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M H Nathanson

Publications and source records attributed to M H Nathanson.

At least 73 records · Page 4Linked to original sources
PubMed1994

Fc- and non-Fc-mediated phagocytosis of Borrelia burgdorferi by macrophages.

The Fc receptor (FcR) for immunoglobulin has been assigned a major role in the ingestion of Borrelia burgdorferi, the Lyme disease spirochete, by macrophages. Yet macrophages readily take up and kill B. burgdorferi that have not been opsonized. By use of doubly-labeled macrophages infected with spirochetes and analyzed by confocal fluorescence microscopy, simultaneous localization of both FcR and spirochetes (opsonized and unopsonized) was quantified. After infection with unopsonized spirochetes, bacterial surface antigen and the FcR remained distinct, confirming the expectation that unopsonized uptake of B. burgdorferi is largely independent of the FcR. A similar lack of colocalization was seen when opsonized spirochetes were ingested by macrophages whose FcRs were sequestered by an immune complex-coated substrate. Furthermore, comparable efficiency of uptake was observed whether or not the FcR was engaged.

Animals↗
PubMed1994

Mechanism of desensitization of the cloned vasopressin V1a receptor expressed in Xenopus oocytes.

The vasopressin V1a receptor exerts its effects by G protein-mediated increases in cytosolic Ca2+ (Cai2+) and activation of protein kinase C. The V1a receptor also undergoes autologous desensitization. To clarify the mechanism of this desensitization, we expressed the cloned receptor in Xenopus oocytes, and vasopressin-induced Cai2+ waves were examined as an index of V1a activation using confocal microscopy. Pretreatment of oocytes with a minimal concentration of vasopressin inhibited further generation of Cai2+ waves upon maximal stimulation. Such pretreatment did not abolish Cai2+ waves induced by subsequent microinjection of inositol trisphosphate, suggesting that this phenomenon represents receptor desensitization rather than depletion of inositol trisphosphate-sensitive Cai2+ stores. Pretreatment with phorbol dibutyrate, ionomycin, or 8-bromoadenosine 3',5'-cyclic monophosphate had no effect on vasopressin-induced Cai2+ waves. Oocytes recovered from desensitization within 1 h, but the microtubule inhibitor methyl-5-[2-thienylcarbonyl]-1H-benzimiidazol-2-yl)-carbamate (nocodazole) inhibited this recovery. Receptor binding sites were reduced by over 50% within 10 min of exposure to vasopressin, with no associated change in the Kd for the V1a receptor. These findings indicate that 1) expression of the cloned V1a receptor in Xenopus oocytes, coupled with subcellular Cai2+ imaging, provides a useful system to examine mechanisms of V1a desensitization, 2) the V1a receptor undergoes autologous desensitization in this experimental system, and 3) protein kinase C, Cai2+, and adenosine 3',5'-cyclic monophosphate do not appear responsible for this desensitization, but 4) microtubule-dependent recycling of the receptor is preserved in this system and may be important for receptor desensitization.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Multistep mechanism of polarized Ca2+ wave patterns in hepatocytes.

The spatial organization of cytosolic Ca2+ (Ca2+i) signals is thought to be important for regulation of cell function. In epithelial cells, the involvement of inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release in evoking Ca2+i signals is appreciated, but the location of IP3-sensitive Ca2+ stores and the role of Ca(2+)-induced Ca2+ release (CICR) for Ca2+ signaling are less defined. Here, we demonstrate that IP3 receptors are localized to the apical region in hepatocytes. We also show that hormone-induced Ca2+i waves propagate across the cell at a rate that depends on mobilization of Ca2+ stores that are sensitive to caffeine, ryanodine, and dantrolene, and that these agents, at concentrations that inhibit CICR, decrease the magnitude of Ca2+i signals. Furthermore, Ca2+i wave speed is not reduced in Ca(2+)-free medium. These findings suggest that Ca2+i signals in epithelial cells begin as apical-to-basal Ca2+i waves that result from sequential release of Ca2+, first from IP3-sensitive stores in the apex and then from Ca(2+)-sensitive stores distributed across the remainder of the cell.

Animals↗
PubMed1993

Endothelin-1 induces cholestasis which is mediated by an increase in portal pressure.

Endothelin-1 (ET-1) is a potent vasoactive peptide which generally exerts its effect on target cells by increasing [Ca++]i. Both vasoconstriction (resulting in an increase in perfusion pressure) and increased [Ca++]i are actions of ET-1 that may result in cholestasis. Single-pass isolated perfused rat liver (IPRL) were used, and [Ca++]i was measured in both populations of hepatocytes and single cells. ET-1 (0.1-100 nM) induced a dose-dependent increase in perfusion pressure and decrease in bile flow. Perfusion pressure increased by 112% (p < 0.001) and bile flow decreased by 17% (p < 0.008) in response to 2 nM ET-1. At this concentration of ET-1, but not at higher concentrations, the cholestasis was abolished (p > 0.18 vs basal) and the rise in perfusion pressure was decreased (by 62%; p < 0.002) by the vasodilator papaverine. This ET-1 concentration also had no measurable effect on [Ca++]i in isolated hepatocytes. Taken together these findings indicate that ET-1 inhibits bile flow in IPRL and suggests that this effect is mediated by vasoconstriction and not by changes in hepatocyte cytosolic Ca++.

Animals↗
PubMed1993

The fate of Borrelia burgdorferi, the agent for Lyme disease, in mouse macrophages. Destruction, survival, recovery.

The macrophage is a known reservoir for a number of infectious agents, and is therefore a likely candidate site for persistence of Borrelia burgdorferi, the Lyme spirochete. We report that unopsonized B. burgdorferi enter macrophages rapidly, resulting mainly in degradation but occasionally in apparent intracellular persistence. We studied uptake of spirochetes by macrophages by simultaneously labeling infected cells with antibodies to B. burgdorferi and with sequential components of the endocytic pathway, and we examined optical sections (0.5-1.0 micron in thickness) of these cells by confocal fluorescence microscopy at multiple time points after infection. We found that only 5 min of incubation at 37 degrees C were required for nearly 100% of B. burgdorferi to enter a lysosomal glycoprotein-positive compartment, whereas 60 min were required for 90% of the spirochetes to appear in a cathepsin L-positive compartment under the same conditions. We also labeled infected living cells with acridine orange to distinguish live from killed intracellular organisms. Although the large majority of spirochetes within a given cell were dead, we saw occasional live ones up to 24 h (the longest interval examined) after all extracellular organisms had been lysed in distilled water. Moreover, we can reculture spirochetes from macrophages after infection. Persistence of spirochetes within macrophages provides a possible pathogenetic mechanism for chronic or recurrent Lyme disease in man.

Animals↗
PubMed1993

Effects of tauroursodeoxycholic acid on cytosolic Ca2+ signals in isolated rat hepatocytes.

BACKGROUND: Tauroursodeoxycholic acid (TUDCA) is of potential benefit in cholestatic disorders. However, the effects of TUDCA on cytosolic free calcium [(Ca2+)i], which regulates hepatocyte secretion, are unknown. METHODS: The effect of TUDCA on (Ca2+)i was investigated in groups of isolated rat hepatocytes by microspectrofluorometry and in single cells by confocal line scanning microscopy. RESULTS: Administration of TUDCA (5-50 mumol/L) induced a nearly fourfold increase of basal levels of (Ca2+)i. After a 15 minute treatment period, the TUDCA (10 mumol/L)-induced change in (Ca2+)i was higher than that of other mono-, di-, and trihydroxy bile acids at equimolar concentrations. Pretreatment with TUDCA (10 mumol/L) markedly reduced or abolished increases in (Ca2+)i induced by phenylephrine (1 mumol/L), the microsomal Ca(2+)-translocase inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (25 mumol/L), or taurolithocholic acid (10-25 mumol/L). In Ca(2+)-free medium, TUDCA caused only a reduced and transient increase in (Ca2+)i. TUDCA (10 mumol/L) induced Ca2+ oscillations in all single cells that responded. However, levels of inositol-1,4,5-trisphosphate (IP3) in hepatocytes were not increased by treatment with TUDCA (10 mumol/L). CONCLUSIONS: TUDCA at physiological concentrations potently modulates (Ca2+)i signals in hepatocytes by (1) mobilizing microsomal IP3-sensitive Ca2+ stores by an IP3-independent mechanism, (2) initiating Ca2+ oscillations, and (3) inducing influx of extracellular Ca2+.

Animals↗
PubMed1993

Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca++ mechanisms defective in cholestasis.

To assess the effects of tauroursodeoxycholic acid (TUDCA) on bile excretory function, we examined whether TUDCA modulates vesicular exocytosis in the isolated perfused liver of normal rats in the presence of high (1.9 mM) or low (0.19 mM) extracellular Ca++ and in cholestatic rats 24 h after bile duct ligation. In addition, the effects of TUDCA on Ca++ homeostasis were compared in normal and in cholestatic hepatocytes. In the isolated perfused rat liver, TUDCA (25 microM) stimulated a sustained increase in the biliary excretion of horseradish peroxidase, a marker of the vesicular pathway, in the presence of high, but not low extracellular Ca++ or in the cholestatic liver. In contrast, TUDCA stimulated bile flow to the same extent regardless of the concentration of extracellular Ca++ or the presence of cholestasis. In indo-1-loaded hepatocytes, basal cytosolic free Ca++ ([Ca++]i) levels were not different between normal and cholestatic cells. However, in cholestatic cells [Ca++]i increases induced by TUDCA (10 microM) and its 7 alpha-OH epimer taurochenodeoxycholic acid (50 microM) were reduced to 22% and 26%, respectively, compared to normal cells. The impairment of TUDCA-induced [Ca++]i increase in cholestatic cells could be mimicked by exposing normal cells to low extracellular Ca++ (21%) or to the Ca++ channel blocker NiCl2 (23%). These data indicate that (a) dihydroxy bile acid-induced Ca++ entry may be of functional importance in the regulation of hepatocellular vesicular exocytosis, and (b) this Ca++ entry mechanism across the plasma membrane is impaired in cholestatic hepatocytes. We speculate that the beneficial effect of ursodeoxycholic acid in cholestatic liver diseases may be related to the Ca+(+)-dependent stimulation of vesicular exocytosis by its conjugate.

Acetylglucosaminidase↗
PubMed1992

Mechanisms of subcellular cytosolic Ca2+ signaling evoked by stimulation of the vasopressin V1a receptor.

Receptor activation may result in distinct subcellular patterns of Ca2+ release. To define the subcellular distribution of Ca2+i signals induced by stimulation of the vasopressin V1a receptor, we expressed the cloned receptor in Xenopus oocytes. Oocytes were then loaded with fluo-3 and observed using confocal microscopy. Vasopressin induced a single concentric wave of increased Ca2+ that radiated inward from the plasma membrane. With submaximal stimulation, however, regions of the Ca2+ wave spontaneously reorganized into repetitive (oscillatory) waves. Focal stimulation of a small part of the plasma membrane resulted in a Ca2+ wave which began at the point of stimulation, radiated toward the center of the cell, then reorganized into multiple foci of repetitive, colliding waves and spirals of increased Ca2+i. The pattern of Ca2+ signaling induced by focal or global stimulation was not altered in Ca(2+)-free medium, although signals did not propagate as fast. Finally, subcellular Ca2+ signaling patterns induced by vasopressin were inhibited by caffeine, while neither vasopressin nor microinjection of inositol trisphosphate blocked caffeine-induced increases in cytosolic Ca2+. Thus, stimulation of the V1a receptor in this cell system induces a complex pattern of Ca2+ signaling which is influenced by (1) the magnitude of the stimulus, (2) the distribution of the surface receptors that are stimulated, and (3) mobilization of Ca2+ from the extracellular space as well as from two distinct endogenous Ca2+ pools. The manner in which a single type of receptor is activated may represent an important potential mechanism for subcellular Ca2+i signaling.

Animals↗
PubMed1992

Mechanism of Ca2+ wave propagation in pancreatic acinar cells.

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca(2+)-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar Cl- secretion, we examined whether such waves also are important for amylase secretion. Ca2+ wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2+ waves which usually travelled in an apical-to-basal direction. Both caffeine and ryanodine, at concentrations that inhibit Ca(2+)-induced Ca2+ release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over 3-fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-induced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca(2+)-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells.

Acetylcholine↗
PubMed1992

Effects of Ca2+ agonists on cytosolic Ca2+ in isolated hepatocytes and on bile secretion in the isolated perfused rat liver.

The effects of increases in cytosolic Ca2+ on hepatocyte bile secretion are unknown. A number of agents that alter levels of cytosolic Ca2+ in the hepatocyte also produce hepatic vasoconstriction and activate protein kinase C, which complicates interpretations of their effects on bile secretion. To better understand the role of cytosolic Ca2+ in bile secretion, we examined the effect of the Ca2+ ionophore A23187 (0.1 mumol/L), the Ca2+ agonist vasopressin (10 nmol/L) and the Ca(2+)-mobilizing agent, 2,5-di(tert-butyl)-1,4-benzohydroquinone (25 mumol/L) on cytosolic Ca2+ in isolated hepatocytes and on bile flow in the isolated perfused rat liver, using vasodilators and inhibitors of protein kinase C and Ca2+ influx. Single-pass perfused livers were used, and cytosolic Ca2+ was measured by luminescent photometry in isolated hepatocytes loaded with the Ca(2+)-sensitive photoprotein aequorin. After A23187 perfusion, a sustained 74% +/- 10% (mean +/- S.D.) decrease in bile flow and a sustained 271% +/- 50% increase in perfusion pressure was observed. Simultaneous pretreatment with the vasodilator papaverine (25 mumol/L) and the protein kinase C inhibitor H-7 (50 mumol/L) abolished the pressure increase but not the decrease in bile flow, whereas pretreatment with Ni2+ (25 mumol/L) to block the influx of extracellular Ca2+ markedly reduced both the pressure increase and the decrease in bile flow. Vasopressin produced a transient (mean = 6 min) 75% +/- 4% decrease in bile flow and a sustained 7% +/- 4% increase in perfusion pressure. Pretreatment with H-7 alone corrected the vasopressin-induced pressure increase but also failed to eliminate the decrease in bile flow, whereas pretreatment with Ni2+ decreased the magnitude of the decrease by two-thirds without affecting the increase in perfusion pressure, 2,5'-di(tert-butyl)-1,4-benzohydroquinone produced a transient 65% +/- 20% decrease in bile flow and a transient 56% +/- 15% increase in perfusion pressure. In isolated hepatocytes, bromo-A23187, the nonfluorescent form of the ionophore, produced a sustained 56% +/- 32% increase in the cytosolic Ca2+ signal, whereas vasopressin resulted in a transient 241% +/- 75% increase and 2,5-di(tert-butyl)-1,4-benzohydroquinone resulted in a sustained 149% +/- 66% increase. The ionophore-induced increase in Ca2+ was abolished completely by pretreatment of the hepatocytes with Ni2+, whereas the vasopressin-induced increase was reduced by 38%.(ABSTRACT TRUNCATED AT 400 WORDS)

Aequorin↗
PubMed1992

Subcellular distribution of cytosolic Ca2+ in isolated rat hepatocyte couplets: evaluation using confocal microscopy.

Ca2+ agonists induce Ca2+ waves and other non-uniform Ca2+ patterns in the cytosol of epithelial cells. To define subcellular Ca2+ transients in the cytosol of hepatocytes we examined Fluo-3-loaded isolated rat hepatocyte couplets using confocal microscopy. Optical sections of less than 1 micron in thickness were observed in couplets, and fluorescence from cytosolic Ca2+ signals was readily distinguished from nuclear, mitochondrial, and lysosomal fluorescence. The nature of the noncytosolic components of the fluorescent images was verified by double labelling with the mitochondrial dye DiOC6(3) and with the lysosomal marker acridine orange. Using the line scanning mode of confocal microscopy, measurements of cytosolic Ca2+ were made with a frequency of up to 250 Hz and without significant bleaching. It was found that phenylephrine-induced Ca2+ signals generally began at the basal pole of the hepatocytes, then spread to the canaliculus at average speeds of 80 micron/s. These findings demonstrate the utility of confocal line scanning microscopy for detecting rapid changes in the subcellular distribution of cytosolic Ca2+ in hepatocyte couplets, and suggest that phenylephrine-induced Ca2+ waves radiate in a basal-to-apical direction in this cell type.

Animals↗
PubMed1992

Coordination of hormone-induced calcium signals in isolated rat hepatocyte couplets: demonstration with confocal microscopy.

Excitable cells often display rapid coordination of hormone-induced intracellular calcium signals. Calcium elevations that begin in a single epithelial cell also may spread to adjacent cells, but coordination of hormone-induced signals among epithelial cells has not been described. We report the use of confocal microscopy to determine the inter- and intracellular distribution of cytosolic calcium in isolated rat hepatocyte couplets, an isolated epithelial cell system in which functional polarity is maintained. Both vasopressin and phenylephrine evoked sequential coordinated calcium signals in the couplets, even during cytosolic calcium oscillations. The coupling was abolished by closure of intercellular gap junction channels by treatment with octanol. These observations demonstrate that hormone-induced intracellular calcium signals are coordinated among hepatocytes and suggest that gap junction channels mediate this intercellular integration of tissue responsiveness.

Animals↗
PubMed1992

Hormonal regulation of paracellular permeability in isolated rat hepatocyte couplets.

Many hormones and drugs exert their effects on cells by increasing cytosolic Ca2+ (Cai2+) and activating protein kinase C (PKC). Each of these actions results in cholestasis in the isolated perfused rat liver, but the responsible mechanisms are unclear. We used isolated rat hepatocyte couplets to observe the direct effects of increased Cai2+ and PKC activation on permeability of the hepatocyte tight junction and canalicular volume, two possible determinants of hepatocyte bile secretion. Couplets were stimulated with the Ca2+ agonist vasopressin (10(-8) M) in the absence and presence of the Ca2+ influx antagonist Ni2+ (5 x 10(-3) M) or with the PKC activator phorbol dibutyrate (10(-6) M). Cai2+ was determined by ratio microspectrofluorometry of indo-1, permeability of the couplet tight junctions was assessed by exclusion of horseradish peroxidase from the canalicular space, and changes in canalicular volume over time were measured directly by optical planimetry. Canalicular volume increased by 1.6 +/- 2.5%/min (mean +/- SD) under basal conditions. In response to vasopressin, there was a rapid 15-fold increase in Cai2+, followed first by an increase in paracellular permeability, then by canalicular collapse (15.9 +/- 5.9%/min). Pretreatment with Ni2+ markedly decreased the vasopressin-induced increase in Cai2+ and abolished both the increase in paracellular permeability and the canalicular collapse. Phorbol dibutyrate also increased paracellular permeability but resulted in neither increased Cai2+ nor canalicular collapse. The PKC inhibitor H-7 reversed the effects of both vasopressin and phorbol dibutyrate on tight junction permeability. Bile secretory pressure, measured in isolated perfused rat liver preparations, was acutely increased by vasopressin, but the increase was augmented rather than inhibited by Ni2+.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin.

Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Regulation of intestinal iron absorption and mucosal iron kinetics in hereditary hemochromatosis.

In hereditary hemochromatosis (HH), increased intestinal iron absorption leads to the development of iron overload. To examine the abnormal regulation of iron absorption in this disorder, we analyzed mucosal iron kinetics in six patients with HH and in five normal subjects by using a compartmental model of intestinal iron absorption and systemic ferrokinetics. Subjects were given simultaneous oral and intravenous tracer doses of iron 59-labeled citrate and iron 55-labeled transferrin, respectively. Plasma and whole-body radioactive iron levels were then monitored serially during the next 2 weeks, and mucosal iron transport rate constants were estimated by non-linear least-squares fit of the model to these data. Iron absorption was inversely related to serum ferritin concentration in both normal subjects and patients with HH but was higher in relation to serum ferritin level among the latter (p less than 0.0002). Analysis of mucosal iron kinetics demonstrated a similar inverse relationship between the rate constant for mucosal iron uptake and serum ferritin among all subjects combined, but the mean uptake rate constant in patients with HH did not differ from that of normal subjects (p = 0.71). The mean rate constant for incorporation of iron into the mucosal storage pool in patients with HH also was comparable to that of normal subjects (p = 0.94). In contrast, the rate constant for transfer of mucosal iron to the plasma was higher in patients with HH than in normal subjects for any given serum ferritin level (p less than 0.0001), and the transfer rate constant accounted for 87% of the variability in iron absorption among all subjects. We conclude that the increased iron absorption in HH is mediated primarily by an increase in the rate constant for transfer of mucosal iron to the plasma.

Ferritins↗