No abstract

1. Tissue blood flow and blood pressure are regulated by the spontaneous, myogenic, contraction developed by resistance arteries. However, the cellular mechanisms underlying myogenic contraction are not understood. In this study, the mechanisms of myogenic contraction in cerebral resistance arteries were investigated. 2. The vasoconstriction observed in response to increased pressure in cerebral resistance arteries (myogenic reactivity) was dependent on Ca2+ entry through voltage‐dependent Ca2+ channels, since it was abolished by Ca2+ removal and by dihydropyridine antagonists of voltage‐dependent Ca2+ channels. 3. Myogenic reactivity persisted in a high‐K+ saline, with reduced Ca2+, where membrane potential is presumed to be clamped. Therefore, membrane depolarization alone does not fully account for the increased voltage‐dependent Ca2+ channel opening. 4. Voltage‐dependent Ca2+ currents in single smooth muscle cells isolated from the resistance artery were substantially increased by applying positive pressure to the patch electrode evoking membrane stretch. 5. Myogenic reactivity remained unaffected by ryanodine and therefore was independent of internal ryanodine‐sensitive Ca2+ stores. 6. The myofilament Ca2+ sensitivity was not increased by elevated pressure in alpha‐toxin‐permeabilized arteries. However, pharmacological activation of protein kinase C or G proteins did increase the myofilament Ca2+ sensitivity. 7. Myogenic contraction over the pressure range 30‐70 mmHg could be accounted for by an increase in [Ca2+]i from 100 to 200 nM. 8. It is concluded that modest increases in [Ca2+]i within the range 100‐200 nM can account for that myogenic contraction, and that stretch‐evoked modulation of Ca2+ currents may contribute to the myogenic response.

The two apoptosis receptors of mammalian cells, i.e. the 55 kDa TNF receptor (TNF-R1) and CD95 (Fas/APO1) are activated independently of each other, however, their signaling involves a variety of ICE-related proteases [I]. We used a cell-permeable inhibitor of ICE-like protease activity to examine in vivo whether post-receptor signaling of TNF and CD95 are fully independent processes. Mice pretreated with the inhibitor, Z-VAD-fluoromethylketone (FMK) were dose-dependently protected from liver injury caused by CD95 activation as determined by plasma alanine aminotransferase and also from hepatocyte apoptosis assessed by DNA fragmentation (ID50 = 0.1 mg/kg). A dose of 10 mg/kg protected mice also from liver injury induced by TNF-alpha. Similar results were found when apoptosis was initiated via TNF-alpha or via CD95 in primary murine hepatocytes (IC50 = 1.5 nM) or in various human cell lines. In addition to prevention, an arrest of cell death by Z-VAD-FMK was demonstrated in vivo and in vitro after stimulation of apoptosis receptors. These findings show in vitro and in vivo in mammals that CD95 and the TNF-alpha receptor share a distal proteolytic apoptosis signal.

The interleukin-1 beta-converting enzyme is a heterodimeric cysteine protease that is produced as a 45-kDa precursor. The full-length precursor form of the enzyme was expressed in Escherichia coli as insoluble inclusion bodies. Following solubilization and refolding of the 45-kDa protein, autoproteolytic conversion to a heterodimeric form containing 10- and 20-kDa subunits was observed. This enzyme had catalytic activity against both natural (interleukin-1 beta precursor) and synthetic peptide substrates. The inclusion of a specific inhibitor (SDZ 223-941) of the converting enzyme in the refolding mixture prevented proteolytic processing to the 10-/20-kDa form. Similarly, refolding under nonreducing conditions also prevented processing. Time course experiments showed that the 10-kDa subunit was released from the 45-kDa precursor before the 20-kDa subunit, implying that the N-terminal portion of the precursor is released last and may play a regulatory role.

By contrast, dexamethasone, cycloheximide, and the Na ؉ /H ؉ antiporter inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride, had no effect on nigericin-induced release of IL-1␤. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1␤ is dependent upon activation of p45 ICE processing. So far, the mechanism by which reduced intracellular potassium ion concentration triggers p45 ICE processing is not known, but further investigation in this area could lead to the discovery of novel molecular targets whereby control of IL-1␤ production might be effected. Interleukin-1␤ (IL-1␤)1 is produced as an inactive 31-kDa precursor protein through the enzymatic cleavage of IL-1␤-converting enzyme (ICE), which cleaves the IL-1␤ precursor between Asp-116 and Ala-117 (1). ICE itself is produced as a 45-kDa precursor, which has recently been shown to be converted autocatalytically to an active p10/p20 heterodimer (2). The physiological control of ICE processing, and hence IL-1␤ conversion and secretion, is still unknown. Studies by Perregaux et al. (3,4) suggest that IL-1␤ processing is controlled by intracellular potassium ion concentration. Mouse peritoneal macrophages stimulated with LPS produce massive amounts of cell-associated, 31-kDa IL-1␤. Upon addition of the K ϩ /H ϩ ionophore, nigericin, rapid and complete processing of intracellular IL-1␤ occurred with the appearance of mature 17-kDa IL-1␤ in the medium. Similar effects were reported using human peripheral blood monocytes. Although in these studies marked leakage of the cytoplasmic enzyme, lactic acid dehydrogenase (LDH) occurred, suggesting substantial cell damage, it was argued that the effect of nigericin was not due simply to lysis, inasmuch as, unlike the effects of hypotonic shock, at no time were significant levels of proIL-1␤ detected in the culture medium. Furthermore, the nigericin-induced 17-kDa IL-1␤ was shown to have the expected N-terminal sequence. These results, together with studies by Walev et al. (5) showing that high extracellular concentrations of K ϩ or combinations of K ϩ -channel blockers prevented the physiological release of IL-1␤, suggest that a net reduction of intracellular K ϩ ion concentration is necessary for the processing of proIL-1␤. Both Perregaux et al.