Properties of guanylate cyclase in adult rat liver and several Morris hepatomas.

Guanylate cyclase (GTP pyrophyosphate-lyase (cyclizing), EC 4.6.1.2) activity was examined in preparations from normal rat liver and a series of Morris hepatomas. Homogenate gyanylate cyclase activites were 3.2, 1.6 and 1.2 nmol cyclic GMP formed per min/g tissue ihe non-substrate analogs of IMP were weak inhibitors of this enzyme, GMP and four of its analogs had Ki values ranging from 30 to 80 muM. The GMP analogs (8-azaGMP, 7-deaza-8-azaGMP, 2'-dGMP and beta-D-arabinosylGMP) and GMP were competitive inhibitors with respect to GTP.

A role for protein kinase C delta in the differential sensitivity of MCF-7 and MDA-MB 231 human breast cancer cells to phorbol ester-induced growth arrest and p21(WAFI/CIP1) induction.

The goal of this study was to investigate the differential sensitivity of estrogen receptor (ER) positive MCF-7 and ER negative MDA-MB 231 breast cancer cells to phorbol myristate acetate (PMA)-dependent growth arrest. MCF-7 cells were growth arrested by 80% while MDA-MB 231 cells were arrested by 20% in response to seven days of treatment with 10 nM PMA. Coincident with the increased sensitivity of MCF-7 cells to be growth arrested by the protein kinase C (PKC) activator PMA, PMA induced 9-fold higher levels of the cyclin dependent kinase (Cdk) inhibitor p21(WAF1/GIP1) in MCF-7 compared to MDA-MB 231 cells. A comparison of the PKC isoforms expressed in MCF-7 versus MDA-MB 231 cells showed that only the PMA-sensitive PKC delta and eta isoforms were expressed at markedly (> or =10-fold) elevated levels in MCF7 versus MDA-MB 231 cells. These results suggested that the differential sensitivity to growth arrest and induction of p2l(WAFl/CIPl) could reflect, at least in part, increased expression of PMA-dependent PKC isoforms delta and/or eta. Direct evidence to support this hypothesis was provided by the ability of transient transfections into MCF-7 cells of constitutively active PKC delta but not of PKC's eta or alpha or epsilon to enhance p21(WAFl/CIP1) promoter activity. These results suggest that PKC delta plays a fundamental role in the regulation of growth in estrogen receptor positive breast cancer cells.

Free versus liposome-entrapped streptomycin sulfate in treatment of infections caused by Salmonella enteritidis.

Streptomycin sulfate liposomes were prepared by the vortex dispersion method. The liposomes were formulated from a mixture of L-alpha-dipalmitoyl phosphatidyl choline (DPPC), cholesterol with or without (neutral) a charge inducing agent. Two phospholipid molar ratios were considered, namely, DPPC cholesterol 7:2 and 7:4. The amount of streptomycin sulfate entrapped was estimated, microbiologically, and found to range from 0.080 to 1.323% of the initial amount of drug used for preparation of liposomes, depending on the surface charge of the liposomal vesicles. Particle size analysis, measured by the coulter counter, showed a mean particle diameter ranging from 4.417-8.424 microns. Drug targeting experiments were done using Swiss mice as the experimental animals. The in-vivo results indicated that the streptomycin sulfate concentration targeted to the liver and spleen by the liposome encapsulated drug was 2-3 times that exhibited by the free drug. This effect occurred after one day of liposome injection, but it decreased over time from one to seven days. The amount of streptomycin sulfate targeted to the lung, by the liposome formulation 7:2:1 was more than that exhibited by the free drug. This is true only after 7 d from injection. On the other hand, the liposomes of molar ratio 7:4:1 showed much less effect even when compared to the free drug. The survival rate experiments indicated a definite protection against Salmonella enteritidis, exhibited by the liposome-encapsulated streptomycin compared to the free drug.

Solubilization of guanylyl cyclase from bovine rod outer segments and effects of lowering Ca2+ and nitro compounds.

Guanylyl cyclase from bovine rod outer segments was solubilized using Triton X-100 and a high concentration of KCl, and its regulation was studied. The efficiency of solubilization was about 50-90% of total activity. When the Ca2+ content was lowered (less than 80 nM), guanylyl cyclase was activated about 2-fold. In the presence of higher concentrations of Ca2+ (greater than 140 nM), the activity was decreased. The regulation by Ca2+ was also demonstrated with solubilized preparations. In the presence of 186 nM Ca2+ which inhibited guanylyl cyclase, La3+ activated the enzyme about 2-fold, suggesting that the Ca2(+)-binding protein similar to other Ca2(+)-binding proteins associates with guanylyl cyclase regulation. Sodium nitroprusside and nitric oxide which are activators of soluble guanylyl cyclase in other tissues also activated the retinal guanylyl cyclase. Maximum activation by sodium nitroprusside was 20-fold using Mg2+ as a cofactor. Activation by nitric oxide and related compounds suggests that retinal guanylyl cyclase contains a heme prosthetic group that may participate in a novel regulatory mechanism for this enzyme.

Activation of guanylate cyclase from rat liver and other tissues by sodium azide.

Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.

Characterization of the receptor for heat-stable enterotoxin from Escherichia coli in rat intestine.

The receptor for the heat-stable enterotoxin (ST) from Escherichia coli was solubilized with Lubrol-PX from rat intestinal brush-border membranes and characterized. The binding kinetics and analog specificity of the solubilized receptor were virtually identical to those obtained with the membrane-bound receptor. Furthermore, the regulation of the receptor's affinity by cations was also maintained after solubilization, indicating a conservation of the toxin-binding site after removal of the receptor from its membrane environment. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 5.5 nm and a sedimentation coefficient of 7.0 S for the solubilized receptor. The isoelectric point of the receptor was determined as 5.5 using Sephadex isoelectric focusing electrophoresis. In all of these separation techniques, the ST receptor showed a single peak of activity that was clearly separated from that of guanylate cyclase. When 125I-ST was cross-linked to brush-border membranes with disuccinimidyl suberate, the affinity-labeled receptor solubilized with 0.1% Lubrol-PX eluted at a similar position as the native receptor on gel filtration chromatography. Analysis of the affinity-labeled receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent and by autoradiography revealed the presence of three specifically labeled polypeptides with apparent molecular weights of 80,000, 68,000, and 60,000. These results suggest that the ST receptor is solubilized by Lubrol-PX in an active form with preservation of its regulation by cations. Also, the ST receptor is separable from particulate guanylate cyclase indicating that the receptor is coupled to the activation of guanylate cyclase by an as yet undefined mechanism. Three subunit peptides may constitute a binding region of the receptor.

Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase.

A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa. Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX. Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes. The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes. Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction. This activity was completely resistant to solubilization with a variety of detergents and chaotropes. Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively. However, ST failed to activate particulate guanylate cyclase in detergent extracts. In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures. The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored. ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose. Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography. Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST. These data suggest that guanylate cyclase and the ST receptor are independent proteins coupled by cytoskeletal components in membranes of intestinal mucosa.