The flickery block of ATP-dependent potassium channels of skeletal muscle by internal 4-aminopyridine

We have examined the effects of 4-aminopyridine (4-AP) on single ATP-dependent potassium channels in patches excised from frog skeletal muscle. 4-AP applied to the internal face of the membrane caused a flickery block. We could not detect any flickery block when 10 mM 4-AP was applied to the external surface of the membrane. The reduction in mean unitary current by internal 4-AP was consistent with 11 binding with a K _d of 3.3 mM at 0 mV. The block was voltage-dependent, increasing with depolarization with an effective valency of 0.57. Rate constants for blocking and unblocking by 4-AP were obtained by fitting functions to the distribution of current amplitudes. Both rate constants were voltage-dependent. At 0 mV they were 17 mM^–1 ms^–1 and 61 ms^–1. Simulation of the block using these rate constants produced a flickery block very similar to that observed experimentally.     

Arborization of axons in oculomotor nucleus identified by vestibular stimulation and intra-axonal injection of horseradish peroxidase

Summary Axons in the medial rectus (MR) subdivisions of the oculomotor nucleus were identified by horizontal rotation and by electrical stimulation of the vestibular nerves and abducens nuclei. Three types of axons (vestibular type I and II and abducens interneurons) were then injected intra-axonally with horseradish peroxidase (HRP). Each injected axon was reconstructed under the microscope in the frontal and horizontal planes and terminal arborization and boutons contacting with MR motoneurons were studied. The MR motoneurons were identified by retrograde uptake of HRP, HRP being injected in the MR muscle prior to the intra-axonal experiment.The main types of horizontal canal-related axons were as follows: (1) ATD-unilateral termination axons: Most type I axons were of this type. Axons ascended in ascending tract of Deiters (ATD) to the oculomotor nucleus and terminated in ipsilateral MR area. (2) ATD-bilateral termination axons: Very few secondary canal responsive axons were in this group. Axons ascended in ATD to the oculomotor nucleus and terminated in MR motoneuron areas bilaterally and in the Edinger-Westphal nucleus. (3) MLF-bilateral termination axons: Most type II neurons were in this group. Axons went up in the contralateral MLF and into both oculomotor nuclei. Their branches distributed to several motoneuron areas but only infrequently to the MR area; and to the Edinger-Westphal nucleus. (4) AB interneuron axons: Axons ascended in the MLF contralateral to cells of origin and terminated in the contralateral MR motoneuron area.Supported by USPHS Grant No. 06658     

Endogene Natriuretische und Ouabain-ähnliche Faktoren

Summary The existence of an endogenous natriuretic hormone and ouabain-like factors (OLF) has been postulated for many years. This postulate was based on our original observation that a small M.W. fraction in the serum after acute expansion of the extracellular fluid volume (ECFV) not only exhibited natriuretic activity but also inhibited the Na-K-ATPase enzyme in vitro similar to ouabain. Since then, numerous studies confirmed the presence of OLFs in serum, urine, cerebrospinal fluid, and various organs including the heart and hypothalamus. Some of these OLFs are well-known endogenous compounds, such as free unsaturated fatty acids, which inhibit in vitro transmembranous sodium transport, Na-K-ATPase and³H-ouabain binding to its membrane receptor or crossreact with digoxin antibodies. Chemically yet undefined OLFs of potentially hypothalamic origin were detected in various models of experimental and clinical hypertension and are suggested to play a pathophysiological role especially in salt- and volume-dependent forms of hypertension. Our results show that OLFs isolated from the urine of salt-loaded healthy subjects strongly enhance basal and vasopressin-stimulated release of calcium in vascular smooth muscle cells and platelets similar to the effects we had observed with endothelin. This urine fraction also exhibits natriuretic activity which increases in parallel with sodium intake. Further chromatographic separation and amino acid analysis confirmed the peptidic nature (M.W.<1000) of the natriuretic factor(s). However, the two biological activities, namely natriuretic and ouabain-like activities, reside in distinct and chemically different compounds. In face of the previous discovery of the atrial natriuretic peptides (ANP) it is of special interest that very recent observations strongly suggest a natriuretic factor of non-cardiac origin to play an important role in the natriuresis that follows ECFV expansion. In addition, numerous experimental data point to an interaction between the ANP and OLF systems. They should stimulate once again the final identification of these yet unknown endogenous natriuretic and ouabain-like factors.

Die in dieser übersicht zitierten eigenen Untersuchungen wurden von der Deutschen Forschungsgemeinschaft, Bonn, dem Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen (FA-2914, FA-8871, IVA6-402-046-87), Düsseldorf, und der Konrad-Adenauer-Stiftung, Bonn, unterstützt     

An ER export signal accelerates the surface expression of shal potassium channels in pyloric neurons of the lobster stomatogastric ganglion

The shal gene encoding the transient potassium current, I _A, plays important roles in shaping the firing properties of neurons in the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus. However, when we overexpressed the shal protein in pyloric dilator (PD) neurons, the effect of increased I _A was compensated by a parallel upregulation of the hyperpolarization activated inward current (I _h). In an attempt to temporally separate the overexpression of shal from the compensatory up-regulation of I _h channels, we inserted an endoplasmic reticulum (ER) export signal sequence, FCYENE, into the shal gene. This signal sequence accelerated the surface expression of shal protein in Xenopus oocytes and PD neurons. However, the accelerated expression of shal still did not alter the firing properties of the injected neuron, suggesting that the compensatory upregulation of I _h occurs simultaneously with the upregulation of I _A.     

Hyperpolarization of hypothalamic parvocellular neurons by 17 β-estradiol and their identification through intracellular staining with procion yellow

Summary Intracellular recordings and injections of procion yellow (PY) were made in parvocellular neurons in hypothalamic slices of female guinea pigs. Eighty-five neurons, with an average resting membrane potential of -35 mV, were recorded in the arcuate (ARC) ventromedial (VM), and in the cellpoor zones between the ARC and VM. Eleven of the ARC neurons and four neurons from the cell-poor zone could be driven antidromically by median eminence (ME) stimulation, nine other neurons from the three areas could be driven orthodromically by stria terminalis (ST) stimulation.Twenty-eight parvocellular neurons were tested with 17 -estradiol (E₂), which was applied in the bathing medium as the free steroid. Eleven neurons (nine ARC and two cell-poor-zone neurons) were hyperpolarized 2 to 24 mV by 10^–10 M E₂ concentrations. 10^–8 M estrone concentration was without effect on three of these cells. Through the intracellular injection of PY, the estrogen-sensitive neurons (N = 11) were identified as small fusiform cells with few dendrites. Spine-like appendages were found on only one of these cells. None of the larger pyramidal-like neurons of these areas responded to the application of E₂.Postdoctoral research fellow of the National Institute of Neurological and Communicative Disorders and Stroke     

The activation of protein kinase C prevents PGE2-induced inhibition of AVP-dependent cAMP accumulation in the rat outer medullary collecting tubule

Previous studies have demonstrated that prostaglandin E₂ (PGE₂) inhibits arginine vasopressin-(AVP)dependent adenosine 3,5-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE₂ was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30°C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP). These compounds were present also with the agonists tested during the incubation period (4 min, 35°C). In contrast to PGE₂, activators of PKC did not decrease AVP-dependent cAMP accumulation (mean ±SEM): 1nM AVP=47.1±6.8 fmol · mm^–1· 4 min^–1; AVP + 0.3 M PGE₂=20.1±2.7, P<0.01 versus AVP; AVP + 10 nM PMA=42.0±4.7, NS versus AVP; AVP + 50 g/ml OAG=44.1±4.8. NS versus AVP, N= 5 experiments. However, 10 nM PMA prevented PGE₂-induced inhibition: AVP + PGE₂= 44.2±3.5% of the response to AVP and 90.3±3.2% without and with PMA respectively, N= 16. Similar results were obtained with either 50 g/ml OAG or 25 g/ ml DOG (AVP + PGE₂ + OAG=92.9±6.6% of the response to AVP, N= 8; AVP + PGE₂ + DOG=94.1 ±5.3%, N= 7). OAG, DOG, PMA or PMA + PGE₂ had no intrinsic agonist activity in the rat OMCD and the addition of an inactive phorbol ester did not prevent PGE₂-induced inhibition. SSP, 50 nM or 0.1 M, did not affect the inhibition due to PGE₂ but abolished the reversion by PMA of PGE₂-induced inhibition. A similar regulation was observed on forskolin-(FK)dependent cAMP accumulation: 5 M FK + 0.3 M PGE₂= 37.7±6.2% of the response to FK; FK + PGE₂ + 10 nM PMA=89.5±6.7%; FK + PGE₂ + PMA + 0.1 M SSP=43.1±7.9%, N= 4. The inhibition induced by an ₂-adrenergic agonist, clonidine 1 M, was not blocked by the activation of PKC. In fura-2-loaded OMCD samples, 10nM PMA decreased by 63.3±5.0% and by 57.2±7.1% the peak and plateau phases, respectively, of the increase in intracellular calcium concentration ([Ca²⁺]_i) obtained with PGE₂ when compared to control responses in the same tubules (n=12) and did not affect the increase in [Ca²⁺]_i induced by 0.1 mM carbachol. It is concluded that: (1) in the rat OMCD the activation of PKC by PMA or analogues of diacylglycerol did not reproduce PGE₂-induced inhibition of AVP- or FK-dependent cAMP accumulation, but prevented specifically this inhibitory action; and (2) this reversion might be the consequence of the effect of PKC activation which impaired the rises in [Ca²⁺]_i induced by PGE₂.     

Dimethyl sulfoxide elevates intracellular Ca2+ and mimics effects of increased light intensity in a photoreceptor

A 1% (v/v) solution of dimethyl sulfoxide (DMSO) added to the saline bath of isolated Balanus eburneus photoreceptors increased receptor potential amplitude by 40–50% and shortened time to peak amplitude and latency by 20–25%. The light-sensitive membrane current of voltage-clamped cells was increased systematically as DMSO concentration was increased from 1% to 10%. The null potential of the light sensitive current was unaffected by DMSO with short pulses of light, indicating that DMSO has no direct effect on ion selectivity of the light-sensitive channel. Absorbance changes of cell injected with the calcium indicator arsenazo III show that DMSO elevates intracellular Ca²⁺ (Ca_i). Current-voltage relations in darkness reveal that DMSO induces a small sustained inward current (5 nA) which has a null potential similar to the lightinduced current. DMSO may activate the light-sensitive conductance via the increase in Ca_i However, the altered kinetics and increased amplitude of the receptor current are opposite to the desensitizing effects normally observed with increased Ca_i.     

Properties of the inactivating outward current in single smooth muscle cells isolated from the rat anococcygeus

The properties of the voltage- and time-dependent outward current in single smooth muscle cells isolated from the rat anococcygeus were studied. The outward current was activated by depolarizations to membrane potentials positive to –40 mV. Activation followed third order kinetics; at + 20 mV, the time for the current to reach half its maximal amplitude was around 55 ms. The current inactivated with a time course that could best be described by a single exponential with a time constant around 1500 ms. The steady-state inactivation curve was voltage dependent over the range –110 to –30 mV, with a half-inactivation point of –67 mV. Recovery from inactivation followed an exponential time course with a time constant of around 770 ms at –90 mV. Deactivating tail current analysis revealed that a 10-fold change in the extracellular potassium ion concentration resulted in a 42 mV change in the reversal potential of the current. The current was blocked by 4-aminopyridine, tetraethylammonium, quinine and verapamil with IC₅₀'s — the concentrations producing 50% inhibition of the peak current — of 2 mM, 4 mM, 12 M and 20 M respectively. The current was not blocked by Toxin I (100 nM) or glibenclamide (10 M). The current was still present in cells containing 5 mM EGTA; in these cells, replacing extracellular calcium with cadmium depressed the peak current by around 12%. This could be explained, at least in part, by a negative shift in the voltage dependence of inactivation.     

Intracellular recordings from isolated rabbit retinal Müller (glial) cells

Müller (glial) cells were isolated from rabbit retinae by papaine and mechanical dissociation. The cells were fixed on a gelatine-covered glass slide by means of concanavalin A, and the slide was mounted in a perfusion chamber under a light microscope with modified optics. Besides the recording microelectrode, two other micropipettes could be adjusted with their tips near the cell. These micropipettes were used for application of test solutions into the environment of the cells. On application of high K⁺ solutions, the cell depolarized strongly but during prolonged application there was a marked repolarization. After the end of high K⁺ application the cells showed a hyperpolarization which was enhanced in both amplitude and duration with prolongation of the K⁺ exposure. Both repolarization and afterhyper-polarization disappeared under ouabain. Ouabain application itself caused a small reversible depolarization. Na⁺ free solution caused hyperpolarization. The results suggest the existence of an active membrane pump mechanism in our cells. This pump seems to be electrogenic under our experimental conditions and seems to be activated even in the absence of sodium. The cell membrane is demonstrated to contain a significant Na⁺ conductance.     

Cockroach allergen extract stimulates protease-activated receptor-2 (PAR-2) expressed in mouse lung fibroblast

Objective: To investigate whether cockroach allergen extract can stimulate Protease-activated receptor 2 (PAR-2) expressed in mouse lung fibroblast.Materials: We established an immortalized lung fibroblast cell line, DM5, from PAR-2 deficient mice. By stable transfection with either an empty vector (DM5/EV) or an expression vector encoding mouse PAR-2 cDNA (DM5/Par2), a pair of lung fibroblast cell lines with or without functional PAR-2 expression were prepared.Treatment: The cells were exposed to cockroach allergen extract {up to 800 protein nitrogen unit (PNU)/ml}, trypsin (up to 100 nM), SLIGRL agonist peptide (up to 500 M), and trans-cinnamoyl-LIGRO agonist peptide (up to 400 M).Methods: The cells were loaded with Fluo-3 calcium indicator and mobilization of intracellular calcium with the stimuli was monitored by a fluorometric plate reader. Extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blot analysis using an anti-phospho ERK antibody.Results: The cockroach extract induced intracellular calcium transients in a concentration dependent manner in DM5/Par2 but not in DM5/EV. The activity was abolished when the cockroach extract was heat denatured or pre-incubated with PMSF (phenylmethanesulfonyl fluoride) prior to the assay. Concomitantly, ERK phosphorylation was seen in DM5/Par2 with the cockroach extract but not with a heat-denatured extract. The responses were sensitive to an inositol-1,4,5 triphosphate antagonist (2-APB) indicating that calcium was mobilized from intracellular store.Conclusions: Cockroach allergen extract can activate PAR-2 and thereby stimulate mouse lung fibroblasts likely through protease(s). The present study proposes a potential mechanism for cockroach antigens, similar to house dust mite antigens, in the etiology of respiratory diseases.Received 29 February 2004; returned for revision 12 April 2004; accepted by M. Katori 22 April 2004