Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release.
1. The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and 'sensory-efferent' (tachykininergic) 35SO4 labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP-regulation of cholinergic neural secretion using glibenclamide (an ATP-sensitive K+ (K(ATP)) channel inhibitor), iberiotoxin (a large conductance calcium activated K+ (BK(ca)) channel blocker), and apamin (a small conductance K(ca) (SK(ca)) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]-choline overflow as a marker for acetylcholine (ACh) release. 2. Exogenous VIP (1 and 10 microM) alone increased 35SO4 output by up to 53% above baseline, but suppressed (by up to 80% at 1 microM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 microM each). Endogenous VIP accounted for the minor increase in non-adrenergic, non-cholinergic (NANC), non-tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP. 3. Iberiotoxin (3 microM), but not apamin (1 microM) or glibenclamide (0.1 microM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion. 4. Both endogenous VIP (by use of the VIP antibody; 1:500 dilution) and exogenous VIP (0.1 microM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 microM). 5. We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is via inhibition of neurotransmitter release, consequent upon opening of BK(Ca) channels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i-NANC nerves in ferret trachea. (+info)
Differential effects of apamin- and charybdotoxin-sensitive K+ conductances on spontaneous discharge patterns of developing retinal ganglion cells.
The spontaneous discharge patterns of developing retinal ganglion cells are thought to play a crucial role in the refinement of early retinofugal projections. To investigate the contributions of intrinsic membrane properties to the spontaneous activity of developing ganglion cells, we assessed the effects of blocking large and small calcium-activated potassium conductances on the temporal pattern of such discharges by means of patch-clamp recordings from the intact retina of developing ferrets. Application of apamin and charybdotoxin (CTX), which selectively block the small and large calcium-activated potassium channels, respectively, resulted in significant changes in spontaneous firings. In cells recorded from the oldest animals [postnatal day 30 (P30)-P45], which manifested relatively sustained discharge patterns, application of either blocker induced bursting activity. With CTX the bursts were highly periodic, short in duration, and of high frequency. In contrast, with apamin the interburst intervals were longer, less regular, and lower in overall spike frequency. These differences between the effects of the two blockers on spontaneous activity were documented by spectral analysis of discharge patterns. Filling cells from which recordings were made with Lucifer yellow revealed that these effects were obtained in all three morphological classes of cells: alpha, beta, and gamma. These findings provide the first evidence that apamin- and CTX-sensitive K+ conductances can have differential effects on the spontaneous discharge patterns of retinal ganglion cells. Remarkably, the bursts of activity obtained after apamin application in more mature neurons appeared very similar to the spontaneous bursting patterns observed in developing neurons. These findings suggest that the maturation of calcium-activated potassium channels, particularly the apamin-sensitive conductance, may contribute to the changes in spontaneous firings exhibited by retinal ganglion cells during the course of normal development. (+info)
Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes.
The biophysical characteristics and alpha subunits underlying calcium-independent transient outward potassium current (Ito) phenotypes expressed in ferret left ventricular epicardial (LV epi) and endocardial (LV endo) myocytes were analyzed using patch clamp, fluorescent in situ hybridization (FISH), and immunofluorescent (IF) techniques. Two distinct Ito phenotypes were measured (21-22 degrees C) in the majority of LV epi and LV endo myocytes studied. The two Ito phenotypes displayed marked differences in peak current densities, activation thresholds, inactivation characteristics, and recovery kinetics. Ito,epi recovered rapidly [taurec, -70 mV = 51 +/- 3 ms] with minimal cumulative inactivation, while Ito,endo recovered slowly [taurec, -70 mV = 3,002 +/- 447 ms] with marked cumulative inactivation. Heteropoda toxin 2 (150 nM) blocked Ito,epi in a voltage-dependent manner, but had no effect on Ito,endo. Parallel FISH and IF measurements conducted on isolated LV epi and LV endo myocytes demonstrated that Kv1.4, Kv4.2, and Kv4.3 alpha subunit expression in LV myocyte types was quite heterogenous: (a) Kv4.2 and Kv4.3 were more predominantly expressed in LV epi than LV endo myocytes, and (b) Kv1.4 was expressed in the majority of LV endo myocytes but was essentially absent in LV epi myocytes. In combination with previous measurements on recovery kinetics (Kv1.4, slow; Kv4.2/4.3, relatively rapid) and Heteropoda toxin block (Kv1.4, insensitive; Kv4.2, sensitive), our results strongly support the hypothesis that, in ferret heart, Kv4.2/Kv4.3 and Kv1.4 alpha subunits, respectively, are the molecular substrates underlying the Ito,epi and Ito,endo phenotypes. FISH and IF measurements were also conducted on ferret ventricular tissue sections. The three Ito alpha subunits again showed distinct patterns of distribution: (a) Kv1.4 was localized primarily to the apical portion of the LV septum, LV endocardium, and approximate inner 75% of the LV free wall; (b) Kv4. 2 was localized primarily to the right ventricular free wall, epicardial layers of the LV, and base of the heart; and (c) Kv4.3 was localized primarily to epicardial layers of the LV apex and diffusely distributed in the LV free wall and septum. Therefore, in intact ventricular tissue, a heterogeneous distribution of candidate Ito alpha subunits not only exists from LV epicardium to endocardium but also from apex to base. (+info)
Recombinant influenza A virus vaccines for the pathogenic human A/Hong Kong/97 (H5N1) viruses.
Recombinant reassortment technology was used to prepare H5N1 influenza vaccine strains containing a modified hemagglutinin (HA) gene and neuraminidase gene from the A/Hong Kong/156/97 and A/Hong Kong/483/97 isolates and the internal genes from the attenuated cold-adapted A/Ann Arbor/6/60 influenza virus strain. The HA cleavage site (HA1/HA2) of each H5N1 isolate was modified to resemble that of "low-pathogenic" avian strains. Five of 6 basic amino acids at the cleavage site were deleted, and a threonine was added upstream of the remaining arginine. The H5 HA cleavage site modification resulted in the expected trypsin-dependent phenotype without altering the antigenic character of the H5 HA molecule. The temperature-sensitive and cold-adapted phenotype of the attenuated parent virus was maintained in the recombinant strains, and they grew to 108.5-9.4 EID50/mL in eggs. Both H5N1 vaccine virus strains were safe and immunogenic in ferrets and protected chickens against wild-type H5N1 virus challenge. (+info)
Coronary microvascular protection with mg2+: effects on intracellular calcium regulation and vascular function.
The use of Mg2+-supplemented hyperkalemic cardioplegia preserves microvascular function. However, the mechanism of this beneficial action remains to be elucidated. We investigated the effects of Mg2+ supplementation on the regulation of intracellular calcium concentration ([Ca2+]i) and vascular function using an in vitro microvascular model. Ferret coronary arterioles (80-150 micrometer in diameter) were studied in a pressurized (40 mmHg) no-flow, normothermic (37 degrees C) state. Simultaneous monitoring of internal luminal diameter and [Ca2+]i using fura 2 were made with microscopic image analysis. The microvessels (n = 6 each group) were divided into four groups according to the content of MgCl2 (nominally 0, 1.2, 5.0, and 25.0 mM) in a hyperkalemic cardioplegic solution ([K+] 25.0 mM). After baseline measurements, vessels were subjected to 60 min of hypoxia with hyperkalemic cardioplegia (equilibrated with 95% N2-5% CO2) containing each concentration of Mg2+ ([Mg2+]) and were then reoxygenated. During hyperkalemic cardioplegia, [Ca2+]i increased in a time-dependent manner in all groups. In the lower [Mg2+] cardioplegia groups, [Ca2+]i was significantly increased at the end of the 60-min cardioplegic period (247 +/- 44 nM and 236 +/- 49 nM in [Mg2+] 0 and 1.2 mM groups, respectively; both P < 0.05 vs. baseline) with 19.6-17.2% vascular contraction. Conversely, there was no significant [Ca2+]i increase in the higher [Mg2+] cardioplegia groups and less vascular contraction (5.4-4.1%, both P < 0.05 vs. [Mg2+] 1.2 mM group). After reperfusion, agonist (U-46619, thromboxane A2 analog)-induced vascular contraction was significantly enhanced in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was normalized in the higher [Mg2+] cardioplegia groups. Intrinsic myogenic contraction was significantly decreased in the lower [Mg2+] cardioplegia groups (both P < 0.05 vs. control) but was preserved in the higher [Mg2+] cardioplegia groups. These results suggest that supplementation of the solution with >5.0 mM [Mg2+] may prevent hyperkalemic cardioplegia-related intracellular Ca2+ overloading and preserve vascular contractile function in coronary microvessels. (+info)
Development and organization of ocular dominance bands in primary visual cortex of the sable ferret.
Thalamocortical afferents in the visual cortex of the adult sable ferret are segregated into eye-specific ocular dominance bands. The development of ocular dominance bands was studied by transneuronal labeling of the visual cortices of ferret kits between the ages of postnatal day 28 (P28) and P81 after intravitreous injections of either tritiated proline or wheat germ agglutinin-horseradish peroxidase. Laminar specificity was evident in the youngest animals studied and was similar to that in the adult by P50. In P28 and P30 ferret kits, no modulation reminiscent of ocular dominance bands was detectable in the pattern of labeling along layer IV. By P37 a slight fluctuation in the density of labeling in layer IV was evident in serial reconstructions. By P50, the amplitude of modulation had increased considerably but the pattern of ocular dominance bands did not yet appear mature. The pattern and degree of modulation of the ocular dominance bands resembled that in adult animals by P63. Flat mounts of cortex and serial reconstructions of layer IV revealed an unusual arrangement of inputs serving the two eyes in the region rostral to the periodic ocular dominance bands. In this region, inputs serving the contralateral eye were commonly fused along a mediolateral axis, rostral to which were large and sometimes fused patches of ipsilateral input. (+info)
Interference with the development of early generated neocortex results in disruption of radial glia and abnormal formation of neocortical layers.
Early generated layers of neocortex are important factors in forming the subsequent architecture of the cerebral cortex. To further explore the role of early generated cortex, we disrupted formation of an early generated cohort of cells by intraperitoneal injections of the mitotic inhibitor methylazoxymethanol (MAM) into pregnant ferrets timed to coincide with generation of subplate neurons in the ventricular zone. Our studies demonstrate that if early development of the neocortex is interrupted by injection of MAM during embryogenesis (on embryonic day 24 or 28; E24 or E28), a distinct laminar pattern fails to form properly in the parietal cortex. A reduced number of MAP2-positive cells were observed in the region of the subplate when compared with the number of MAP2-positive cells found in normal animals. Interference with the superficial neocortical layers that form later during development (on embryonic day 33) by appropriately timed MAM injections does not result in a severely disrupted laminar pattern. The interrupted laminar pattern that arises after early MAM injections coincides with distorted radial glial cells (identified by immunoreactivity to the intermediate filament protein, vimentin), which occur after early, but not late, MAM injections. Further analysis suggests that interference with early development of neocortex leads to premature differentiation of radial glial cells into astrocytes, as demonstrated by the presence of glial fibrillary acidic protein (GFAP). Experiments involving injections of the thymidine analog, bromodeoxyuridine (BRDU), demonstrated that 4 days after E24 MAM injection cells are generated and migrate into the thin cortical plate. By E38, however, cells continue to be generated in animals treated with MAM on E24 but do not reach their normal positions in the cortical plate. In addition, immunoreactivity using the CR50 antibody, which identifies presumptive Cajal-Retzius cells present in layer 1, demonstrates that the CR50-positive cells, normally precisely located in the outer portion of layer 1, are distributed in disarray throughout the thickness of the neocortex and intermediate zone in early MAM-treated animals, but not in those treated with MAM injections later during gestation. These findings are consistent with the idea that early generated layers are important in providing factors that maintain the environment necessary for subsequent neuronal migration and formation of neocortical layers. (+info)
The vomeronasal organ of the male ferret.
The vomeronasal organ (VNO) is known to play a major role in sexual behavior in many mammals. This study is the first report that the adult male ferret has a VNO, which is considerably smaller and morphologically different from the usually crescent-shaped epithelium in several mammalian species, particularly rodents. There were no differences in the size or structure of the ferret VNO between the mating season in spring and the sexually quiescent season in autumn, although plasma testosterone, testis size and brain size are dramatically increased in spring and behavior changes significantly. The histological data suggest that the VNO might be not as important a structure in male ferret sexual behavior as in rodents. (+info)