl-CoA:retinol acyltransferase activities. Parenchymal cells accounted for about 75-80% of the total hepatic content of these two enzyme activities, with the rest located in stellate cells. On a cell protein basis, the concentrations of both of these activities were much greater in stellate cells than in parenchymal cells. In contrast, cholesteryl oleate and triolein hydrolase activities were fairly evenly distributed in all types of liver cells. Large amounts of cellular retinol binding proteins were also found in parenchymal and stellate cells. Although parenchymal cells accounted for more than 90% of hepatic cellular retinol binding protein, the concentration of the protein in stellate cells (per unit protein) was 22 X greater than that in parenchymal cells. Stellate cells were also enriched in cellular retinoic acid binding protein. Thus, both parenchymal and stellate cells contain substantial amounts of retinoids and of the enzymes and intracellular binding proteins involved in retinol metabolism. Stellate cells are particularly enriched in these several components.     

Evidence for a sodium/calcium exchanger and voltage-dependent calcium channels in adipocytes

The objective of these studies is to identify and characterize Ca2+-transport systems that may be of potential importance in the action of Ca2+-mobilizing hormones in the adipocyte. Using the Ca2+-sensitive photoprotein, aequorin, [Ca2+]i was estimated to be 0.15 microM, assuming an intracellular [Mg2+] of 1 mM. Substitution of Na+ with choline+ caused a transient increase in [Ca2+]i which was inversely related to extracellular [Na+], consistent with operation of a mediated Na+-Ca2+ exchange system. The stoichiometry was 3Na+:Ca2+. Elevation of extracellular K+ caused an increase in [Ca2+]i that was blocked by the Ca2+ channel antagonist, diltiazem, by omitting extracellular Ca2+, or by substituting Sr2+ for Ca2+. These findings indicate the presence of an Na+-Ca2+ exchanger and voltage-sensitive Ca2+ channels in adipocytes.