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PubMed2001

Effects of berberine on intracellular free calcium in smooth muscle cells of Guinea pig colon.

AIM: To investigate the effects and mechanism of berberine (Ber) on the intracellular free calcium concentration ([Ca(2+)](i)) in the smooth muscle cells of guinea pig colon. METHODS: The changes of [Ca(2+)](i) were assayed by the biwavelength spectrophotometry with Fura 2-AM in the cell suspension of the smooth muscle cells, which were freshly isolated from guinea pig colon. RESULTS: In the resting state, [Ca(2+)](i) in the HEPES-Ringer solution (CaCl(2) 1.5 mmol.l(-1)) was (108 +/- 9.4) nmol.l(-1) (n = 7). Ber had no significant effects on the resting [Ca(2+)](i), but markedly inhibited the increase in [Ca(2+)](i )induced by 60 mmol.l(-1) KCl in a concentration-dependent manner. The value of IC(50 )was 34.09 micromol.l(-1). 30 and 100 micromol.l(-1) Ber also inhibited the elevation of [Ca(2+)](i) evoked by 10 micromol.l(-1) Ach in a dose-dependent fashion in the presence or absence of extracellular Ca(2+). In addition, Ber inhibited the elevation of [Ca(2+)](i) stimulated by cyclopiazonic acid (CPA) in a dose-dependent manner. This effect was more potent in the HEPES-Ringer solution (IC(50) = 37.79 micromol.l(-1)) than Ca(2+)-free medium (IC(50) = 49.70 micromol.l(-1)). CONCLUSIONS: Ber possessed an inhibitory effect on the influx of extracellular Ca(2+) and Ca(2+)-release from intracellular stores in the smooth muscle cells of colon. That is to say Ber may be a blocker of Ca(2+) channels.

Acetylcholine↗
PubMed1985

Regional variation in norepinephrine-stimulated calcium uptake in rabbit aorta.

This study was conducted to determine if there is a difference in the calcium handling mechanism of the thoracic and abdominal aortic regions of the rabbit aorta. Isolated segments of aorta from both regions were studied in HEPES buffer at 37 degrees C and bubbled with 100% oxygen using identical procedures. Intracellular calcium levels were determined after segments had been exposed to 45Ca-labeled buffer during experimental procedures and subsequently washed with a 'quench' solution of zero calcium, 2 mM EGTA, and HEPES buffer, maintained at 0 degree C. The results suggest that the thoracic region is capable of taking up more calcium under resting conditions compared to the abdominal aorta. During stimulation with norepinephrine, both regions show significantly increased calcium influx rates after 10 min of exposure to the agonist, but only in the abdominal region is this effect maintained after 60 min of norepinephrine stimulation. Net (summation of calcium influx and efflux) intracellular calcium levels are significantly increased by norepinephrine in the thoracic but not in the abdominal aorta. Taken together, these data suggest that, compared to the thoracic aorta, the abdominal aorta may possess a more effective calcium efflux mechanism which may be able to fully compensate for the norepinephrine-stimulated increase in calcium influx.

Animals↗
PubMed1988

15-Hydroxyeicosatetraenoic acid (15-HETE) specifically inhibits LTB4-induced chemotaxis of human neutrophils.

15-Hydroxyeicosatetraenoic acid (15-HETE), a 15-lipoxygenase (15-LO) product of arachidonic acid has the potential to inhibit leukotriene formation. In the present study the effect of 15-HETE on leukotriene B4 (LTB4)-induced polymorphonuclear leukocytes (PMN) and monocyte chemotaxis was investigated. LTB4-induced chemotaxis of PMNs was inhibited in a dose-dependent manner by 15-HETE. Maximum inhibition (68%) occurred at a 15-HETE concentration of 10(-4) M. The 15-LO product of eicosapentaenoic acid (15-HEPE) was approximately 10 times less potent in inhibiting LTB4-induced PMN chemotaxis. LTB4-induced chemotaxis of monocytes was unaffected by both 15-HETE and 15-HEPE. Using N-formyl-methionyl-leucyl-phenylalanine (FMLP) and complement split product C5a as chemoattractants, 15-HETE did not decrease PMN chemotaxis. Furthermore, 15-HETE itself did not affect random migration of leukocytes. The present results demonstrate that 15-HETE inhibits LTB4-induced chemotaxis of PMNs in vitro in a specific and selective way. Because 15-HETE not only inhibits formation, but also the effect of LTB4, it may be important in regulating LTB4-induced inflammation.

Adult↗
PubMed1992

HCO3(-)-dependent intracellular pH regulation in the premature myocardium.

This study investigated developmental changes in Na(+)-H+ exchange and HCO3(-)-Cl- exchange activities in newborn and adult rabbit hearts. pHi was measured using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in isolated myocytes. Myocardial mechanical function was measured in the isolated ventricular preparation. Intracellular acidosis with normal pHo was induced by an NH4Cl (10 mM) prepulse technique. Upon removal of NH4Cl, pHi fell transiently and then recovered toward the control level. In the HCO3-/CO2-buffered solution, the rate of recovery of pHi in the newborn was greater than in the adult. In the HCO3-/CO2-buffered solution, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na(+)-H+ exchange, inhibited the recovery of pHi completely in the adult. In the newborn, however, significant recovery of pHi was observed in the presence of EIPA. In the presence of both EIPA and 4-acetamido-4'-isothiocyanatostilbene-2',2'-disulfonic acid (SITS), an inhibitor of HCO3(-)-Cl- exchange, the recovery of pHi was not observed in the two age groups. In the HEPES-buffered solution that did not contain HCO3-/CO2, the rate of recovery of pHi after NH4Cl removal was similar in the two age groups. In the HEPES-buffered solution, the recovery of pHi was completely inhibited by EIPA in the two age groups. In the presence of EIPA in the HCO3-/CO2-buffered solution, contractile function decreased during acidosis after NH4Cl removal and did not recover in the adult. In the newborn, significant recovery of contractile function was observed after NH4Cl removal in the presence of EIPA. The recovery of mechanical function observed in the presence of EIPA in the newborn was inhibited by SITS. These data suggest that, although there is no developmental change in the Na(+)-H+ exchange activity, HCO3(-)-Cl- exchange is more active in the premature myocardium. The presence of the HCO3(-)-Cl- exchanger is important in maintaining myocardial contractile function during acidosis, especially when Na(+)-H+ exchange is inhibited and may partly explain the greater resistance of the premature myocardium to acidosis.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1995

pHi regulation in myocardium of the spontaneously hypertensive rat. Compensated enhanced activity of the Na(+)-H+ exchanger.

To elucidate the mechanisms controlling pHi in myocardium of the spontaneously hypertensive rat (SHR), experiments were performed in papillary muscles (isometrically contracting at 0.2 Hz) from SHR and age-matched normotensive Wistar-Kyoto (WKY) rats loaded with the pH-sensitive fluorescent probe BCECF-AM. An enhanced activity of the Na(+)-H+ exchanger was detected in the hypertrophic myocardium of SHR. This conclusion was based on the following: (1) The myocardial pHi was more alkaline in SHR (7.23 +/- 0.03) than in WKY rats (7.10 +/- 0.03) (P < .05) in HEPES buffer. (2) SITS (0.1 mmol/L in HEPES buffer) did not alter pHi in the SHR (pHi 7.26 +/- 0.03 and 7.28 +/- 0.03 before and after SITS, respectively). (3) The fall in pHi observed after 20 minutes of Na(+)-H+ exchanger inhibition [5 mumol/L 5-(N-ethyl-N-isopropyl)amiloride (EIPA)] was greater in SHR (-0.16 +/- 0.01) than in WKY rats (-0.09 +/- 0.02, P < 0.05). (4) The velocity of pHi recovery from an intracellular acid load was faster in SHR than in WKY rats (0.068 +/- 0.02 versus 0.014 +/- 0.002 pH units/min at pHi 6.99, P < .05). (5) After EIPA inhibition, the rate of pHi recovery from the same acid load decreased to a similar value in both rat strains (0.0032 +/- 0.002 pH units/min in SHR and 0.0032 +/- 0.002 pH units/min in WKY rats). Under the more physiological HCO3(-)-CO2 buffer, no significant difference in steady state myocardial pHi was detected between rat strains (7.15 +/- 0.03 in SHR and 7.11 +/- 0.05 in WKY rats).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Cl(-)-HCO3- exchanger isoform AE2 is restricted to the basolateral surface of alveolar epithelial cell monolayers.

We investigated the polarized distribution and isoform specificity of anion exchange (Cl(-)-HCO3- exchange) in alveolar epithelial cell monolayers. Rat alveolar type II epithelial cell monolayers were grown in primary culture on detachable tissue culture-treated nuclepore filters. Each filter was mounted in a cuvette containing two fluid compartments (apical and basolateral) separated by the monolayer, the cells loaded with pH-sensitive dye, and intracellular pH (pHi) measured spectrofluorometrically. To assay for Cl(-)-HCO3- exchange, monolayers were incubated in medium containing 24 mM HCO3-/5% CO2 and 140 mM NaCl at pH 7.4 and acutely alkalinized by replacement of the fluid by HCO3(-)-free buffer containing Hepes (6 mM) at pH 7.4. Monolayers exhibited basolateral (but not apical) Cl(-)-dependent, Na(+)-independent recovery from an alkaline load that was abolished when Cl- was substituted by equimolar gluconate in the basolateral fluid, or if DIDS (500 microM) was present basolaterally. Substitution of gluconate for Cl- in the basolateral fluid, but not the apical fluid, resulted in a rise in steady-state pHi that was reversible on replacement of the basolateral fluid with Cl(-)-containing buffer, which occurred in HCO3(-)- but not Hepes-buffered medium. These data indicate that alveolar epithelial cells express basolateral membrane domain of these cells. Northern analysis of alveolar epithelial cell mRNA using anion exchanger (AE) isoform-specific cDNA probes indicates that alveolar epithelial cells express the AE2 isoform predominantly, if not exclusively, and do not express detectable AE1 (i.e., band-3 protein) or AE3.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1988

Bicarbonate absorption stimulates active calcium absorption in the rat proximal tubule.

To evaluate the effect of luminal bicarbonate on calcium reabsorption, rat proximal tubules were perfused in vivo. Perfusion solution contained mannitol to reduce water flux to zero. Total Ca concentration was measured by atomic absorption spectrometry, Ca ion concentration in the tubule lumen (CaL2+) and the peritubular capillary (CaP2+), and luminal pH (pHL) with ion-selective microelectrodes and transepithelial voltage (VTE) with conventional microelectrodes. When tubules were perfused with buffer-free Cl-containing solution, net Ca absorption (JCa) averaged 3.33 pmol/min. Even though VTE was 1.64 mV lumen-positive, CaL2+, 1.05 mM, did not fall below the concentration in the capillary blood, 1.07 mM. When 27 mM of Cl was replaced with HCO3, there was luminal fluid acidification. Despite a decrease in VTE and CaL2+, JCa increased to 7.13 pmol/min, indicating that the enhanced JCa could not be accounted for by the reduced electrochemical gradient, delta CCa. When acetazolamide or an analogue of amiloride was added to the HCO3 solution, JCa was not different from the buffer-free solution, suggesting that HCO3-stimulated JCa may be linked to acidification. To further test this hypothesis, we used 27 mM Hepes as the luminal buffer. With Hepes there was luminal fluid acidification and JCa was not different from the buffer-free solution but delta CCa was significantly reduced, indicating enhanced active calcium transport. We conclude from the results of the present study that HCO3 stimulates active Ca absorption, a process that may be linked to acidification-mediated HCO3 absorption.

Absorption↗
PubMed1993

Effect of secretion on intracellular pH regulation in isolated rat bile duct epithelial cells.

The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pHi) and HCO3- excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pHi was measured with 2,7-bis(carboxy-ethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECF-AM) using a microfluorimetric method. Basal pHi of BDE was 7.04 +/- 0.06 in Hepes and 7.16 +/- 0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not affected by this hormone. After acute Cl- removal, pHi rose 0.24 +/- 0.08 pHU at a maximal rate of 0.125 +/- 0.06 pHU/min (H+ flux rates = 6.02 +/- 3.27 mM/min) and recovered after Cl- readmission (0.188 +/- 0.08 pHU/min; H+ flux rates = 11.82 +/- 5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl- removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3- influx. Secretin significantly increased both the maximal rate of alkalinization after Cl- removal (P < 0.012) and of pHi recovery after Cl- readmission (P < 0.025), indicating stimulation of Cl-/HCO3- exchange activity. These findings were reproduced with N6,2'-O-Dibutyryladenosine-3',5'-cyclic monophosphate (DBcAMP). The Cl- channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 microM) significantly decreased the effects of secretin and DBcAMP on the pHi changes promoted by acute Cl- removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3- exchanger in BDE cells, probably by activating Cl- channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na+/HCO3- symport. The cell depolarization induced by Cl- channel activation should enhance HCO3- entrance through electrogenic Na+/HCO3- symport, which in turn stimulates the Cl-/HCO3- exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile.

Animals↗
PubMed1991

Induction of calcium phosphate precipitation by titanium dioxide.

Titanium powder and various titanium dioxides were tested for their capacity to reduce the induction time for calcium phosphate precipitation from supersaturated solutions. Only after a pretreatment aimed at increasing its oxide surface layer was titanium powder found to accelerate the precipitation from solutions containing 2 mmol/L CaCl2, 2 mmol/L KH2PO4, 50 mmol/L Hepes, pH 7.2, and to induce precipitation from metastable solutions containing 1.2 mmol/L CaCl2, 1.2 mmol/L KH2PO4, 50 mmol/L Hepes, pH 7.2, at 37 degrees C. Even stronger effects were found when suspensions of the titanium dioxides anatase or rutile (10-50 micrograms/mL) were added to these solutions. TiO2 appeared to serve as a reactive substrate for secondary nucleation at a wide range of calcium-to-phosphate ratios and concentrations, even in the presence of 40 mg/mL bovine serum albumin, which completely inhibited precipitation in control incubations. These results suggest that the oxide surface layer of titanium implants may induce calcium phosphate precipitation in the metal-to-bone interface, which may play a role in the integration of such implants in bone.

Calcium Phosphates↗
PubMed1992

Silica-induced precipitation of calcium phosphate in the presence of inhibitors of hydroxyapatite formation.

The promotion and the inhibition of hydroxyapatite formation by various substances were determined by measurement of the induction time of spontaneous precipitation (ti) from supersaturated solutions. Silica was found to decrease ti in Hepes-buffered (pH 7.2) supersaturated solutions with a wide range of calcium-to-phosphate ratios and concentrations. Also, in suspensions of the oral bacteria S. mutans or C. matruchotii in 1 mmol/L calcium, 7.5 mmol/L phosphate, and 50 mmol/L Hepes (pH 7.2), silica was capable of stimulating precipitation. Macromolecules derived from these bacteria by freezing and thawing appeared to be strong inhibitors of calcium phosphate precipitation. In the presence of silica, the effects of these bacterial inhibitors could be partially overcome, which supports the idea that silica in dental plaque is a promoter of calculus formation. In contrast, inhibition of calcium phosphate precipitation by a low-molecular-weight inhibitor, pyrophosphate, could not be counteracted by silica.

Bacterial Proteins↗
PubMed2001

Hypertonic saline alters ion transport across the human airway epithelium.

Aerosolized hypertonic saline is currently being investigated as a new agent for the treatment of impaired mucociliary clearance which occurs in many respiratory diseases. Mannitol aerosols, in particular dry powder inhalers, have been proposed as an alternative treatment to saline, offering the same osmotic load with other benefits. However, the effects of these hypertonic aerosols on airway epithelial ion transport processes have not been tested in human subjects in vivo. This report examines the effect of these solutions on airway ion transport using the nasal potential difference (PD) technique. Seven healthy nonsmoking adult volunteers were studied. On different days, a dose-response curve was constructed for the saline added to Krebs N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulphonic acid] (HEPES) diluent. The reversibility of this saline effect was measured, and the response to additional saline (500 mM) and mannitol (1 M) compared. Hypertonic saline decreased nasal PD in a dose-related manner, with mean (SEM) decreases in PD (less negative) of 6.6 (1.5), 7.6 (1.6), 10.0 (2.0), 13.1 (2.9) and 14.8 (3.2) mV (n =4) for addition of 150 mM, 250 mM, 500 mM, 1,200 mM and 2,000 mM NaCl to the Krebs HEPES diluent, respectively. The effect of hypertonic saline was fully reversible with washout for 3 min (presaline 15.9 (0.5) mV, postwashout 15.8 (1.1) mV, (n=4)). The hypertonic saline response was rapid in onset, sustained for at least 4 min, and decreased PD from 13.7 (1.7) mV to 5.1 (1.3) mV (n = 7, p < 0.001). In contrast, addition of mannitol to the perfusate did not significantly alter nasal PD, with a nonsignificant trend towards an increase (more negative) in the PD, (premannitol 13.9 (1.6) mV, postmannitol 15.3 (2.0) mV, n=7). As the osmotic stimulus of the 1 M mannitol is similar to that of the 500 mM sodium chloride, the divergent nasal potential difference responses suggest that the response to the saline was specific to the sodium chloride itself and not the simultaneous change in osmolarity. This demonstrates that the human airway epithelium in vivo can respond to topical hypertonic saline independent of the altered osmolarity.

Administration, Intranasal↗
PubMed1983

Impaired cerebroside sulfate hydrolysis in fibroblasts of sibs with "pseudo" arylsulfatase A deficiency without metachromatic leukodystrophy.

Low arylsulfatase A levels are reported in two siblings, one with a neurologic disability not typical for metachromatic leukodystrophy, the other a healthy 18-year-old female with a normal developmental history. In both individuals, arylsulfatase A levels in white blood cells were 7-8% of control values. Cultured fibroblasts gave low values (8-10% of normal) for both cerebroside sulfatase and arylsulfatase A activities. Other family members had enzyme levels consistent with heterozygote or normal status. Cerebroside sulfate loading tests of cultured fibroblasts in 199-CO2 media were normal for all family members who were tested. In MEM-HEPES media, however, cells from the two arylsulfatase A deficient siblings showed attenuated sulfolipid catabolism. Additional clinical and laboratory studies on these individuals failed to demonstrate any features suggestive of metachromatic leukodystrophy, i.e., normal nerve conduction velocities, normal sural nerve biopsy results, and normal urinary sulfatide excretion. It is concluded that the neurologic abnormalities in the one sibling are not the result of the low enzyme activity and that both individuals represent examples of pseudo arylsulfatase A deficiency (arylsulfatase A deficiency without metachromatic leukodystrophy). These results thus call into question the ability of the high-sensitivity cerebroside sulfate loading test as carried out in MEM-HEPES media to differentiate pathologically significant defects i.e., metachromatic leukodystrophy from benign "pseudo-deficiencies."

Adolescent↗
PubMed1982

Insulin radioreceptor assay on murine splenic leukocytes and peripheral erythrocytes.

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH4Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free [125I]iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 X 10(7)/ml) consisted of incubation with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific [125I]iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 X 10(9)/ml) were incubated with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific [125I]iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner. This should facilitate direct comparisons of these cells with classical target cells in a variety of mouse model diseases.

Ammonium Chloride↗
PubMed2000

Regulation of the formation of tumor cell pseudopodia by the Na(+)/H(+) exchanger NHE1.

The Na(+)/H(+) exchanger NHE1 is involved in intracellular pH homeostasis and cell volume regulation and accumulates with actin in the lamellipodia of fibroblasts. In order to determine the role of NHE1 following epithelial transformation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) transformed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV). Expression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with b-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-mediated (22)Na uptake to ethylisopropylamiloride, a specific inhibitor of NHE1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intracellular pH induced upon EIPA treatment were also of higher magnitude in MSV-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 microM ethylisopropylamiloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells, the effect of ethylisopropylamiloride treatment was more pronounced and associated with the increased reversible detachment of the cells from the substrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minutes of addition, ethylisopropylamiloride induced pseudopodial retraction and inhibited cell motility. The ability of ethylisopropylamiloride to prevent nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO(3) medium, showing that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesion and pseudopodial protrusion of motile, invasive tumor cells.

Actin Cytoskeleton↗
PubMed1990

Greatly improved activity of staphylococcal ribosomes in polyadenylate directed polylysine synthesis: as an assay system for investigating their sensitivity to macrolide antibiotics.

In polyadenylate directed polylysine synthesis, homologously cell-free extracts containing ribosomes and S-100 (105,000 x g supernatant) from staphylococcal cells have less than one-half (one-tenth, when the extracts were stored at -80 degrees C within a few weeks) of the activity of the extracts from Escherichia coli Q13. The present study is concerned with further improving the activity of staphylococcal ribosomes. The polylysine-synthesizing ability by staphylococcal ribosomes increased up to about two times as much as that by E. coli Q13 ribosomes, when S-100 from E. coli Q13 was mixed with staphylococcal ribosomes which had been washed with a high salt HEPES buffer containing 10 mM HEPES, 1 mM EGTA, 16 mM magnesium acetate, 1.0 M ammonium chloride and 0.1 mM dithiothreitol (pH7.6). Polylysine synthesis by the heterologous extracts has an advantage over polyuridylate-directed polyphenylalanine synthesis in the analysis of ribosome sensitivity for macrolide antibiotics, especially erythromycin.

Anti-Bacterial Agents↗
PubMed1981

Artificial buffers do not inhibit contractile responses in the smooth muscle of rat portal vein and guinea pig taenia coli.

Effects of substitution for NaHCO3 (and 5% CO2) in the physiological solution with equimolar N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or morpholinopropane sulfonic acid (MOPS) (and 100% O2) at various pH levels on the contractility of smooth muscle were examined. At pH 7.4, spontaneous contraction in rat portal vein was not inhibited by the artificial buffer solutions, as compared to findings in the case of bicarbonate buffer solution. Norepinephrine-dose response curve in the case of rat portal vein and the histamine-dose response curve in cases of guinea pig taenia coli remained unchanged in the artificial buffer solutions. At pH 7.2 and 7.0, the spontaneous contraction in portal vein was reversibly inhibited either in artificial or in bicarbonate buffer solutions. The norepinephrine- and histamine-dose response curve shifted downwards and/or to the right in these low pH solutions. Thus, HEPES and MOPS had no inhibitory effect on the smooth muscle contractility. Since low pH strongly inhibited the contractility, attention should be directed to the temperature-dependent decreases in pKa of the artificial buffers (i.e., if pH of the solution is adjusted at room temperature and then warmed, pH decreases).

Animals↗
PubMed1976

[Studies on the biosynthesis and secretion of parathyroid hormone in monolayer cultures of bovine parathyroid cells (I) (author's transl)].

We have developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3 yields to 0.5 percent collagenase at 37 degrees C for 60 min. Dispersed cells were cultured at 37 degrees C in MEM-Hanks' BSS containing 10 percent fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1 percent BSA-MEM-Hanks-HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10(-7) yields to 10(-5)M in the former and 10(-5) yields to 10(-3)M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3 yields to 4.4 mM.

Animals↗
PubMed1996

Effect of cooling mouse spermatozoa to 4 degrees C on fertilization and embryonic development.

Attempts to freeze mouse spermatozoa in liquid nitrogen (-196 degrees C) have met with limited success. In an attempt to identify the factor(s) that damage mouse spermatozoa during cryopreservation, the effect of slow cooling to 4 degrees C was examined. Epididymal spermatozoa were collected into a variety of media at 37 degrees C, cooled slowly to 4 degrees C over 4 h and warmed in a water bath at 37 degrees C for 5 min. Survival of spermatozoa was assessed by motility, membrane integrity and acrosome status. Labelling with chlortetracycline showed that > 80% of spermatozoa were capacitated and had intact acrosomes immediately after warming compared with < 20% of freshly collected (control) spermatozoa. The rate of fertilization in vitro was similar using spermatozoa cooled in Dulbecco's phosphate-buffered saline and then mixed with oocytes immediately after warming and with control spermatozoa incubated for 2 h before mixing with oocytes (85%). Fewer oocytes were fertilized with spermatozoa cooled in either a modified HEPES-buffered Tyrode's medium or a simple HEPES-buffered medium with a high osmolarity (D3), 63% and 58%, respectively. Two-cell embryos were transferred to the oviducts of pseudopregnant recipients. Implantation was similar in all groups (81-88%) and 54-74% of embryos formed normal late stage fetuses.

Acrosome↗