Cranberries (Vaccinium macrocarpon Aiton) in dog nutrition: influence on diet digestibility and palatability and in the course of urinary tract infections / [Cranberrys (Vaccinium macrocarpon Aiton) na nutrição de cães: influência na digestibilidade e palatabilidade da dieta e no curso de infecções do trato urinário

The objective was to evaluate the effects of cranberry on blood and urinary parameters of dogs (experiment I), digestibility of nutrients (experiment II), palatability of diet (experiment III) and the influence of cranberry on E. coli UPEC-MRHA fimbriae in vitro (experiment IV). For experiment I and II, ten dogs were fed with diets containing 0% or 0.4% cranberry for 30 days. Experiment III compared the diets containing 0% and 0.4% cranberry using 16 adult dogs. There were no statistical differences (P>0.05) in the blood parameters evaluated. Dogs consuming cranberry presented lighter color and appearance of urine, compared to the control group (P<0.05). The diet containing cranberry showed higher digestibility of dry matter, organic matter, ether extract, higher metabolizable energy (P<0.05) and reduced fecal sialic acid concentration (P<0.05) compared to the control diet. There was no influence of cranberry on the formation of fimbriae of E. coli UPEC-MRHA. There was a lower intake ratio of the diet containing cranberry (P<0.05). The inclusion of 0.4% cranberry increases the digestibility of nutrients and influences the color and appearance of urine of dogs. However, it reduces diet palatability and does not alter the adhesion of E. coli UPEC-MRHA in vitro.(AU)

O objetivo foi avaliar os efeitos do cranberry nos parâmetros sanguíneos e urinários de cães (experimento I), na digestibilidade dos nutrientes (experimento II), na palatabilidade da dieta (experimento III) e a influência do cranberry sobre E. coli UPEC-MRHA fimbriae in vitro (experimento IV). Para os experimentos I e II, 10 cães foram alimentados com dietas contendo 0% ou 0,4% de cranberry por 30 dias. O experimento III comparou as dietas contendo 0% e 0,4% de cranberry usando 16 cães adultos. Não houve diferenças estatísticas (P>0,05) nos parâmetros sanguíneos avaliados. Cães que consumiram cranberry apresentaram cor e aparência mais claras da urina, em comparação com o grupo controle (P<0,05). A dieta contendo cranberry apresentou maior digestibilidade da matéria seca, extrato etéreo, matéria orgânica, maior energia metabolizável (P<0,05) e menor concentração de ácido siálico fecal (P<0,05) comparada à dieta controle. Não houve influência do cranberry na formação de fímbrias de E. coli UPEC-MRHA. Houve uma menor taxa de ingestão da dieta contendo cranberry (P<0,05). A inclusão de 0,4% de cranberry aumenta a digestibilidade dos nutrientes, influencia a cor e a aparência da urina dos cães. No entanto, reduz a palatabilidade da dieta e não altera a adesão de E. coli UPEC-MRHA in vitro.(AU)

Angiotensin II type 1 receptor-mediated inhibition of K+ channel subunit kv2.2 in brain stem and hypothalamic neurons.

Angiotensin II (Ang II) has powerful modulatory actions on cardiovascular function that are mediated by specific receptors located on neurons within the hypothalamus and brain stem. Incubation of neuronal cocultures of rat hypothalamus and brain stem with Ang II elicits an Ang II type 1 (AT1) receptor-mediated inhibition of total outward K+ current that contributes to an increase in neuronal firing rate. However, the exact K+ conductance(s) that is inhibited by Ang II are not established. Pharmacological manipulation of total neuronal outward K+ current revealed a component of K+ current sensitive to quinine, tetraethylammonium, and 4-aminopyridine, with IC50 values of 21.7 micromol/L, 1.49 mmol/L, and 890 micromol/L, respectively, and insensitive to alpha-dendrotoxin (100 to 500 nmol/L), charybdotoxin (100 to 500 nmol/L), and mast cell degranulating peptide (1 micromol/L). Collectively, these data suggest the presence of Kv2.2 and Kv3.1b. Biophysical examination of the quinine-sensitive neuronal K+ current demonstrated a macroscopic conductance with similar biophysical properties to those of Kv2.2 and Kv3.1b. Ang II (100 nmol/L), in the presence of the AT2 receptor blocker PD123,319, elicited an inhibition of neuronal K+ current that was abolished by quinine (50 micromol/L). Reverse transcriptase-polymerase chain reaction analysis confirmed the presence of Kv2.2 and Kv3.1b mRNA in these neurons. However, Western blot analyses demonstrated that only Kv2.2 protein was present. Coexpression of Kv2.2 and the AT1 receptor in Xenopus oocytes demonstrated an Ang II-induced inhibition of Kv2.2 current. Therefore, these data suggest that inhibition of Kv2.2 contributes to the AT1 receptor-mediated reduction of neuronal K+ current and subsequently to the modulation of cardiovascular function.

Re-examination of Na+-dependent regulation of cAMP-dependent Cl- currents in the heart.

Cardiac Cl- channels may play an important role in both the physiological as well as the pathophysiological regulation of the heart. Due to the present controversy regarding the role that Na+ may play in the regulation of the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent Cl- channels in the heart, we have re-examined the effects of intracellular and extracellular Na+ on ICl,cAMP in isolated guinea-pig ventricular myocytes. In conventional, whole-cell patch-clamp experiments, exchanging of extracellular Na+ for N-methyl-d-glucamine (NMG) did not have a significant effect on the magnitude of the ICl,cAMP activated by external forskolin (FSK) in the presence of beta-adrenergic and muscarinic receptor blockade compared with normal rundown of the current with time. Utilization of the amphotericin B-perforated-patch technique prevented rundown of FSK-stimulated ICl, cAMP, and failed to reveal alterations in this conductance when NMG was substituted for extracellular Na+. Finally, intracellular dialysis of cells during whole-cell experiments with both 0 and 10 mM Na+-containing solutions failed to show any significant effect of intracellular Na+ on the magnitude of ICl,cAMP. We therefore conclude that intracellular or extracellular Na+ plays little or no direct regulatory role in modulation of cAMP-dependent Cl- channels in heart.

Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

CFTR chloride channels in human and simian heart.

OBJECTIVES: The cAMP-dependent Cl- conductance in heart is believed to be due to cardiac expression of the cystic fibrosis transmembrane conductance regulator (CFTR). While CFTR expressed in rabbit and guinea-pig heart (CFTRcardiac) is an alternatively spliced isoform of the epithelial gene product, little information is known regarding possible expression of CFTR in primate heart. In this study, we examined molecular expression of CFTR in human and simian atrium and ventricle and functional expression of cAMP-dependent Cl- currents in isolated human atrial and simian ventricular cells. METHODS: The reverse transcription polymerase chain reaction (RT-PCR) was performed on human and simian atrial and ventricular mRNA using primers designed to border regions of the CFTR gene product corresponding to transmembrane segments I-VI (TSI-VI), the first nucleotide binding domain (NBD1), transmembrane segments VII-XII (TSVII-XII), and the large cytoplasmic domain which includes the regulatory (R) domain and NBD1. Functional expression of CFTR Cl- channels in human atrial and simian ventricular myocytes was determined using whole-cell and giant inside-out patch-clamp techniques. RESULTS: Southern blot analysis of these RT-PCR products demonstrated expression of CFTR transcripts in human and simian atrial and ventricular tissue and revealed a novel pattern of expression compared to most animal species studies: both the exon 5 plus (unspliced) and exon 5 minus (spliced) CFTR transcripts are co-expressed in human and simian atrium and ventricle. Whole-cell experiments demonstrated a Cl- sensitive time-independent background conductance in both human atrial and simian ventricular myocytes that was activated by forskolin (FSK) and insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In inside-out patches utilizing the giant patch technique on human atrial myocytes, unitary Cl- sensitive channels resembling CFTR Cl- channels (approximately 14 pS conductance) were activated by the catalytic subunit of protein kinase A (PKA) in 3/12 patches examined. CONCLUSIONS: These results clearly demonstrate the molecular expression of CFTR Cl- channels and provide electrophysiological evidence consistent with functional expression of these channels in human atrial and simian ventricular myocardium.

A case of bleeding requiring hospitalization that was likely caused by an interaction between warfarin and levamisole.

Warfarin is one of the most commonly used oral anticoagulants. Many medications have been shown to influence the prothrombin time in patients treated with warfarin by various mechanisms. We observed a major bleeding episode that resulted from a probable interaction between levamisole (Ergamisol), 5-fluorouracil, and warfarin. It is conceivable that many patients who are predisposed to thromboembolism because of cancer and surgery will be taking this combination of medications.

Molecular physiology of CFTR Cl- channels in heart.

Recent electrophysiological data suggests a number of similarities in the properties of cAMP-dependent Cl- channels in heart and cAMP-dependent Cl- channels encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in various epithelial cells. We tested the hypothesis that cAMP-dependent Cl- channels in heart may be due to cardiac expression of CFTR by amplification and sequencing of several regions of CFTR from myocardial tissue derived from various species and areas of the heart. Regions corresponding to the first nucleotide binding domain (NBD1), transmembrane segments I-VI (TS I-VI), transmembrane segments VII-XII (TS VII-XII), and the regulatory domain (R domain) were amplified and sequenced from rabbit ventricle (see Fig. 1). Comparison of the known amino acid sequence of human epithelial CFTR with the deduced sequence from rabbit heart indicated deletion of exon 5 in the first cytoplasmic loop of TS I-VI suggesting that CFTR is an alternatively spliced isoform in rabbit ventricle. Outside of the alternatively spliced region, the heart CFTR Cl- channel isoform displayed greater than 95% identity to human epithelial CFTR Cl- channels. We have also compared the molecular distribution of the CFTR gene product to the distribution of cAMP-dependent Cl- channels in native cardiac myocytes derived from various species and areas of the heart. Amplification of regions corresponding to NBD1, R domain, and TS VII-XII from atrium and ventricle of guinea pigs, rabbit, and dog hearts exhibited a distribution which closely matched the distribution of cAMP-dependent Cl- channels assessed using electrophysiological techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct measurement of the internal viscosity of sickle erythrocytes as a function of cell density.

The rotational dynamics of TEMPAMINE can be used to study directly the intracellular environment. The extracellular signal from TEMPAMINE is broadened away by the use of potassium ferricyanide which does not enter the cell. The EPR signal which results when 1 mM TEMPAMINE, 120 mM ferricyanide, and erythrocytes are mixed together arises from TEMPAMINE only in the intracellular aqueous space. The relative viscosity measured by the motion of TEMPAMINE in various control environments is: water at 37 degrees C = 1; human plasma at 37 degrees C = 1.1; internal aqueous environment of washed erythrocytes or whole blood at 37 degrees C = 4.92 +/- 0.32. Erythrocytes can be fractionated by density. In sickle-cell anemia (SS), the percentage of cells we find with density greater than 1.128 g/ml is 15-40%, in normals (AA) and sickle trait (AS) 1%. By direct spin-label measurements with TEMPAMINE we show, for the first time, that the relative internal viscosity (eta mu) of these dense erythrocytes is markedly elevated and density-dependent. Our results show that (1) eta mu increases with increasing cell density; (2) eta mu obtained from sickle cells is higher than eta mu obtained from normal cells at a given density, and this effect is greater at 37 degrees C than at 20 degrees C; (3) eta mu is proportional to MCHC, but eta mu in erythrocytes is higher than eta mu obtained from in vitro preparations of hemoglobin S at equivalent concentrations. We conclude that the relative internal viscosity of erythrocytes is affected by three factors: the state of cell hydration, the amount of hemoglobin polymer present, and the potential interactions of the cell membrane with intracellular hemoglobin.

Sequestrocytes: a manifestation of transcellular cross-bonding of the red cell membrane in sickle cell anemia.

An unusual form of red cells (called sequestrocytes) that circulate in the peripheral blood of patients with sickle cell anemia has been identified. In wetmount light microscopy preparations, sequestrocytes appear as massively vacuolated erythrocytes. We have shown by transmission electron microscopy and scanning electron microscopy that the morphology of sequestrocytes can be accounted for on the basis of transcellular cross-bonding of the cell membrane. Initially, endocytotic vesicles span the cytoplasmic space, forming putative cytoskeletal fusion zones. Points of fusion of the membrane skeleton bridging the cytoplasmic compartment expand laterally to form linear fusion zones that entrap lakes of hemoglobin within pseudovacuoles. Sequestrocytes can be isolated in the densest layer (1.159 g ml-1 or greater) of an arabinogalactan density gradient. These cells can be generated in increased numbers in sickle cell blood by incubating samples in 1.5 mM-acetylphenylhydrazine (APH) solution for two hours at 37 degrees C. Their formation is partially blocked by incubation with the reducing agent, dithiothreitol (DTT). Our results suggest that these cells represent an expression of oxidative membrane injury in sickle cell anemia.

Altered amount and activity of superoxide dismutase in sickle cell anemia.

The amount and activity of superoxide dismutase (SOD) (EC 1.15.1.1) were measured in red cells collected from 50 white controls, 101 black controls, 50 patients with sickle hemoglobin (SS Hb), 12 with sickle trait, and 11 with other sickling hemoglobinopathies. Red cells from normal black subjects had more SOD amount and activity than normal whites (1.77 U/mg Hb and 2.96 micrograms/mg Hb vs. 1.47 U/mg Hb and 2.64 micrograms/mg Hb, respectively) or blacks with SS Hb or other sickling hemoglobinopathies. Patients with more severe manifestations of SS Hb had lower levels of SOD activity than those with milder symptoms but had the same amount of enzyme protein. Individuals with sickle trait had amounts and activities of SOD comparable to black controls. An alteration in defense to free radical oxygen may play a role in the severity of symptoms experienced by patients with homozygous sickle cell disease.

Painful crisis and dense echinocytes: effects of hydration and vasodilators.

We have conducted a series of studies using discontinuous arabinogalactan density gradient ultracentrifugation of erythrocytes from the peripheral blood of: patients with sickle cell anemia (SCA), in and out of pain crisis; hydrated SCA, hemoglobin SC, and normal individuals all of whom were pain-free; and patients with SCA given short courses of oral vasodilator compounds. Our results indicate that in pain crisis patients develop an echinocytic change that is most prominent in the denser layers (specific gravity greater than 1.128 g/ml) of the discontinuous gradient and effects both irreversibly sickled cells (ISC) and non-ISC. This echinocytic change, we have previously shown, correlates with a decrease in erythrocyte reduced glutathione, a major intraerythrocyte anti-oxidant compound. We could find no consistent change in the percentage of dense cells in pain crisis versus out of crisis. However, out of crisis, hydration led to a marked increase in the percentage of dense erythrocytes in sickle cell anemia and in a HbSC patient, compared to the same individual out of crisis and on ad lib fluids. There was no consistent change in echinocytic forms. Because hydration may be expected to have produced an increase in intravascular volume and vasodilation, we determined whether short courses of three different vasodilators would increase the dense fraction in patients with SCA who were pain-free. There was no change in percentage of dense erythrocytes or in the percentage of echinocytes. We concluded that painful crisis occurs in association with an echinocytic change that may be induced by oxidant injury and that in the pain-free state, hydration, but not short courses of vasodilator drugs, increased the percentage of dense erythrocytes but not the degree of echinocytosis they displayed. The differential effect of hydration, with respect to painful crisis, may indicate that these dense cells are bound to vascular endothelium or trapped in blood vessels at the time of crisis but mobilized by hydration in the out-of-crisis state.

The phosphoproteins of the sickle erythrocyte membrane.

The uptake of 32P from exogenous 32Pi into membrane proteins of sickle erythrocytes has been analyzed. The phosphorylation of spectrin is normal in sickle cells. There is, however, a substantial increase in 32P in the sialoproteins of the membrane, which can be demonstrated after fractionation or selective proteolysis. Normal and sickle erythrocytes were separated on Stractan gradients and average cell age was determined using the remaining pyruvate kinase activity as a marker. The altered phosphorylation of sickle cells was not seen in young normal cells, suggesting that it was not related to cell age. The altered phosphorylation was also not correlated with the level of reticulocytes in these fractions. This result is further evidence for abnormalities in the sialoproteins of sickle erythrocytes and is the first demonstration of altered sialoprotein phosphorylation in the red cell.

Elimination of the settling artifact in the nuclear magnetic resonance spectroscopy of living cells.

Nuclear magnetic resonance spectroscopy has become a powerful tool for metabolic investigations on living cell suspensions. However, unless mechanical means are used to maintain the cells in dispersion, settling occurs during the NMR experiment. Because high packed-cell volumes are generally used to produce maximum NMR signals, settling may be inapparent to the eye, leading to unrecognized artifactual changes in NMR spectra. Such artifacts include time-dependent loss of signal intensity when the sample volume approximates the sensitive volume of the NMR probe, and time-dependent increase in signal intensity when the sample volume exceeds the sensitive volume. Through the addition of the polysaccharide arabinogalactan, increasing the buoyant density of the suspending medium to approach that of the cells, we have eliminated cell settling and improved the quality of 31P NMR spectra of human erythrocytes.

Density ultracentrifugation of sickle cells during and after pain crisis: increased dense echinocytes in crisis.

An increase in the number of irreversibly sickled cells (ISCs) in pain crisis has been found by some investigators but not others. We have used the technique of discontinuous arabinogalactan density-gradient ultracentrifugation of whole blood to study ISCs from patients with sickle cell anemia (SCA) during pain crisis and again when pain free (5-331 days after crisis). Nine patients have been studied through ten episodes of pain crisis. Five layers with densities from 1.128 g/mL to 1.158 g/mL have been used. Careful classification of the cells using Nomarsky optics demonstrated highly significant changes occurring in the layers of the gradient. The changes involve the appearance of an increased percentage of echinocytic ISCs and echinocytic cells that were not ISCs, especially in the denser gradient layers, during crisis, and their replacement by normal-appearing discocytes in the pain-free state. There was no change in ISCs that were not echinocytic. Data collected previously demonstrated that reduced glutathione activity correlated with increased echinocytosis in our gradient layers. This indicates that the echinocytic change may occur as a result of oxidant stress. Hemoglobin F levels and the percentage of hemoglobin F from reticulocytes showed no consistent change to coincide with the rise in normal-appearing discocytes in the lightest layers after crisis. Our data indicate that pain crisis occurs in association with an echinocytic change, which may be induced by oxidant injury. The rise in normal-appearing cells after crisis may reflect increased hemoglobin F production in some patients but mainly relates to the disappearance of these echinocytic erythrocytes.

Relationship of glutathione levels and Heinz body formation to irreversibly sickled cells in sickle cell anemia.

To assess the possibility that the membrane lesion seen in sickle cells involves oxidant damage, we investigated the relationship between irreversibly sickled cells and glutathione levels and Heinz body production. We found that the mean level of red cell glutathione, whether expressed as micromoles per gram of hemoglobin or micromoles per milliliter of cell water, was significantly decreased in 39 patients with sickle cell anemia. A weak but significant negative correlation between glutathione levels and irreversibly sickled cell counts in the patients was observed. Stractan density gradient studies were performed to determine glutathione differences in normal- and abnormal-appearing sickle cells. The percent of abnormal cells in each of the five stractan gradient fractions was negatively correlated with both the micromole per gram of hemoglobin and micromole per milliliter of cell water levels of glutathione. Another indicator of susceptibility to oxidant stress, Heinz body formation, was increased in patients with sickle cell anemia vs controls in response to oxidant stress. Surprisingly, we found that the majority of Heinz bodies were in the non-ISC fraction of cells, rather than in the irreversibly sickled cells.

Hypertension and a seizure following transfusion in an adult with sickle cell anemia.

A 20-year-old black man with known sickle cell anemia was admitted to the hospital with the signs and symptoms of right middle lobe pneumonia. Because of a hemoglobin value of 3.9 g/dL, he received a transfusion of 3 units of packed erythrocytes. Seven days later, hypertension, headache, and one grand mal seizure occurred. Evaluation of his condition gave normal findings. Similar events, some terminating in cerebral hemorrhage and death, have been reported in patients with hemolytic anemia after they received multiple transfusions. To our knowledge, this patient represents the first adult with sickle cell anemia who had this syndrome. Prompt antihypertensive therapy may have been of benefit.

Calmodulin level in whole blood correlates with the percentage of reticulocytes.

We studied the calmodulin-like activity (CaM) level in hemolyzed samples of whole blood after determining the percentage and absolute number of reticulocytes present. Twenty-six samples from 25 people with a range of reticulocyte counts were studied. CaM levels correlated with the percentage (r = 0.63, P less than .01) and absolute number (r = 0.64, P less than .01) of reticulocytes present, indicating that the calmodulin-like activity level in whole blood was inversely related to erythrocyte age.