Preparation and Physicochemical Characterization of Gelatin-Aldehyde Derivatives.

The present study aimed at preparing novel free-radical scavenging and water-soluble compounds derived from gelatin. Specifically, gelatin−syringaldehyde, gelatin−anisaldehyde, and gelatin−vanillin were synthesized and thoroughly studied for their physicochemical properties. In particular, the compounds were characterized by UV-Vis spectroscopy, Fourier-transform infrared spectroscopy, and scanning electron microscopy. Notably, as demonstrated by thermogravimetry and differential scanning calorimetry, all three derivatives exhibited higher thermal stability than gelatin itself. Free-radical scavenging activities of the examined compounds were explored by (i) a standard spectrophotometric ABTS assay and (ii) an assay of oxidative degradation of hyaluronic acid monitored by rotational viscometry. We found that gelatin and gelatin−syringaldehyde demonstrated the highest efficacy in scavenging •OH radicals, whereas gelatin−anisaldehyde was the least effective. The efficacy of scavenging alkyloxy- and alkylperoxy-type free radicals via hydrogen-atom-transferring property was in the following order: gelatin > gelatin−vanillin > gelatin−syringaldehyde > gelatin−anisaldehyde. Electron-donor properties determined using the ABTS assay revealed the following order in one-electron reduction of ABTS•+: gelatin > gelatin−anisaldehyde > gelatin−vanillin > gelatin−syringaldehyde.

Enhanced Anti-Inflammatory Effect of the Combination of Lactiplantibacillus plantarum LS/07 with Methotrexate Compared to Their Monotherapies Studied in Experimental Arthritis.

The gut microbiome (GM) of rheumatic arthritis (RA) patients is often altered in composition and function. Moreover, methotrexate (MTX), one of the most frequently used disease-modifying antirheumatic drugs, is known to negatively affect GM composition. The modulation of immune system activity is one of the therapeutic benefits of probiotics. The aim of the current investigation was to determine the impact of MTX therapy combined with one of the Lactobacillus strains, Lactoplantibacillus plantarum LS/07 (LB), on adjuvant arthritis (AA) in rats. Methods focused on biometric and inflammatory parameters in AA, particularly on plasmatic levels of IL-17A, MMP-9, and MCP-1, and the activities of gamma-glutamyl transferase in the spleen and joints were applied. Enhancing the effect of MTX, LB positively influenced all biometric and inflammatory parameters. The findings of the present study may be of help in proposing novel therapeutic strategies for RA patients.

Improved tissue integrity after alginate treatment in rat spinal cord injury: evidence from ex vivo diffusion tensor imaging.

Diffusion tensor imaging (DTI) is a magnetic resonance imaging technique used to characterize fibrous structures such as white matter in the central nervous system, including normal and spinal cord injury (SCI) conditions. Our aim was to evaluate the effect of alginate treatment in the rat SCI by DTI parametric measures. Ex vivo DTI data were collected by spin echo sequence with following parameters TR/TE: 2500 ms/32 ms and b-value of 1500 s/mm2. Main significant changes were found in fractional anisotropy (FA), and radial diffusivity (RD), between the saline- and alginatetreated group at the level of individual sections and whole spinal cord. Results indicate that ex vivo DTI can be used as a tool for tissue structure characterisation and both FA and RD as promising prognostic parameters of SCI treatment.

Diffusion Tensor Imaging: Tool for Tracking Injured Spinal Cord Fibres in Rat.

Spinal cord injury (SCI) is a severe disorder of the CNS leading to tissue damage and disability. Because it is critical to understand the pathological processes, it is important to find efficient ways to diagnose the severity of injured spinal cord tracts in situ from beginning up to a certain level of recovery following therapeutic interventions. In the current study, we set-up the criteria for diffusion tensor imaging (DTI) in order to capture changes of nerve fibre tracts in rat spinal cord compression injury. We tested four DTI parameters, such as fractional anisotropy, mean diffusivity, axial diffusivity and radial diffusivity at the lesion site, in time course of 7 weeks. Afterwards, we compared DTI data with histological results and locomotor outcomes to examine their consistency and capability of reflecting the lesion development in time. Our data confirm that DTI is a valuable in vivo imaging tool capable to distinguish damaged white matter tracts after mild SCI in rat. Fractional anisotropy showed decreased values for injury site, while the mean diffusivity had higher values, with increased both axial and radial diffusivity in comparison to control subjects. Thus, the combination of DTI parameters can reflect the severity of lesion in time and may correlate with histological evaluation of spared tissue, but not with locomotor recovery following mild injury associated with spontaneous recovery.

Membrane depolarization and aberrant lipid distributions in the neonatal rat brain following hypoxic-ischaemic insult.

Neonatal hypoxic-ischaemic (HI) encephalopathy is among the most serious complications in neonatology. In the present study, we studied the immediate (0 hour), subacute (36 hours) and late (144 hours) responses of the neonatal brain to experimental HI insult in laboratory rats. At the striatal level, the mass spectrometry imaging revealed an aberrant plasma membrane distribution of Na+/K+ ions in the oedema-affected areas. The failure of the Na+/K+ gradients was also apparent in the magnetic resonance imaging measurements, demonstrating intracellular water accumulation during the acute phase of the HI insult. During the subacute phase, compared with the control brains, an incipient accumulation of an array of N-acylphosphatidylethanolamine (NAPE) molecules was detected in the HI-affected brains, and both the cytotoxic and vasogenic types of oedema were detected. In the severely affected brain areas, abnormal distributions of the monosialogangliosides GM2 and GM3 were observed in two-thirds of the animals exposed to the insult. During the late stage, a partial restoration of the brain tissue was observed in most rats in both the in vivo and ex vivo studies. These specific molecular changes may be further utilized in neonatology practice in proposing and testing novel therapeutic strategies for the treatment of neonatal HI encephalopathy.

In vivo magnetic resonance approach to trimethyltin induced neurodegeneration in rats.

Trimethyltin (TMT) is commonly used to induce neurodegeneration in mice and rats; however, only scarce data of in vivo magnetic resonance (MR) spectroscopy and imaging characterizing TMT neurotoxicity are available. Our aim was to assess brain metabolite changes and brain atrophy by in vivo MR in the rat model of neurodegeneration induced by TMT. Adult male Wistar rats exposed to TMT (8mg/kg, i.p.) were used in the study. Proton MRS was applied on the dorsal hippocampus to reveal changes in neurochemical profile, and MR imaging was used to assess the volume of the entire hippocampus, ventricles and whole brain. Hippocampal levels of N-acetylaspartate (NAA), glutamate (Glu), total creatine (tCr) and taurine (Tau) significantly decreased, while the levels of myo-Inositol (mIns) and glutamine (Gln) significantly increased in TMT treated rats compared to controls. No changes in choline metabolites (tCho), glutathione (GSH), and GABA were observed. MR volumetry revealed a substantial loss of hippocampal mass, cerebral volume shrinkage and ventricular enlargement in the TMT treated group in comparison to the control group. To the best of our knowledge, this is the first study characterizing TMT induced neurodegeneration in the rat by in vivo MRS. Our findings suggest that TMT exposed rats may serve as a reliable animal model of neurodegeneration and MR based parameters could serve as potential in vivo biomarkers of therapeutic response.

Neonatal brain injury as a consequence of insufficient cerebral oxygenation.

Neonatal brain hypoxic-ischemic injury represents a serious health care and socio-economical problem since it is one of the most common causes of mortality and morbidity of newborns. Neonatal hypoxic-ischemic encephalopathy is often associated with signs of perinatal asphyxia, with an incidence of about 2-4 per 1,000 live births and mortality rate up to 20%. In about one half of survivors, cerebral hypoxic-ischemic insult may result in more or less pronounced neuro-psychological sequelae of immediate or delayed nature, such as seizures, cerebral palsy or behavioural and learning disabilities, including attention-deficit hyperactivity disorder. Hypoxic-ischemic injury develops as a consequence of transient or permanent restriction of blood supply to the brain. Severity of hypoxic-ischemic encephalopathy varies depending on the intensity and duration of hypoxia-ischemia, on the type and size of the brain region affected, and on the maturity of the foetal/neonatal brain. Though a primary cause of hypoxic-ischemic injury is lack of oxygen in the neonatal brain, underlying mechanisms of subsequent events that are critical for developing hypoxic-ischemic encephalopathy are less understood. Their understanding is however necessary for elaborating effective management for newborns that underwent cerebral hypoxic-ischemic insult and thus are at risk of a negative outcome. The present paper summarizes current knowledge on cerebral hypoxic-ischemic injury of the neonate, fundamental processes involved in etiopathogenesis, with a special focus on cellular and molecular mechanisms and particular attention on certain controversial aspects of oxidative stress involvement.

Influence of tiopronin, captopril and levamisole therapeutics on the oxidative degradation of hyaluronan.

The ability to protect hyaluronic acid (HA) from oxidative degradation by cupric ions and ascorbate (production of (•)OH and peroxy-type radicals) during acute phase joint inflammation has been investigated using the following drugs: tiopronin, captopril, and levamisole. Radical scavenging activity, i.e. the propensity for donation of electrons was assessed for the drugs by ABTS and DPPH assays. The kinetics of HA degradation have been measured in the presence of each drug using rotational viscometry. The results of ABTS and DPPH assays show the highest radical scavenging activity for captopril, followed by tiopronin. For levamisole, no effect was observed. Captopril and tiopronin prevented HA degradation induced by (•)OH radicals in a similar manner, while tiopronin was more effective in scavenging peroxy-type radicals. On the other hand, levamisole was shown to be a pro-oxidant. Recovered HA fragments were characterized using FT-IR analysis, the incorporation of a sulphur atom from captopril and tiopronin but not from levamisole into the HA molecule was demonstrated.

Anthracycline-dependent cardiotoxicity and extracellular matrix remodeling.

The mechanisms of anthracycline-dependent cardiotoxicity have been studied widely, with the suggested principal mechanism of anthracycline damage being the generation of reactive oxygen species by iron-anthracycline complexes, leading to lipid peroxidation and membrane damage. An increasing number of researchers studying cardiovascular events associated with anthracycline-based chemotherapy are addressing cardiac extracellular matrix (ECM) remodeling. The heart is an efficient muscular pump, with the cardiomyocytes and intramural coronary vasculature of the heart tethered in an ECM consisting of a network of fibrillar, structural proteins, mostly collagens. Increasing evidence suggests that the ECM plays a complex and diverse role in the processes initiated by anthracycline-class drugs that lead to cardiac damage. This review discusses adverse myocardial remodeling induced by anthracyclines and focuses on their mechanisms of action.

Biological importance of reactive oxygen species in relation to difficulties of treating pathologies involving oxidative stress by exogenous antioxidants.

Findings about involvement of reactive oxygen species (ROS) not only in defense processes, but also in a number of pathologies, stimulated discussion about their role in etiopathogenesis of various diseases. Yet questions regarding the role of ROS in tissue injury, whether ROS may serve as a common cause of different disorders or whether their uncontrolled production is just a manifestation of the processes involved, remain unexplained. Dogmatically, increased ROS formation is considered to be responsible for development of the so-called free-radical diseases. The present review discusses importance of ROS in various biological processes, including origin of life, evolution, genome plasticity, maintaining homeostasis and organism protection. This may be a reason why no significant benefit was found when exogenous antioxidants were used to treat free-radical diseases, even though their causality was primarily attributed to ROS. Here, we postulate that ROS unlikely play a causal role in tissue damage, but may readily be involved in signaling processes and as such in mediating tissue healing rather than injuring. This concept is thus in a contradiction to traditional understanding of ROS as deleterious agents. Nonetheless, under conditions of failing autoregulation, ROS may attack integral cellular components, cause cell death and deteriorate the evolving injury.

Free-radical degradation of high-molar-mass hyaluronan induced by ascorbate plus cupric ions: testing of stobadine and its two derivatives in function as antioxidants.

Stobadine·2HCl and its two hydrophilic derivatives SM1dM9dM10·2HCl and SME1i-ProC2·HCl were tested in the function of antioxidants on hyaluronan (HA) degradation induced by the Weissberger oxidative system [ascorbate plus Cu(II)]. As a primary method, rotational viscometry was applied, where the substance tested was added before or 1 h after the initiation of HA degradation. The most effective scavengers of •OH and peroxy-type radicals were recorded to be stobadine·2HCl and SME1i-ProC2·HCl, respectively. The most effective scavenger, determined by applying the ABTS assay, was stobadine·2HCl.

Free-radical degradation of high-molar-mass hyaluronan induced by ascorbate plus cupric ions: evaluation of antioxidative effect of cysteine-derived compounds.

Based on our previous findings, the present study has focused on free-radical-mediated degradation of the synovial biopolymer hyaluronan. The degradation was induced in vitro by the Weissberger's system comprising ascorbate plus cupric ions in the presence of oxygen, representing a model of the early phase of acute synovial joint inflammation. The study presents a novel strategy for hyaluronan protection against oxidative degradation with the use of cysteine-derived compounds. In particular, the work objectives were to evaluate potential protective effects of reduced form of L-glutathione, L-cysteine, N-acetyl-L-cysteine, and cysteamine, against free-oxygen-radical-mediated degradation of high-molar-mass hyaluronan in vitro. The hyaluronan degradation was influenced by variable activity of the tested thiol compounds, also in dependence of their concentration applied. It was found that L-glutathione exhibited the most significant protective and chain-breaking antioxidative effect against the hyaluronan degradation. Thiol antioxidative activity, in general, can be influenced by many factors such as various molecule geometry, type of functional groups, radical attack accessibility, redox potential, thiol concentration and pK(a), pH, ionic strength of solution, as well as different ability to interact with transition metals. Antioxidative activity was found to decrease in the following order: L-glutathione, cysteamine, N-acetyl-L-cysteine, and L-cysteine. These findings might be beneficial in future development of potential drugs in the treatment of synovial hyaluronan depletion-derived diseases.

Free-radical degradation of high-molar-mass hyaluronan induced by Weissberger's oxidative system: potential antioxidative effect of bucillamine.

OBJECTIVES: Hyaluronan (HA), one of the main components of extracellular matrix, is a glycosaminoglycan composed of repeating disaccharide units of N-acetyl-d-glucosamine and d-glucuronic acid linked by ß-(1→4) and ß-(1→3) glycoside bonds. High-molar-mass HA was used as a model for studying its oxidative degradation. In the present paper protective effects of bucillamine against the free-radical degradation of HA were investigated. The HA fragments generated were characterized as well. METHODS: To induce free-radical-mediated degradation of high-molar-mass HA under aerobic conditions, we applied Weissberger's oxidative system, comprising biogenic compounds in relevant pathophysiological concentrations, i.e. 100 µM ascorbate plus 1 µM Cu(II). Time-dependent decreases of dynamic viscosity of the HA solutions were recorded by rotational viscometry. Electron donor behaviors of bucillamine were studied by a standard ABTS test method and a chemiluminescence (CL) assay. Ability of incorporation of generated bucillamine thiyl radicals into the biopolymer was verified by Fourier-transform infrared spectroscopy (FT-IR) and size exclusion chromatography with a multi-angle light scattering photometer (SEC-MALS). RESULTS: Decrease of HA viscosity reflected HA degradation. The drug tested was applied in two arrangements: to prevent •OH radical generation (1) and ROO• type radicals propagation (2). Bucillamine, which acted as an efficient •H donor, is also a proper electron donor, as proved by ABTS and CL assays. FT-IR and SEC-MALS methods showed that the drug tested did not incorporate into the biopolymer chains. CONCLUSION: Bucillamine significantly protected high-molar-mass HA against free-radical degradation in vitro, and supposedly this positive action of the drug may be involved in its beneficial effect observed in clinical practice.

Pro-oxidative effect of peroxynitrite regarding biological systems: a special focus on high-molar-mass hyaluronan degradation.

Current understanding on the role of peroxynitrite in etiology and pathogenesis of some human diseases, such as cardio-vascular diseases, stroke, cancer, inflammation, neurodegenerative disorders, diabetes mellitus and diabetic complications has recently led to intensive investigation of peroxynitrite involvement in physiology and pathophysiology. Mechanism of cytotoxic effects of peroxynitrite involve its reactions with lipids, DNA/RNA, proteins, and polysaccharides, thus triggering cellular responses ranging from subtle changes of cell functioning to severe oxidative damage of the affected macromolecules leading to necrosis or apoptosis. The present work is aimed at providing a brief overview of i) peroxynitrite biosynthesis and reaction pathways in vivo, ii) its synthetic preparation in vitro, and iii) to reveal its potential damaging role in vivo, on actions studied via monitoring in vitro hyaluronan degradation. The complex biochemical behavior of peroxynitrite is determined by a number of variables, such as chemistry of the reaction itself, depending mostly on the involvement of conformational structures of different energy states, concentration of the species involved, content of reactive intermediates and trace transition metal ions, contribution of carbon dioxide, presence of trace organics, and by the reaction kinetics. Recently, in vitro studies of oxidative cleavage of hyaluronan have, in fact, been the subject of growing interest. Here we also describe our experimental set-up for studying peroxynitrite-mediated degradation of hyaluronan, a system, which may be suitable for testing prospective pharmacological substances.

Aurothiomalate as preventive and chain-breaking antioxidant in radical degradation of high-molar-mass hyaluronan.

The potential anti- or pro-oxidative effects of a disease-modifying antirheumatic drug, aurothiomalate, to protect high-molar-mass hyaluronan against radical degradation were investigated along with L-glutathione - tested in similar functions. Hyaluronan degradation was induced by the oxidative system Cu(II) plus ascorbate known as the Weissberger's oxidative system. The time- and dose-dependent changes of the dynamic viscosity of the hyaluronan solutions were studied by the method of rotational viscometry. Additionally, the antioxidative activity of aurothiomalate expressed as a radical-scavenging capacity based on a decolorization 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay was inspected. At the higher concentrations tested, L-glutathione showed excellent scavenging of (.) OH and peroxyl-type radicals, however, at the lowest concentration applied, its pro-oxidative effect was revealed. The effects of aurothiomalate on hyaluronan degradation were similar to that of L-glutathione, however, at the lowest concentration tested, no significant pro-oxidant effect was observed.

Free-radical degradation of high-molecular-weight hyaluronan induced by ascorbate plus cupric ions. Testing of bucillamine and its SA981-metabolite as antioxidants.

High-molecular-weight hyaluronan (HA) samples were exposed to free-radical chain-degradation reactions induced by ascorbate in the presence of Cu(II) ions - the so-called Weissberger's oxidative system. The concentrations of both reactants [ascorbate, Cu(II)] were comparable to those that may occur during an early stage of the acute phase of joint inflammation. The time-dependent changes of the viscosity of the HA solution in the absence of the substance tested were monitored by rotational viscometry. However, when the anti- or pro-oxidative effects of the antioxidants/drugs were investigated, their dose-dependency was also examined. Additionally, the anti-oxidative activities of these substances were screened by the well-established ABTS and DPPH decolorization assays. The actions of the disease-modifying anti-rheumatic drugs, namely bucillamine and D-penicillamine, were compared to those of L-cysteine and of SA981, the oxidized metabolite of bucillamine. The results indicated that bucillamine was the most efficient scavenger of hydroxyl- and/or peroxyl-type radicals, even at the lowest drug concentration. In contrast, SA981 demonstrated no scavenging activity against the aforementioned free radicals. D-Penicillamine and L-cysteine showed a dual effect, i.e. a pronounced anti-oxidative effect was, after a given time period, followed by a significant pro-oxidative effect.

Carnosine inhibits degradation of hyaluronan induced by free radical processes in vitro and improves the redox imbalance in adjuvant arthritis in vivo.

OBJECTIVE: New ways of supplementary or combinatory therapy of rheumatoid arthritis (RA) are of great importance. The aim is to find an additive to classical RA therapy with natural molecules without side effects possessing anti-inflammatory and anti-oxidative properties. In this study we investigated the anti-oxidative and anti-inflammatory properties of the endogenous natural compound carnosine (CARN) in vitro and in vivo. Moreover, we tested also the inhibitory properties of the drug methotrexate (MTX) on dynamic viscosity of hyaluronan (HA) solutions in the same manner. METHODS: For in vitro testing of the inhibitory properties of CARN against degradation of HA solutions, we used the model of degradation of hyaluronan (HA) induced by free radicals. Both substances, CARN and MTX, were compared to glutathione (GSH). Rotational viscometry was used in evaluation of protective properties of compounds studied. The ability of CARN to restore the redox imbalance occurring in adjuvant arthritis (AA) of rats was also tested. We monitored the effect of CARN on hind paw volume (HPV) and on the levels of protein carbonyls, and thiobarbituric acid reacting substances (TBARS) in AA. RESULTS: In the reaction system with the prevalence of •OH and/or peroxy-type radicals, CARN in 200 µmol/L concentration tested was shown to exert a protective action on HA degradation. MTX was less effective than CARN in preventing HA degradation. Its ability to protect HA against radical degradation was evident only at the highest concentration of 400 µmol/L. In AA, carnosine significantly reduced TBARS and protein carbonyls in plasma, and also decreased the HPV of animals most effectively on the day 14. CONCLUSIONS: CARN proved its inhibitory properties against degradation of HA solutions at experimental conditions in vitro and showed its beneficial efficiency in vivo. Moreover, it reduced also HPV, the clinical marker of inflammation in AA.

Ascorbate and Cu(II)-induced oxidative degradation of high-molar-mass hyaluronan. Pro- and antioxidative effects of some thiols.

OBJECTIVE: This study presents the results of antioxidative and pro-oxidative efficacy of cysteamine and D-penicillamine (D-pen) in comparison to L-glutathione (L-GSH) on high-molar-mass hyaluronan (HA) degradation by cupric ions plus ascorbic acid. METHODS: The substance tested was applied in the degradative system cupric ions plus ascorbate: (i) before the reaction onset or also (ii) 1 h after the reaction started. The results obtained were compared with that one recorded by using the degradative system in the absence of the substance tested. To monitor HA degradation kinetics, rotational viscometry was applied. Moreover, the standard ABTS and DPPH assays were used. RESULTS: By using the method of rotational viscometry, D-pen showed dual effect: initial inhibitory effect on •OH radicals was changed to a pro-oxidative one in the dose and time dependent manner. Both L-GSH and cysteamine were recorded to be more effective scavengers of •OH radicals than D-pen. Cysteamine demonstrated to be an excellent scavenger also of alkoxyl- and peroxyl- type radicals. Based on IC50 values, gained by ABTS assay, it is evident that D-pen showed higher radical scavenging capacity compared to cysteamine. Similar results were observed also in DPPH assay, although in this assay less effective radical scavenging capacities of both substances tested were recorded. CONCLUSIONS: On the basis of the results obtained, it can be stated that D-pen can produce hydrogen peroxide or •OH radicals and can inhibit the production of these oxidants. Our results showed that both L-GSH and cysteamine are similarly effective in inhibiting of HA degradation. Moreover, cysteamine demonstrated to be a significant inhibitor of alkoxyl- and peroxyl- type radicals generated from C-type macroradical of HA.

Cerebral hypoxia-ischemia: focus on the use of magnetic resonance imaging and spectroscopy in research on animals.

Cerebral hypoxic-ischemic injury represents a serious health problem and is the third leading cause of mortality in developed countries. Early diagnosis of hypoxic-ischemic injury to the brain is inevitable for timely and efficient treatment. However, routinely applied cranial ultrasonography or computed tomography is often not sensitive enough to detect cerebral hypoxic-ischemic injury in its early stages. Therefore searching for a more effective diagnostic tool has been an intensive process in many laboratories within the last decades. Nowadays, magnetic resonance imaging (MRI) and spectroscopy (MRS) are the most promising non-invasive and non-destructive tools working in-real-time. These magnetic resonance-based techniques are progressively utilized in neurological and neonatology departments to confirm or refute cerebral hypoxic-ischemic injury in adults and neonates. The purpose of the present paper was therefore (i) to provide a brief overview on mechanisms of hypoxic-ischemic injury to the brain and (ii) to summarize main findings of both clinical reports and experimental studies, performed on various animal models of brain hypoxia-ischemia, with a particular focus on the monitoring of the evolving cerebral hypoxic-ischemic injury by means of in vivo MRI and MRS.

Protein kinase C regulates functional coupling of beta1-adrenergic receptors to Gi/o-mediated responses in cardiac myocytes.

The effect of protein kinase C (PKC) activation on beta1-adrenergic receptor (beta1-AR) regulation of the cardiac L-type Ca2+ current (ICa,L) was studied using the whole-cell patch clamp technique. Treatment of guinea pig ventricular myocytes with phorbol-12,13-dibutyrate (PDBu) caused a significant decrease in ICa,L sensitivity to stimulation by submaximal beta1-AR activation using isoproterenol (Iso). This decrease in sensitivity was also associated with the ability of higher concentrations of Iso to directly inhibit the stimulatory response. PDBu treatment produced similar effects on H2 histamine receptor-mediated ICa,L responses. In the presence of PDBu, higher concentrations of Iso inhibited the histamine stimulated ICa,L, and this effect was blocked by a selective beta1-AR antagonist. Higher concentrations of histamine also inhibited the Iso stimulated ICa,L, and this effect was blocked by a selective H2 receptor antagonist. The effects of PDBu were blocked by the PKC inhibitor bisindolylmaleimide I, and they were not mimicked by the inactive phorbol ester 4alpha-phorbol-12,13-didecanoate. The inhibitory effects of Iso and histamine were significantly reduced when Gi/o mediated responses were blocked with pertussis toxin. These results suggest that PKC promotes coupling of cardiac beta1-adrenergic and H2 histamine receptors to Gi/o mediated inhibitory responses.