A Galactoside-Binding Protein Tricked into Binding Unnatural Pyranose Derivatives: 3-Deoxy-3-Methyl-Gulosides Selectively Inhibit Galectin-1.

Galectins are a family of galactoside-recognizing proteins involved in different galectin-subtype-specific inflammatory and tumor-promoting processes, which motivates the development of inhibitors that are more selective galectin inhibitors than natural ligand fragments. Here, we describe the synthesis and evaluation of 3-C-methyl-gulopyranoside derivatives and their evaluation as galectin inhibitors. Methyl 3-deoxy-3-C-(hydroxymethyl)-ß-d-gulopyranoside showed 7-fold better affinity for galectin-1 than the natural monosaccharide fragment analog methyl ß-d-galactopyranoside, as well as a high selectivity over galectin-2, 3, 4, 7, 8, and 9. Derivatization of the 3-C-hydroxymethyl into amides gave gulosides with improved selectivities and affinities; methyl 3-deoxy-3-C-(methyl-2,3,4,5,6-pentafluorobenzamide)-ß-d-gulopyranoside had Kd 700 µM for galectin-1, while not binding any other galectin.

Glycointeractome of Neisseria gonorrhoeae: Identification of Host Glycans Targeted by the Gonococcus To Facilitate Adherence to Cervical and Urethral Epithelial Cells.

Neisseria gonorrhoeae is a significant threat to global health for which a vaccine and novel treatment options are urgently needed. Glycans expressed by human cells are commonly targeted by pathogens to facilitate interactions with the host, and thus characterization of these interactions can aid identification of bacterial receptors that can be exploited as vaccine and/or drug targets. Using glycan array analysis, we identified 247 specific interactions between N. gonorrhoeae and glycans representative of those found on human cells. Interactions included those with mannosylated, fucosylated, and sialylated glycans, glycosaminoglycans (GAGs), and glycans terminating with galactose (Gal), N-acetylgalactosamine (GalNAc), and N-acetylglucosamine (GlcNAc). By investigating the kinetics of interactions with selected glycans, we demonstrate that whole-cell N. gonorrhoeae has a high affinity for mannosylated glycans (dissociation constant [KD ], 0.14 to 0.59 µM), which are expressed on the surface of cervical and urethral epithelial cells. Using chromatography coupled with mass spectrometric (MS) analysis, we identified potential mannose-binding proteins in N. gonorrhoeae Pretreatment of cells with mannose-specific lectin (concanavalin A) or free mannose competitor (α-methyl-d-mannopyranoside) substantially reduced gonococcal adherence to epithelial cells. This suggests that N. gonorrhoeae targets mannosyl glycans to facilitate adherence to host cells and that mannosides or similar compounds have the potential to be used as a novel treatment option for N. gonorrhoeaeIMPORTANCE Multidrug-resistant strains of Neisseria gonorrhoeae are emerging worldwide, and novel treatment and prevention strategies are needed. Glycans are ubiquitously expressed by all human cells and can be specifically targeted by pathogens to facilitate association with host cells. Here we identify and characterize the N. gonorrhoeae host-glycan binding profile (glycointeractome), which revealed numerous interactions, including high-affinity binding to mannosyl glycans. We identify gonococcal potential mannose-binding proteins and show that N. gonorrhoeae uses mannosyl glycans expressed on the surface of cervical and urethral epithelia to facilitate adherence. Furthermore, a mannose-binding lectin or a mannoside compound was able to reduce this adherence. By characterizing the glycointeractome of N. gonorrhoeae, we were able to elucidate a novel mechanism used by this important pathogen to interact with human cells, and this interaction could be exploited to develop novel therapeutics to treat antibiotic-resistant gonorrhea.

Antiallergic activity of novel isoflavone methyl-glycosides from Cordyceps militaris grown on germinated soybeans in antigen-stimulated mast cells.

Isoflavones are known to possess immunomodulating and antiallergic activities. Previously we identified novel isoflavone methyl-glycosides (daidzein 7-O-ß-d-glucoside 4″-O-methylate (CDGM), glycitein 7-O-ß-D-glucoside 4″-O-methylate (CGLM), genistein 7-O-ß-D-glucoside 4″-O-methylate (CGNMI) and genistein 4'-O-ß-D-glucoside 4″-O-methylate (CGNMII)) from Cordyceps militaris grown on germinated soybeans (GSC). The biological activity of novel isoflavone methyl-glycosides, however, remains unknown. In this study, CGNMII showed the strongest inhibition of degranulation. Additionally, the release of interleukin (IL)-4 and tumor necrosis factor (TNF)-α was decreased by CGNMII in antigen-stimulated RBL-2H3 cells. To elucidate the antiallergic mechanism of CGNMII, we examined whether it affected levels of signaling molecules responsible for degranulation. The levels of activated Lyn, Syk, PLCγ1 and LAT proteins were reduced in CGNMII treated RBL-2H3 cells. CGNMII also inhibited the activation of AKT and ERK1/2 proteins. These results suggest that CGNMII might be used as a therapeutic agent for allergic diseases.

Secondary metabolites and insecticidal activity of Anemone pavonina.

The insecticidal properties of the crude extracts of the leaves and flowers of Anemone pavonina were evaluated on Pheidole pallidula ants and showed significant levels of activity. Bioassay-guided fractionations led to the isolation of the butenolide ranunculin (1) as the active principle. Chemical investigations of the extracts showed them to contain as major components the sitosterol glycopyranoside lipids 2-5 and the glycerides 6-8. The structures of the metabolites were elucidated, following acetylation and hydrolysis of the natural products, by interpretation of their NMR and mass spectral data. The uncommon lipid metabolites 2-8 were isolated for the first time from the genus Anemone and this is the first report of insecticidal activity of the Anemone metabolite ranunculin against ants.

Beta-methyl-xyloside: positive effect on xylanase induction in Cellulomonas flavigena.

Synthesis of extracellular xylanase in Cellulomonas flavigena is induced in the presence of xylan and sugarcane bagasse as substrates. The essential factors for efficient production of xylanase are the appropriate medium composition and an inducing substrate. The increase in xylanase production levels in C. flavigena were tested with a number of carbon sources and different culture conditions. Xylose, arabinose, glycerol and glucose did not induce xylanase production in this microorganism. beta-Methyl-xyloside (beta-mx), a structural analog of xylobiose, also did not induce xylanase when used as the sole carbon source, but when xylan or sugar cane bagasse was supplemented with beta-mx, extracellular xylanase production increased by 25 or 46%, respectively. The response of C. flavigena to xylan plus beta-mx was accompanied by a significant accumulation of reducing sugar, an effect not observed with the combination sugarcane bagasse plus beta-mx as substrate. To our knowledge, this is the first report on the effect of beta-mx on the induction of xylanase in C. flavigena.

Further evidence for the gelation ability-structure correlation in sugar-based gelators.

Eight methyl glycosides of 4,6-O-benzylidene derivatives of the monosaccharides D-glucose, D-mannose, D-allose and D-altrose were synthesized to systematically study the effect of small configurational changes on the ability to gelate organic solvents. Among the beta anomers, only the D-mannose glycoside exhibits a strong gelation ability, whereas in the alpha-series the D-glucose and D-mannose derivatives act as versatile gelators. Also, as a general rule we found that the beta anomers possess a higher ability to gelate solvents than the alpha anomers. The gelation properties are discussed on the basis of SAXS, FTIR, differential scanning calorimetric (DSC) measurements and scanning electron microscopy (SEM) observations. The temperature-dependent SAXS measurements were carried out to elucidate the sol-gel transition temperature. The present study emphasizes that the saccharide family provides, not only valuable information of the structural requirements for the design of new gelators, but also for molecular assembly systems in general.

Adherence of Mycoplasma pneumoniae to human alveolar macrophages.

The human pathogen Mycoplasma pneumoniac causes primary atypical-cold agglutinin-positive pneumonia. Since alveolar macrophages internalize mycoplasma as part of their immune defense, we studied characteristics of the human macrophage receptor for opsonized and nonopsonized M. pneumoniae. The glass-adhering subpopulation of M. pneumoniae attached more than the non-adherent subpopulation. The attachment was dose-dependent and enhanced by opsonization in the presence of human serum. It is inhibited by sulfated compounds such as dextran-sulfate and polyanetholsulfonic acid, but not by dextran or several monosaccharides, suggesting that sulfated glycolipids on the macrophage surface may act as receptors for M. pneumoniae binding. In addition, sialylated compounds, such as fetuin and alpha 1-acid glycoprotein, were found to be potent inhibitors of the attachment, also indicating the role of sialic acid residue in recognition and attachment of M. pneumoniae to human alveolar macrophages.

[The cytotoxicity and action mechanism of ranunculin in vitro].

This paper describes the cytotoxicity of ranunculin (RAN) and its mechanism of action. The IC50 of RAN against the KB and Bel7402 cells in colony test were found to be 0.21 and 0.35 mumol/L respectively. RAN inhibited the incorporation of 3H-labeled precursors into DNA and RNA of L1210 cells. RAN (15 mumol/L) markedly decreased DNA synthesis catalyzed by DNA polymerase I and promoted the generation of superoxide anions in DMSO/KO2 system. In the meantime, SOD and CAT were shown to partly revoke the inhibitory effects of RAN upon the incorporation of 3H-TdR into DNA. No direct reaction between RAN and DNA template was observed and no effect of RAN on DNA TOPO II or RNA polymerase was found. Our results suggest that the cytotoxicity of RAN in vitro may be due to inhibition of DNA polymerase and increase of oxygen free radicals.

[Antimutagenic activity and metabolic transformation of ranunculin by rat liver microsomes].

Ranunculin (RAN) was shown to be an antimutagenic agent selectively against mitomycin C (MMC) or methyl methane sulfanate (MMS) treated Salmonella typhimminum TA100/TA102. It decreased the formation of micronucleus of MMC induced polychromatic erythrocytes (PEC) from 46 +/- 9.2% to 20 +/- 6% in mice. The inhibition of RAN on the incorporation of 3H-TdR into DNA disappeared after incubation with rat liver microsomes and cytoplasm since RAN was found to be metabolized by rat liver microsomes in vitro, resulting in a new absorbance peak at 258 nm, determined by RP-HPLC. The data show that RAN may have both antimutagen and antitumor activity, but the latter action may disappear by metabolic transformation.

Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.

Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.

Studies on pH regulatory mechanisms in cultured astrocytes of DBA and C57 mice.

pH regulatory mechanisms in primary cultures of astrocytes from the cerebral cortex of neonatal audiogenic-seizure-susceptible DBA/2J (DBA) and genetically controlled C57BL/6J (C57) mice were studied with [14C]dimethyloxazolidine-2-4-dione (DMO) and [3H]-methyl-D-glucose (MDG). Effects of changing the concentration of Na+, K+, HCO3- or Cl- in medium, and/or of different transport blockers and metabolite inhibitor on intracellular pH (pHi) of cultured astrocytes were also studied. In nominal HCO3(-)-free HEPES-buffered Hanks' balanced salt solution (HEPES HBSS), when the pH of medium (pHo) was maintained at 7.4, the steady-state pHi of cultured astrocytes from DBA mice was 6.98 +/- 0.03, and that from C57 mice was 7.01 +/- 0.03. When the cells were incubated in HBSS containing 25 mM HCO3- and equilibrated with 5% CO2 (HCO3- HBSS, pHo = 7.4), pHi of both DBA and C57 astrocytes was approximately 0.1-0.15 pH units higher than that in HEPES HBSS. Reducing the pH or the Na+ concentration in media (pHo, [Na+]o) of either HEPES HBSS or HCO3- HBSS, pHi of both DBA and C57 astrocytes decreased markedly (0.25-0.45 pH units lower than the controls). The decrease in pHi was greater in HEPES HBSS than in HCO3- HBSS. Reducing the Cl- concentration ([Cl-]o) in either HEPES or HCO3- HBSS, pHi of astrocytes increased by 0.05-0.1 pH units. Increasing the K+ concentration ([K+]o) of or adding Ba2+ to the media increased the pHi of both DBA and C57 astrocytes accordingly. SITS, an anion transport inhibitor, decreased the pHi of both DBA and C57 astrocytes in HCO3- HBSS but not in HEPES HBSS. It enhanced the response of pHi to reduction in pHo. Amiloride, a Na(+)-H+ exchange inhibitor, decreased the pHi of both DBA and C57 astrocytes more in HEPES HBSS than in HCO3- HBSS. It enhanced the response of pHi to reduction in pHo and [Na+]o. Ouabain, an Na+,K(+)-ATPase inhibitor, decreased the pHi of cultured astrocytes in HEPES HBSS, but not in HCO3- HBSS. It also enhanced the response of pHi to changing pHo and [Na+]o in HEPES HBSS. Acetazolamide, a carbonic anhydrase inhibitor, decreased the pHi of astrocytes in both HEPES and HCO3- HBSS. Both bumetanide, an Na+,K+/Cl- cotransport blocker, and KCN, a metabolic inhibitor, produced no significant effect on the steady-state pHi or the response of pHi to changing ionic concentration in media in both DBA and C57 astrocytes.

Free-radical synthesis of 3-(2-cyanoethyl)- and 3-(2-methoxycarbonylethyl)-2,3-dideoxy-alpha-D-erythro-pentofu ranoside and their application in the synthesis of potential antiviral nucleosides.

Free-radical reaction of different carbohydrate educts 2, 5, and 7 with acrylonitrile in the presence of tributyltin hydride and a radical initiator (AIBN) gave the methyl 3-(2-cyanoethyl)-2,3-dideoxypentofuranosides 3a and 6. Similar reaction of 2 with methyl acrylate gave 3-(2-methoxycarbonylethyl)-2,3-dideoxypentofuranose 3b. Nucleoside coupling of 3a with silylated uracil gave an anomeric mixture of beta- and alpha-nucleoside 8 and 9 which were deprotected to give 10 and 11, respectively. Similar reaction of 3b with silylated N4-isobutyrylcytosine gave 12 and 13 which were deprotected to give the final nucleosides 16 and 17, respectively. None of the compounds 10a, 11, 14-17 showed significant activity against HIV.

Derivatives of methyl beta-lactoside as substrates for and inhibitors of beta-D-galactosidase from E. coli.

The 2'-,4'-, and 6'-deoxy derivatives of methyl beta-lactoside have been synthesised by deoxygenation at positions 2', 4', and 6', and the 3'-deoxy derivative was obtained by a glycosylation reaction. The 2'-O-methyl, 2'-O-benzyl, 2'-amino-2'-deoxy, and 1'-deuterio derivatives have been synthesized also. Only the 6'-deoxy and 1'-deuterio derivatives were substrates for the beta-D-galactosidase from E. coli, and the 2'-deoxy- and 2'-amino-2'-deoxy derivatives were potent inhibitors for the hydrolysis of methyl beta-lactoside by the enzyme.

Whole cell recording of sugar-induced currents in LLC-PK1 cells.

Gigaohm-seal whole cell recording techniques were used to monitor function of the Na(+)-coupled sugar transport system in LLC-PK1 cells. The currents coupled to sugar transport were identified as those that are induced by the presence of 10 mM alpha-methylglucoside (AMG) in either the extracellular or intracellular compartment and were inhibited by addition of 320-800 microM phlorizin to the extracellular bathing medium. The sugar-induced currents are small, 15-20 pA, but of the expected magnitude as determined from the known kinetic parameters for Na(+)-coupled sugar transport in LLC-PK1 cells. The phlorizin-sensitive currents are Na+ dependent and can be studied under conditions in which the net Na+ and sugar flux (and consequently the Na+ electrical current) is in either the inward or outward direction. The reversal potential of the sugar-induced currents measured under conditions with high Na+ and AMG concentrations inside the cell is close to values predicted from thermodynamic principles, assuming a coupling stoichiometry of 2 Na+: 1 sugar for the transport system. The reversal potential of the sugar-induced currents with high extracellular Na+ and AMG is not equal to the predicted value, but it is of the polarity expected for inward-imposed solute gradients. Reasons for the observed discrepancy between observed and calculated values are discussed.

Effect of lactose derivatives on metastatic potential of B16 melanoma cells.

The effects of various sugars and sugar derivatives on lung colonization (i.e., metastatic deposition) of the highly metastatic BL6 clone of B16 mouse melanoma cells in syngeneic mice were studied, based on the assumption that carbohydrate structures, particularly those with a Gal terminus, play a crucial role in defining the metastatic potential of B16 cells. After incubation with sugar compounds (usually at 0.1 M concentration), tumor cells were injected via the tail vein into 8-week old female mice. Mice were sacrificed after 18-21 days, and tumor cell colonies in lung were counted under a dissecting microscope. Only methyl beta-D-lactoside and lacto-N-tetraose caused significant reduction (35-45% and 36%, respectively) of metastatic deposition compared to controls. Methyl beta-D-lactoside did not exhibit a growth inhibitory effect on BL6 tumor cells, as determined by several methods: in vitro [3H]thymidine incorporation assay, in vitro plating in RPMI-1640 medium culture under physiological conditions followed by cell counting, and in vivo subcutaneous inoculation of age-matched C57/BL mice followed by tumor measurement. These results indicate that the inhibitory effect of methyl beta-D-lactoside on tumor deposition was not related to its effect on tumor cell growth.

Sugar-dependent selective induction of mouse jejunal disaccharidase activities.

1. Sugar-containing diets chosen not to affect intestinal structure or enterocyte turnover have been fed to mice previously maintained on a low carbohydrate diet in order to determine their ability to induce disaccharidase enzymes in the small intestine. 2. Glucose-, fructose- and 3-O-methyl-glucose-containing diets increased sucrase and maltase but not lactase activities in mouse jejunal homogenates. These effects were either absent or negligible in more distal regions of the small intestine. 3. Placing mice on glucose-, fructose- or 3-O-methyl-glucose-containing diets was further shown, by quantitative cytochemistry, to cause a 1.6-, 2.6- and 3.2-fold increase in the initial rate at which alpha-glucosidase activity (sucrase + maltase) appeared in the brush-border membrane of developing enterocytes. 4. The time during which alpha-glucosidase activity increased in enterocyte brush-border membranes fell from 30 h for low carbohydrate fed mice to 21, 19 and 17 h in mice fed glucose, fructose and 3-O-methyl-glucose respectively. Change of diet had no effect on the kinetics of lactase expression by developing enterocytes. 5. Maximal alpha-glucosidase activity detected in enterocyte brush-border membranes is equal to RT, where R is the initial rate of enzyme appearance and T is the time during which this rate operates. The ability of sugars to increase R selectively, but only at the expense of T, defines unexpected limits to the capacity of enterocytes to adapt to changes in luminal nutrition. 6. The above results are discussed in relation to other aspects of enterocyte differentiation recently subjected to quantitative analysis. The need to standardize other aspects of intestinal physiology and redefine the energy content of diets containing non-metabolizable substrates in this type of work is also emphasized.

The role of glucose uptake and metabolism in hyperglycemic exacerbation of neurological deficit in the paraplegic rat.

Previous studies indicate that hyperglycemia, particularly that induced by exogenous glucose administration, exacerbates neurological deficits in the rat spinal cord ischemic model. The effect of inhibition of glucose uptake (glucose transporter) and initial metabolism (hexokinase) on neurological outcome was evaluated in the present investigation using the competitive inhibitors 2-deoxyglucose (2-DG) and 3-O-methylglucose (3-OMG). Sprague-Dawley rats, weighing 200 to 300 gm each, received either 0.25, 1, or 2 gm/kg 2-DG; 2 gm/kg 3-OMG; 2 gm/kg glucose; or an equivalent volume of 0.9% saline intraperitoneally. Rats were intubated and ventilated with 1% to 1.5% halothane. The aortic arch was exposed and snares were placed on the right and left subclavian arteries and the aorta distal to the left subclavian artery. The three vessels were occluded for 10, 11, 12, or 13 minutes. Lower-extremity neurological deficits were evaluated at 1, 4, 18, and 24 hours postocclusion based on a 15-point scale (normal = 0, severe deficit = 15). Lower-extremity neurological deficits were significantly less severe in the groups treated with 2-DG (0.25 and 1 gm/kg) at 18 and 24 hours postocclusion (p less than 0.05 for 0.25 gm/kg and p less than 0.005 for 1 gm/kg, Student's t-test with Bonferroni correction). The lower 2-DG dose of 0.25 gm/kg did not significantly increase the plasma glucose level, suggesting that the glucose transporter was not markedly inhibited, and that the improved neurological outcome was more likely due to inhibition of hexokinase. The higher 2-DG dose of 1 gm/kg afforded protection despite significantly increasing the plasma glucose level, implying a strong inhibition of both the glucose transporter and hexokinase. Administration of 3-OMG, which only inhibits glucose uptake and not hexokinase, actually worsened the neurological deficit in a manner similar to that observed in rats treated with glucose. The authors conclude that the activity of the glucose transporter by itself does not significantly contribute to hyperglycemic exacerbation of neurological deficits. In contrast, the hexokinase step, at least in combination with the transporter and possibly alone, plays a significant role in hyperglycemic exacerbation of the lower-extremity neurological deficit in the paraplegic rat.

[Studies of the total synthesis of (-)-ranunculin].

The synthesis of (-)-ranunculin from mannitol is described. The oxidation of 1,2,5,6-diacetone d-mannitol with NaIO4 or lead tetraacetate gave 2,3-O-isopropylidene-D-glyceraldehyde with the desired alpha-carbon-(R)-configuration. The Wittig reaction of isopropylidene-D-glyceraldehyde with (C6H5)3P = CHCOOCH3 gave a mixture of Z and E methyl-3-(2,2-dimethyl-1,3-dioxo-lan-4-yl)-2-propenoate with a Z:E ratio of 85:15. The dependence of Z:E ratio upon the solvent present was studied. On careful treatment of the cis isomer with 0.1 mol/L HCl, hydrolysis of the ketal took place along with cyclization to give the optically active aglycone with the expected stereochemistry, which was followed by Koenigs-Knorr reaction to give (-)-ranunculin tetraacetate. Since the glycoside is sensitive to acid and base, hydrolysis was carried out with strongly acidic cation exchange resin which yielded (-)-ranunuculin smoothly. The overall yield from mannitol with all the intermediates purified amounted to 15%.

Effects of inhibition of proteoglycan synthesis on the differentiation of cultured rat Schwann cells.

Schwann cells synthesize two heparan sulfate proteoglycans, one that is a component of the Schwann cell basement membrane and a smaller one that is an integral component of the Schwann cell plasma membrane. To determine the functions of these molecules, Schwann cell-nerve cell cultures were grown in medium containing a specific inhibitor of proteoglycan biosynthesis, 4-methylumbelliferyl-beta-D-xyloside. Treatment with 1 mM beta-D-xyloside caused a 90% reduction in the accumulation of 35SO4-labeled proteoglycans in the cell layer of the cultures. Gel filtration analysis revealed that both the basement membrane and plasma membrane proteoglycans were affected. Inhibition of proteoglycan biosynthesis was accompanied by an inhibition of laminin deposition into extracellular matrix as determined by immunostaining of cultures and by immunoblotting of cell-associated proteins. This occurred even though there was no decrease in the amount of laminin detected in the medium of beta-D-xyloside-treated cultures. Deposition of collagen type IV was similarly affected. In addition, there was no myelin produced in beta-D-xyloside treated cultures. However, when beta-xyloside-treated cultures were supplied with exogenous basement membrane, Schwann cells produced numerous myelin segments. These results indicate that Schwann cell proteoglycans play an essential role in basement membrane assembly, and that the integral plasma membrane proteoglycan is not required for the basement membrane to exert its effects on Schwann cell differentiation.

Prevention of streptozotocin-induced alterations in the rat heart by 3-O-methyl glucose and insulin treatments.

Streptozotocin-induced diabetes has previously been shown to alter the sensitivity and responsiveness of rat myocardial tissues to cardiotonic agonists. The objective of the present study was to determine if these alterations were due to the diabetogenic or possible direct cardiotoxic effects of streptozotocin. One month after streptozotocin treatment the following changes were observed in the rat: decrease in body weight; elevation of blood glucose and glycosylated hemoglobin levels; decrease in spontaneously beating atrial rate; elevation in basal developed force of electrically driven right ventricle; and inotropic subsensitivity of right ventricle to isoproterenol, which was associated with decreased beta-adrenoceptor density and supersensitivity to calcium. Pretreatment with the nonmetabolizable glucose analog 3-O-methyl glucose prevented these alterations. Chronic insulin replenishment also reversed the effects of streptozotocin, with the exception of complete normalization of elevations in blood glucose and basal developed force. Acute exposure to high glucose in the medium preserved the subsensitivity to isoproterenol but resulted in an elevated basal developed force in both control and streptozotocin groups. These observations indicate that myocardial alterations after streptozotocin treatment are not the result of direct cardiotoxic effects but rather a consequence of the drug-induced diabetic state. They also suggest that the increase in basal developed force might be related to elevated glucose concentrations.