Specialized membrane areas in non-activated and thrombin-activated platelets

Filipin, a complex of polyene antibiotics, forms morphologically distinctive complexes with cholesterol in cell membranes under proper experimental conditions. When applied to non-activated, discoid platelets, filipin-induced lesions (FIL) occurred in rows at the platelet equator, suggesting a specialized membrane organization at the platelets' largest circumference. In some thrombin-activated platelets we observed surface membrane blebbing and release of lipid vesicles that predominantly originated from the plasma membrane proper, but some originated from (unidentified) platelet granules. FIL were initially present in high numbers over the entire bleb, they accumulated later at the neck of blebs, while the released vesicle was free of FIL. Absence of intramembrane protein particles (IMP) from the membranes of blebs and vesicles suggests that released vesicles are essentially without cholesterol and intrinsic membrane proteins and may consist predominantly of phospholipids. Membrane blebbing and vesicle release may represent unmasking and release of procoagulant platelet factor 3 activity.     

Caveolae, caveolin and cav-p60 in smooth muscle and renin-producing cells in the rat kidney

AIMS: In vascular smooth muscle cells caveolae are important for signalling mechanisms regulating vascular contraction. In smooth muscle layer of the renal afferent arteriole juxtaglomerular cells (JG cells) are non-contractile renin producing cells that have the capacity to change their phenotype into smooth muscle cells and back again by metaplastic transformation. Signalling mechanisms in JG cells are not fully understood and we therefore investigated if caveolae were present, and thereby could be involved as integrators of cellular signalling in both of these phenotypes of smooth muscle cells. METHODS: Using electron microscopy we compared the number of caveolae in JG cells and smooth muscle cells in the afferent arteriole of the rat kidney. The expression of caveolin and cav-p60 was examined using a combination of immunogold electron microscopy and immunofluorescence microscopy. RESULTS: We found that JG cells have sixfold less caveolae per cell surface sectional length than smooth muscle cells. The expression of cavolin-1 and cav-p60 correlated with the number of caveolae. An examination of the general distribution of caveolae, cav-p60 and caveolins in the rat kidney showed that cav-p60, like caveolin-1, is a specific maker of caveolae. CONCLUSION: The number of caveolae in JG cells is very low, and this makes it unlikely that caveolae are of major importance for the renin secretion specific for JG cells.     

Molecular weight of the membrane C5b-9 complex of human complement: characterization of the terminal complex as a C5b-9 monomer.

The hydrodynamic properties of the detergent-solubilized, terminal membrane complex of serum complement components C5-C9 [C5b-9(m)] were studied to obtain an estimate of its molecular weight. In a solution of Triton X-100/deoxycholate, the protein complex binds 17% Triton X-100 and 11% deoxycholate by weight. The sedimentation coefficient of the protein-detergent complex is 26 S as determined by sucrose density gradient ultracentrifugation, and gel filtration indicated a molecular radius of 11 nm. It was ascertained by electron microscopy that these hydrodynamic parameters apply to mono-dispersed C5b-9(m) complexes, which were observed as nonaggregated, hollow protein cylinders and were identical to the complement "lesions" formed on target membranes. The calculated molecular weight of the protein-detergent complex is approximately 1,286,300 to which the protein moiety contributes approximately 1,000,000. The results indicate that the C5b-9(m) complex formed on biological membranes is a monomer entity of the C5-C9 complement components.     

Frequency-Dependence of the Metabolism of 3H-Noradrenaline Released from Rabbit Isolated Aorta: Effects of a Combination of Cocaine and Corticosterone or Hydrocortisone

Abstract The effect of cocaine (neuronal uptake inhibitor) in combination with either hydrocortisone or corticosterone (extraneuronal uptake inhibitors) on the metabolism of ³H-noradrenaline (³H-NA) released spontaneously or by electrical-field stimulation was studied on the isolated rabbit aorta preloaded with ³H-NA. In the spontaneous outflow ³H-O-methylated and deaminated metabolites (³H-OMDA) accounted for 41% of the total radioactivity whereas ³H-NA represented only 22%. Cocaine (3 × 10^-5M) + hydrocortisone (10^-4 M) neither changed the spontaneous outflow of total tritium nor the distribution of the ³H-outflow on ³H-NA and its ³H-metabolites. However, cocaine (3 × 100^-5M) + corticosterone (4 × 10^-5M) enhanced the passive ³H-outflow, increased the percentage recovered as ³H-3,4-dihydroxyphenylglycol (³H-DOPEG), and reduced the percentage of ³H-OMDA + ³H-NMN. The effect of field-stimulation was investigated during and ofter stimulation at two frequencies: 3 and 10 Hz. The stimulation-induced ³H-overflow consisted mainly of unmetabolized ³H-NA and ³H-OMDA in the sample collected during stimulation. The ratio between ³H-NA and ³H-OMDA was strongly dependent on the frequency of stimulation, Thus it was 1:2 at 3 Hz, but 3:1 at 10 Hz. These ratios were about the same during the poststimulation period. On the other hand, whereas the formation of ³H-DOPEG from ³H-NA released during stimulation at both frequencies was small, it increased rapidly in the poststimulation sample. At 3 Hz, inhibition of neuronal and extraneuronal uptake by cocaine + steroids did not change the stimulation-induced ³H-overflow, but the distribution on ³H-NA and ³H-metabolites was markedly altered. Thus the percentage of ³H-N A was doubled, ³H-OMDA was halved, and ³H-DOPEG was almost abolished. At 10 Hz, however, cocaine + corticosterone decreased the stimulation-induced ³H-overflow, but did not change the proportions of ³H-NA and ³H-OMDA. Only the formation of ³H-DOPEG was markedly reduced. It is concluded that the distribution of stimulation-induced ³H-overflow on ³H-NA and its ³H-metabolites and the effect of neuronal and extraneuronal uptake inhibition on this is strongly influenced by the frequency of stimulation. Furthermore, inhibition of both neuronal and extraneuronal uptake does not fully prevent metabolism of ³H-NA released by electrical-field stimulation.     

Fine structural identification of individual cells subjected to microelectrode recording in perfused cardiac preparations

We recorded transmembrane potentials from atrial, AV nodal and ventricular cells in perfused canine and rabbit heart preparations, and from Purkinje cells in superfused canine false tendons. Recording was maintained during perfusion with an aldehyde fixative. Action potential duration was greatly prolonged, and finally repolarization failed when the cells became fixed. After fixation the microelectrodes were withdrawn. Appropriate tissue blocks were embedded in Epon and serially sectioned at 4 μm. In 24 out of 34 attempts, the very cell recorded from was identified under the light microscope. After remounting and resectioning of the 4 μm sections for electron microscopy, the terminal electrode track was found in 12 instances, the very end of the track in six cases.Two main types of atrial action potentials were recorded: with and without a plateau phase. After retrieval of the individual cells we found no basis for a morphological distinction between different cell types. In the AV node, electrotonic contact between two cells was established for distances up to 320 μm by delivering current pulses to one of two microelectrodes which were simultaneously in an intracellular position. Both “N” and “NH” types of action potentials could originate in the proximal atrioventricular bundle. Possibly the N-type in this location was induced by hypoxia. Impalements of Purkinje fibres and ventricular myocardial cells extended the observations on details of microelectrode penetration and impalement damage.The most conspicuous disorder observed in all cells impaled by a microelectrode was a local or universal hypercontraction of myofibrils. In all cases where stable recordings were obtained, the microelectrode tip was found close to the cell membrane opposite to the site of entrance.     

Antibiotics in periodontal therapy: advantages and disadvantages*

Abstract. Antibiotic treatment of periodontitis aims at eradicating or controlling specific pathogens. Prime candidates for antibiotic therapy are patients with recently diagnosed active periodontitis or a history of recurrent disease who fail to stabilize following mechanical/surgical therapy. Since a variety of microbes with differing antimicrobial susceptibility profiles may cause periodontitis, selection of antimicrobial agents should be based on proper microbial diagnosis and sensitivity testing, as well as consideration of the patient's medical status. The risk of treating chemotherapeutically solely on the basis of clinical features, radiographic findings or a limited microbiological analysis, is failure to control the pathogens or overgrowth of new pathogens. A review of published papers reveals that appropriate systemic antibiotic therapy may enhance healing in patients with recent or high risk of periodontal breakdown. Systemic antibiotic therapy seems more predictable than topical administration in eradicating periodontal pathogens from deep periodontal pockets. Several promising antimicrobial agents for periodontitis treatment need testing in placebo-controlled, double-blind, randomized clinical trials.     

Morphology of electrophysiologically identified junctions between Purkinje fibers and ventricular muscle in rabbit and pig hearts

Purkinje fiber-ventricular muscle (PV) junctions were identified by extracellular recording in isolated, superfused preparations from rabbit and pig hearts. Microelectrode recordings from different cell types at the PV junctions were obtained, and the cells recorded from were retrieved microscopically. To this end 26 tissue blocks were serially sectioned at 4 microns. Microscopic identification of the very cell recorded from was obtained in five of seven Purkinje, five of 16 transitional, and two of two ventricular muscle cell recordings. In addition, some tissue blocks from both junctional and nonjunctional sites identified only by extracellular recording were examined in serial sections. Transitional cells in the rabbit heart are thin, broad bandlike cells (30-35 by 3-5 microns) arranged in one or two sheets in the subendocardium between the Purkinje layer and ventricular mass. Transitional cells are coupled via short, thin strands to both Purkinje and ventricular muscle cells. A second type of PV coupling was observed frequently in the pig, but in only one of 21 cases in the rabbit. Here, a short, linear segment of small transitional cells connected large-diameter Purkinje cells to ventricular muscle cells. Distances found between Purkinje-transitional cell coupling sites and transitional cell-ventricular muscle coupling sites varied from 100 to 1,000 microns in the rabbit heart and from 50 to several hundred micrometers in the pig heart. Action potentials from transitional cells typically showed multiple components in their upstroke. Both our morphological and electrophysiological findings are compatible with the existence of a relatively high-resistance barrier between Purkinje and transitional cells and between transitional and ventricular muscle cells.     

Autonomous hyperparathyroidism in X-linked hypophosphataemia

Four patients with familial hypophosphataemic rickets developed significant hypercalcaemia which persisted after discontinuation of vitamin D therapy. They had increased PTH levels and were operated for hyperparathyroldism at the ages of 18, 20, 24 and 45 years, respectively. Three of the patients had previously received phosphate treatment and one patient developed hyperparathyroldism 7 years after treatment with calcitriol. Histological evaluation revealed different degrees of parathyroid hyperplasia in all patients, with persistently Increased PTH and/or calcium levels after surgery. The possibility of autonomous hyperparathyroldism should be evaluated in the follow-up of patients with X-linked hypophosphataemic rickets.     

Complement activation and attack on autologous cell membranes induced by streptolysin-O.

Streptolysin-O damages mammalian membranes through generation of large transmembrane channels formed by membrane-inserted polymers of the toxin (S. Bhakdi et al., Infect. Immun. 47:52-60, 1985). We here report that the native toxin binds naturally occurring human serum immunoglobulin G antibodies to form immune complexes with potent complement-activating capacity. Nanomolar concentrations of toxin added to antibody-containing serum cause rapid consumption of C4 and C5 hemolytic activity and 30 to 90% C3 conversion within 10 to 60 min at 37 degrees C. After binding to target membranes, streptolysin-O polymers serve as foci for antibody-dependent complement activation, which proceeds to completion with the formation of terminal C5b-9 complexes on the autologous cells. The binding and insertion of a primarily water-soluble bacterial product into a host cell membrane has thus been shown to generate a stable and hyperactive focus for activation of and self-attack by the complement system. We suggest that this process perpetuates local tissue damage, deviates host complement action away from the invading bacteria, and may possibly play a role in the pathogenesis of poststreptococcal disease.