Effects of charge on membrane processing in the proximal nephron.

To examine the effects of molecular charge on membrane processing in renal tubular cells, the distribution of cationic and anionic ferritin was characterized in microperfused proximal nephron segments. During the first 7 min of proximal tubule perfusion, cationic ferritin was observed 1) bound to the brush-border membrane, 2) in apically positioned vesicles and vacuoles, 3) in lysosomes, 4) in vesicles adjacent to the basolateral plasmalemma, and 5) bound to the basolateral plasmalemma. Compared with anionic ferritin, the distribution of cationic ferritin was characterized by 1) a smaller relative grain density for lysosomes, 2) an accumulation of granules in an enlarged pool of apical cytoplasmic vesicles and vacuoles, and 3) a greater number of granules reaching the basolateral plasmalemma. During incubation directly in the presence of isolated renal cortical microvilli, binding of cationic ferritin increased significantly as pH was lowered from 8.0 to 4.5 and was greater than that of anionic ferritin, which varied little with pH. The data indicate that the molecular charge of endocytosed substances affects routing and membrane processing in proximal tubular cells, suggesting that their membrane-binding characteristics may influence transport patterns.

Carrier-mediated transport of pyroglutamyl-histidine in renal brush border membrane vesicles.

These studies were performed to determine if a transmembrane carrier for pyroglutamyl-histidine (pGlu-His) is present in the luminal membrane of renal proximal tubular cells. Previous studies have suggested the intact transepithelial transport of pGlu-His, a dipeptide formed by the hydrolysis of luteinizing hormone-releasing hormone by enzymes associated with the brush border in the proximal nephron. With the use of a renal brush border membrane vesicle preparation, pGlu-His showed H+-stimulated, Na-independent, saturable transport into an osmotically active space. High-pressure liquid chromatographic analysis of both the intravesicular and extravesicular fluids indicated intact uptake of the dipeptide. The transport constant (Kt) and Vmax for pGlu-His transport were 9.3 X 10(-8) M and 6.1 X 10(-12) mol.mg-1.min-1, respectively. Transport of pGlu-His was not inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and N-beta-alanyl-L-histidine, which have been previously shown to be transported into renal brush border vesicles via a single, low-affinity, high-capacity, Na-independent, and H+-stimulated peptide carrier. In addition, the gamma-glutamyl-containing peptides gamma-glutamyl-histidine and N(N-L-gamma-glutamyl-L-cysteinyl)glycine and the tripeptide pyroglutamyl-histidyl-prolinamide were without an inhibitory effect. In contrast, transport of pGlu-His was inhibited by the dipeptide pyroglutamyl-alanine. This study demonstrates the existence of a high-affinity, low-capacity H+ cotransport system for pGlu-His in the proximal tubular luminal plasmalemma, which appears to be specific for pyroglutamyl-containing dipeptides. The data indicate that multiple dipeptide carriers are present in the proximal nephron.

Bacterial peptidoglycans as modulators of sleep. I. Anhydro forms of muramyl peptides enhance somnogenic potency.

Chemically defined muramyl peptides (MPs), derived primarily from enzymatic digests of Neisseria gonorrhoeae peptidoglycan, were used to define the structural determinants of MP-mediated somnogenic activity. One of these, i.e. N-acetylglucosaminyl-N-acetyl-1,6-anhydro-N-acetylmuramyl-alanyl-glutamy l- diaminopimelyl-alanine, was structurally identical to the major naturally occurring MP previously detected in mammalian brain and urine. The somnogenic potency of this MP was similar to that of the corresponding disaccharide pentapeptide containing an additional alanine at the C-terminus and the analogous anhydro-muramic acid-containing monosaccharide tetrapeptide lacking the glucosamine moiety. Infusion of as little as 1 pmol of these highly active MPs increased significantly the percentage of slow-wave sleep in experimental animals. In fact, each of 5 anhydro-muramyl disaccharide peptides tested was somnogenic at a dose of 10 pmol or less and, as far as tested, the activity was affected only slightly by the length or composition of the peptide side chain. However, none of a matched set of analogous MPs, differing only in replacement of the anhydro-muramyl end by a hydrated muramic acid residue, was somnogenic at this dose. A modified form of the hydrated muramyl tripeptide containing a free amide on the diaminopimelic acid residue was completely inactive in amounts up to 1000 pmol. Together, the current data suggested: that the anhydro-muramic acid end (but not the glucosamine moiety) is essential for maximal somnogenic potency; and that amidation of carboxyl groups on the peptide-side chain may block MP-mediated somnogenic activity.

Bacterial peptidoglycans as modulators of sleep. II. Effects of muramyl peptides on the structure of rabbit sleep.

Sleep-promoting substances derived from human urine and rabbit brain were identified as muramyl peptides (MPs). We report in the accompanying paper that in the molecular structure of MPs, the 1,6-anhydro muramic acid moiety of MPs is important for enhancement of slow-wave sleep (SWS) in rabbits. Here, we document more extensively the effects of one MP: 1,6-anhydro-muramyl-alanyl-glutamyl-diaminopimelyl-alanine (AMTP for anhydro-muramyl tetrapeptide) on sleep structure of rabbits. AMTP significantly increased percent of time spent in SWS but its effects on rapid eye movement (REM) sleep were dose-dependent. Brain temperatures were significantly elevated but continued to fluctuate with sleep and wake state transitions indistinguishably from control. Sleep was episodic and animals could be easily aroused. AMTP increased number of SWS episodes and decreased number of REM episodes. There was a shift in the distribution of sleep-wake episode durations: longer waking and REM episodes were decreased, thus increased the proportion of shorter episodes. Increased duration of SWS resulted from a larger number of SWS episodes longer than 8 min. We conclude that AMTP amplifies the SWS compenent of physiological sleep.

Interferon alpha-2 enhances slow-wave sleep in rabbits.

Interferon alpha-2 (IFN) is a leukocyte product with several biological properties including antiviral activity, pyrogenicity and enhancement of immune functions. We report here that an additional facet of IFN activity is its ability to enhance slow-wave sleep (SWS) without greatly altering other aspects of sleep. Intravenous or cerebral intraventricular injections of human IFN into rabbits induced enhancement of SWS, electroencephalographic slow-wave (0.5-4 Hz) activity and brain temperatures. IFN induced slight reductions in rapid-eye movement sleep. Animal behavior, brain temperature changes that occur during the transition from one arousal state to another, and the cyclic nature of states of vigilance remained undisturbed after IFN treatment. The sleep-promoting activity of IFN may be related to feelings of lassitude and sleepiness that often accompany viral disease and interferon therapy. That IFN and other immunoactive substances, e.g. interleukin-1 and muramyl peptides, can enhance sleep suggests that sleep is linked into the immune response.

Enhancement of slow-wave sleep by endotoxin and lipid A.

Some muramyl peptides derived from bacterial peptidoglycan enhance slow-wave sleep (SWS). The purpose of this study was to test whether another cell wall component, lipopolysaccharide (LPS), and its lipid A moiety also have an effect on sleep. When injected intravenously, both LPS and lipid A enhanced the duration of SWS, increased electroencephalogram delta-wave amplitudes, suppressed rapid eye movement (REM) sleep, and induced biphasic fevers. The effects of intravenously administered lipid A and LPS on SWS were present primarily during the first 3 h postinjection. Intraventricular lipid A administration enhanced SWS, did not suppress REM, and induced a monophasic fever; the SWS effect had a 3-h latency, whereas temperature started to rise during the second hour. Regardless of the route of administration, within the dose range used here, sleep was normal by the following criteria: sleep was episodic, animals could be easily aroused, and brain temperature, although elevated to "febrile" levels, continued to fluctuate during sleep-state transitions indistinguishably from control conditions. We conclude that LPS and lipid A are capable of modulating sleep.