Fast and slow blockades of the inward-rectifier K+ channel by external divalent cations in guinea-pig cardiac myocytes

The present patch-clamp study shows that external Mg²⁺, Ca²⁺ and Sr²⁺ decrease the unit amplitude of inward current through the inward-rectifier K⁺ channel in a concentration-dependent manner. Sr²⁺ produces a voltage-dependent flickering block as well, and the fractional electrical distance between the external orifice and the Sr²⁺ binding site () is 0.73. The decrease of unit amplitude is reversible and voltage independent while it does not increase the noise level on the open-channel current. Unit current decreased by Mg²⁺ or Ca²⁺ has a longer mean open time, which is inversely proportional to the unit amplitude. External Mg²⁺ does not decrease the amplitude of unit outward current. A surface potential shift, measured using voltage-dependent Cs⁺ block (=1.60), failed to explain the current decrease. Therefore, we conclude that (1) the external divalent cations cause an extremely fast channel block, which appears as a decreased amplitude of the unit current on the recording system; (2) the blocking site (fast site) is present near the external orifice of the channel, and it is separate from the blocking site (slow site) to which Cs⁺ and Sr²⁺ bind.     

ULTRASTRUCTURAL ASPECTS OF THE ROLE OF THE MEDIA-SMOOTH MUSCLE CELLS IN ARTERIOSCLEROSIS OF MAN AND ANIMALS

In various arterial lesions including atherosclerotic lesions, the main morphological change involves smooth muscle cells. The potential sensitivity is different among the arterial smooth muscle cells, venous smooth muscle cells and smooth muscle cells of other organs. The modified smooth muscle cells characterized by the increase of rough endoplasmic reticula are considered to express their latent ability to synthesize collagen fibers, elastic fibers and other ground substances.
The foam cells noted in atherosclerosis and fatty streak consist of lipid accumulated smooth muscle cells and hematogenous macrophages. Lipid metabolism and synthesis in the latter differ from those in the former. The ratio of the two kinds of foam cells in atheroma or fatty streak varies by the stage of the lesion.
It is possible to suppose that there exists a factor which would selectively attack the media smooth muscle cells of small arteries or arterioles. This is observed electron microscopically as focal cytoplasmic necrosis (cytoplasmolysis) of smooth muscle cells and plays an important role in the histogenesis of fibrinoid necrosis.
In case of experimental periarteritis nodosa the early stage begins with cytoplasmolysis of smooth muscle cells and marked increase of rough endoplasmic reticula in adjacent smooth muscle cells.     

Ultrastructure and synaptic organization of the spinal accessory nucleus of the rat

The accessory nucleus is composed of neurons in the medial column that innervate the sternocleidomastoid muscle, and neurons in the lateral column that innervate the trapezius muscle. We retrogradely labeled these neurons by injection of cholera toxin conjugated horseradish peroxidase into the sternomastoid (SM) or the clavotrapezius (CT) muscles, and investigated fine structure and synaptology of these neurons. Almost all SM and CT motoneurons had the appearance of alpha-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles, Nissl bodies, and a prominent spherical nucleus. More than 60% of the somatic membrane was covered with terminals. The SM motoneurons (34.4 x 52.2 microm, 1,363.1 microm(2) in a section) were slightly larger than the CT motoneurons (32.8 x 54.2 microm, 1,180.8 microm(2)). The average number of axosomatic terminals in a section was 52.2 for the SM, and 54.2 for the CT motoneurons. More than half of them (58.0%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II) with the SM motoneurons, while 57.9% of them contained round vesicles and made asymmetric synaptic contacts (Gray's type I) with the CT motoneurons. A few C-terminals were present on the SM (3.5) and the CT (3.7) motoneurons. About 60% of the axodendritic terminals were Gray's type I in both the SM and the CT motoneurons. A few labeled small motoneurons were also found among the SM and the CT motoneurons. They were small (19.2 x 26.2 microm, 367.0 microm(2)), round cells containing poorly developed organelles with a few axosomatic terminals (9.3). Only 20% of the somatic membrane was covered with the terminals. Thus, these neurons were presumed to be gamma-motoneurons. These results indicate that the motoneurons in the medial and the lateral column of the accessory nucleus have different ultrastructural characteristics.     

Myelin Degeneration in Multiple System Atrophy Detected by Unique Antibodies

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple system atrophy (MSA). However, as we reported previously, the antiserum, which is highly specific for the sequence QDENPVV corresponding to human myelin basic protein residues 82 to 88, failed to recognize any structures in normal human brain. QD-9, a mouse monoclonal antibody raised against human myelin basic protein residues 69 to 88, which also recognizes specifically the epitope QDENPVV, gave the same results as did anti-EP. The unusual epitope recognized by anti-EP/QD-9 antibodies appears to be accessible in areas of myelin degeneration, and the antibodies have been shown to detect such areas in multiple sclerosis and infarcted brains. These antibodies detect myelin degeneration more widely than previous conventional methods. The present study emphasizes the importance of myelin degeneration in the pathogenesis of multiple system atrophy.     

Nucleotide mutations associated with hepatitis B e antigen negativity

One hundred and forty four patients with chronic hepatitis B were tested to identify new mutations associated with hepatitis B e antigen (HBeAg) negativity, using a full genome sequence analysis. All the patients were Chinese and had hepatitis B virus infection of genotype C. Patients with none of the pre-core or core promoter mutations were significantly (P < 0.001) less common in the group with anti-HBe (13%) than in the group with HBeAg (56%). The complete nucleotide sequence was determined in four anti-HBe-positive patients who had neither pre-core nor core promoter mutations and in five HBeAg-positive patients who also had neither of these mutations (the groups were matched for age and sex). Six mutations were found to be significantly more common in the former group than in the latter: G529A (3/4 vs. 0/5), C934A (4/4 vs. 1/5), A1053G (4/4 vs. 1/5), G1915T/A (4/4 vs. 0/5), T2005C/A (4/4 vs. 0/5), and C3026T (3/4 vs. 0/5). Three of the six mutations were significantly more common in the four anti-HBe-positive patients who had neither pre-core nor core promoter mutations, compared to 11 HBeAg-positive patients who had pre-core and core promoter mutations, and also compared to 15 anti-HBe-positive patients who had pre-core and core promoter mutations, suggesting further the specificity of these mutations. Of the six mutations, two resulted in amino acid substitution in the polymerase protein, and one is located near the enhancer I region. The results suggest that the six newly discovered mutations are associated with HBeAg negativity.     

Regulation of the type I IFN induction: a current view

The type I IFN-alpha/beta gene family was identified about a quarter of a century ago as a prototype of many cytokine gene families, which led to the subsequent burst of studies on molecular mechanisms underlying cytokine gene expression and signaling. Although originally discovered for their activity to confer an antiviral state on cells, more evidence has recently been emerging regarding IFN-alpha/beta actions on cell growth, differentiation and many immunoregulatory activities, which are of even greater fundamental biological significance. Indeed, much attention has recently been focused on the induction and function of the IFN-alpha/beta system regulated by Toll-like receptors (TLRs), which are critical for linking the innate and adaptive immunities. The understanding of the regulatory mechanisms of IFN-alpha/beta gene induction by TLRs and viruses is an emerging theme, for which much new insight has been gained over the past few years.     

Mode of regulation of the ACh-sensitive K-channel by the muscarinic receptor in rabbit atrial cells

The mechanism underlying the regulation of the K-channel by the muscarinic receptor was examined with patch-clamp experiments in atrial cells isolated enzymatically from the rabbit heart. The patch-electrode and the recording chamber were perfused with various solutions while the activity of the K-channels in the membrane-patch was recorded continuously. In the absence of muscarinic agonists, opening of K-channels occurred at a low frequency (basal activity). Application of ACh to the bath did not affect the basal activity, but perfusion of the patch electrode with ACh markedly increased the channel activity in the "cell-attached" patch. Application of oxotremorine, i.e. a specific muscarinic agonist, via the pipette also opened K-channels. When the membrane patch was isolated from the cell body ("inside-out" patch), ACh-induced single K-channel currents were still observed, but the frequency was reduced. Perfusion of atropine or scopolamine, two muscarinic antagonists, through the patch-electrode depressed the basal activity. In the case of scopolamine, channel-activity recovered after washing out the drug. The current voltage relationship determined from the basal activity was similar to that of ACh-induced single K-channel currents. The mean open time was 0.49 ms at basal activity and 1.35 ms during the application of 0.1 microM ACh via the patch electrode. Application of oxotremorine via the pipette hardly affected the open-time, it remained at 99 +/- 4% (n = 7) of the control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of cardiac Na+ channel simply through open states as revealed by single-channel analysis in guinea pig ventricular myocytes

Inactivation of the cardiac Na(+) channel was analyzed by recording channel currents from a cell-attached patch containing only one functional Na(+) channel in guinea-pig ventricular myocytes. A two-step test pulse, first to variable levels (Pulse 1) and then to -30 mV (Pulse 2) was applied from a holding potential of -140 mV. When a cumulative histogram was determined for the latency of first opening, the histogram was well fitted with a single exponential function at -70 to -30 mV of Pulse 1. The activation time course of ensemble average was virtually single exponential. Although the ensemble average of 500 sweeps showed various extents of inactivation during Pulse 1, the saturation level of the cumulative first-latency histogram at the end of the two-step pulse was almost constant (0.7-0.8), irrespective of Pulse 1. Even when the interval between successive test pulses was prolonged from 70 to 970 ms, the saturation level of the histogram was not modified. These findings are consistent with inactivation only through the open state. Thus, the apparent "blank sweep inactivation" does not necessarily indicate direct inactivation from closed states. These findings support the hypothesis that the inactivation of cardiac Na(+) channel occurs exclusively through the open state.