Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria.

1. Ouabain-sensitive 86Rb+ uptake by tissue preparations has been used as an estimate of Na+ pump activity. This uptake, however, may be a measure of the Na+ influx rate, rather than capacity of the Na+ pump, since intracellular Na+ concentration is a determinant of the active Na+/Rb+ exchange reaction under certain conditions. This aspect was examined by studying the effect of altered Na+ influx rate on ouabain-sensitive 86Rb+ uptake in atrial preparations of guinea pig hearts. 2. Electrical stimulation markedly enhanced ouabain-sensitive 86Rb+ uptake without affecting nonspecific, ouabain-insensitive uptake. Paired-pulse stimulation studies indicate that the stimulation-induced enhancement of 86Rb+ uptake is due to membrane depolarizations, and hence related to the rate of Na+ influx. 3. Alterations in the extracellular Ca2+ concentration failed to affect the 86Rb+ uptake indicating that the force of contraction does not influence 86Rb+ uptake. 4. Reduced Na+ influx by low extracellular Na+ concentration decreased 86Rb+ uptake, and an increased Na+ influx by a Na+-specific ionophore, monensin, enhanced 86Rb+ uptake in quiescent atria. 5. Grayanotoxins, agents that increase transmembrane Na+ influx, and high concentrations of monensin appear to have inhibitory effects on ouabain-sensitive 86Rb+ uptake in electrically stimulated and in quiescent atria. 6. Electrical stimulation or monensin enhanced ouabain binding to (Na+ + K+)-ATPase and also increased the potency of ouabain to inhibit 86Rb+ uptake indicating that the intracellular Na+ available to the Na+ pump is increased under these conditions. 7. The ouabain-sensitive 86Rb+ uptake in electrically stimulated atria was less sensitive to alterations in the extracellular Na+ concentration, temperature and monensin than that in quiescent atria. 8. These results indicate that the rate of Na+ influx is the primary determinant of ouabain-sensitive 86Rb+ uptake in isolated atria. Electrical stimulation most effectively increases the Na+ available to the Na+ pump system. The ouabain-sensitive 86Rb+ uptake by atrial preparations under electrical stimulation at a relatively high frequency seems to represent the maximal capacity of the Na+ pump in this tissue.     

Bile acid uptake by isolated intestinal mucosa cells of guinea pig

Isolated intestinal mucosa cells of the guinea pig were employed to study intestinal transport of bile acids. Chenodeoxycholate and lithocholate were rapidly taken up into jejunal and ileal cells by diffusion. Taurocholate and cholate however showed only a minor diffusion rate and were preferentially taken up by the ileal bile acid carrier. This uptake was saturable with an apparent K_m of 231 μM and V of 7 nmol/mg protein per min for taurocholate; this bile acid was accumulated 90-fold. Its uptake was strongly inhibited by antimycin A, FCCP, ouabain or Na⁺-deficiency in the medium. Sugars or amino acids did not interfere with uptake. Experimental conditions were optimized with regard to incubation medium, cell amount, cell age and length of preincubation. It is concluded that ileal cells of the guinea pig are superior to other experimental models for characterizing the ileal bile acid carrier, because they allow us to determine initial rates of uptake and have a very efficient energetic coupling.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

Sodium-pump activity and its inhibition by extracellular calcium in cardiac myocytes of guinea pigs

Myocardial sodium-pump activity was examined from ouabain-sensitive ⁸⁶Rb⁺ uptake using myocytes isolated from guinea-pig heart. Either sodium loading or the sodium ionophore, monensin, increased ⁸⁶Rb⁺ uptake by over 400%, indicating that the amount of Na⁺ available to the pump is the primary determinant of its activity, and that the sodium pump has a substantial reserve capacity in quiescent myocytes. Moreover, the degree of the above stimulation is markedly higher than corresponding values reported with multicellular preparations, suggesting that diffusion barriers make it impossible to observe the capacity of the sodium pump in the latter preparations. Removal of extracellular Ca²⁺ increased ouabain-sensitive ⁸⁶Rb⁺ uptake, probably by enhancing turnover of the sodium pump rather than increasing availability of Na⁺ to the pump.     

Purification and characterization of an enkephalin-degrading aminopeptidase from guinea pig serum

An enkaphalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The amino-peptidase had a pH optimum of 7.0 with K_m values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid β-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.     

Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.     

Bile salt sulfotransferase in guinea pig liver

Bile salt sulfotransferase from guinea pig liver is purified by the procedures of ammonium sulfate fractionation, Sephadex G-100 column chromatography, agarose-hexane-adenosine 3′,5′-diphosphate affinity chromatography and polyacrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 6.8, an isoelectric point of 5.6 and a molecular weight of 7600 estimated by gel filtration technique. The apparent K_m values of the enzyme are 7.7·10^?5 M for taurolithocholate and 1.4·10^?6 M for 3′-phosphoadenosine 5′-phosphosulfate. It requires Mg²⁺ and free sulfohydryl group(s) for activity. The enzyme reacts with hydroxy groups of bile salts at both 3α and 3β positions. No activity is found in the kidney of guinea pig. The purified enzyme does not react with estrone, estradiol, testosterone, dehydroepiandrosterone, cholesterol, phenol, tyramine, and serotonin. The results indicate that bile salt sulfotransferase is distinct from other hepatic sulfotransferases.     

Immunological identification of soluble carbonyl reductase with testosterone 17β-dehydrogenase (NADP+) in guinea pig liver and kidney

Antiserum was produced against one of two carbonyl reductases purified from guinea pig liver cytosol to identify the enzymes as testosterone 17β-dehydrogenase isozymes. Immunoelectrophoresis and immunoprecipitation with the antiserum indicated that the two reductases had common antigenic sites. The antiserum inhibited most of both carbonyl reductase and testosterone 17β-dehydrogenase activities in the purified reductases and in cytosols of liver and kidney.     

Time course of pump inhibition by ouabain in amphibian epithelia

Inhibition by ouabain of rheogenic Na⁺ transport across the basolateral membranes of frog skin is found to be manifest within 3–4 min. This rate of pump inhibition is not different from the rate of diffusion through extracellular tissue layers between the serosal bath and the actual site of action, i.e., the epithelial cell layers. It is concluded that the well-known slow time course of decrease in transepithelial current flow is due ionic redistribution and conductance changes of the epithelial membranes secondary to pump inhibition.     

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. ¹⁴C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [¹⁴C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.     

Electron microscopic studies on guinea pig rib cartilage

Summary The guinea-pig rib cartilage consists of chondrocytes dispersed in an intercellular substance composed of collagen fibrils, often characteristically cross-striated, and polygonal granules. Electron-dense membrane-bounded matrix vesicles are also observed intercellularly, especially in the central, partly calcified zone of the cartilage. With respect to their location in a cross-section of the rib, the chondrocytes differ in size, shape and intracellular fine structure. Thus, three separate types of cells are recognized. Peripheral chondrocytes have a flattened shape and are largely occupied by the nucleus. In the cytoplasm, the granular endoplasmic reticulum is the most extensive organelle. Intermediate chondrocytes are oval or round in shape. The endoplasmic reticulum and the Golgi complex are both prominent. Mitochondria and membrane-bounded cytoplasmic dense bodies are more numerous than in the peripheral cells. The ground cytoplasm often contains a few lipid droplets. In the central chondrocytes, such droplets sometimes fill the entire cytoplasm. Concomitantly, the nucleus is usually completely heterochromatic and the cells are therefore regarded as being metabolically inert.After preparations including ruthenium red staining en bloc, the general stainability of the chondrocytes is decreased. Intracellularly, positive ruthenium red staining of granular material within the Golgi vacuoles are to be observed. Extracellularly, the matrix granules are stained with this polyvalent, cationic dye. Extraction of the cartilage with 4 M guanidine-HCl removes all matrix granules and about 70% of the proteoglycans, measured as hexosamine, from the tissue. It is concluded that the matrix granules contain proteoglycan complexes.Financial support was received from the Swedish Medical Research Council (proj. no. 12X-3355), the Swedish Cancer Society (proj. no. 100-K71-05XK), the King Gustaf V 80th Birthday Fund, the Harald and Greta Jeansson Foundation, the C. B. Nathhorst Foundation, and from the funds of Karolinska Institutet.The skilled technical assistance of Mrs. Eva Lundberg and the secretarial assistance of Mrs. Inger Åhrén are gratefully acknowledged. The authors are indebted to Dr. S. Lohmander for helpful suggestions during the progress of the work.     

Ovarian sympathectomy in the guinea pig

Summary The role of ovarian adrenergic nerves in follicular growth was studied in prepubertal guinea pigs by determining the effect of sympathectomy on 1) follicle populations and 2) follicular development following exogenous gonadotropin administration. Selective unilateral ovarian sympathectomy was achieved by injecting 6-hydroxydopamine into a surgically closed periovarian bursa on day 20 postpartum. The contralateral surgically closed ovarian bursa was injected with the vehicle used for 6-hydroxydopamine. On day 25, animals were injected with pregnant mare serum or saline followed by human chorionic gonadotropin or saline 48 h later. All animals were laparotomized on day 28 and blood from utero-ovarian veins was collected bilaterally for androstenedione determination. Ovaries were processed for morphometric analysis of follicles. The sympathectomized ovary in saline-injected animals had a significant decrease in preantral follicles (characterized by 2 layers of granulosa cells without antrum formation), an increase in 310–500 m diameter atretic follicles and an increase in follicles 700 um compared to the contralateral control ovary. There were no differences in androstenedione levels from the two sides, ovarian weights or the total number of follicles per ovary. Neither ovary had corpora lutea. The sympathectomized ovary in animals injected with gonadotropins was not different from the contralateral ovary in any of the parameters measured. Both control and sympathectomized ovaries had newly formed corpora lutea in response to the exogenous gonadotropins. These results suggest that ovarian adrenergic nerves normally participate in follicular development in the prepubertal guinea pig. However, exogenous gonadotropins may override neural influences on the prepubertal ovary.     

Light- and electron microscopy of isolated vestibular hair cells from the guinea pig

Summary Cells isolated from the guinea-pig vestibular sensory epithelia were studied using light- and electron-microscopic techniques. The cells maintained their characteristic shapes when they had been separated. Mammalian vestibular cells are traditionally divided into two classes, type-I and type-II hair cells. It was, however, found that the population of isolated cells consisted of hair cells with a striking variability in shape and size. This was most conspicuous for the type-I hair cells. Isolated hair cells processed for electron microscopy showed that the isolation process caused minor ultrastructural damage but that the separation often was incomplete in that the large calyx-like nerve endings were still attached to type-I cells. The results suggest that the distinction of only two classes might be insufficient to describe mammalian vestibular hair cells.     

Physiological effects and biochemical properties of a serum protein that produces positive inotropic and chronotropic effects on isolated guinea pig atria

Isolated left and right guinea pig atria were used as a bioassay for the detection of an endogenous cardioactive substance in bovine serum. Serum, buffer exchanged to Krebs–Henseleit solution, produced positive inotropic and chronotropic effects on the isolated guinea pig atria. The cardiotonic effects were unaffected by the combined presence of propranolol and methysergide (both 10^–6M) and were also dissimilar in time course from other known cardiotons such as catecholamines and cardiac glycosides. Following ultrafiltration (using XM100A Amicon membranes), activity was found solely in the retentate fractions and was therefore probably due to a large molecular weight (>100 kDa) substance or a small molecule bound to a large protein. The cardioactive factor (CF) in the whole serum was heat labile, sensitive to acidification, exposure to potassium bromide and equilibration to physiological buffers of a low ionic strength. Isolation by conventional protein purification techniques was unsuccessful due to the labile nature of the active molecule(s) when exposed to non-physiological experimental conditions. Physical and biochemical properties of the CF which may help avoid inactivation are discussed for future experiments aimed at elucidating the nature and identity of the cardiotonic principle. (Mol Cell Biochem 261: 201–207, 2004)     

GABA immunoreactivity of calyceal nerve endings in the vestibular system of the guinea pig

Summary Neurotransmitters involved in the vestibular system are largely uncharacterized. On the basis of results of earlier electrophysiological and immunohistochemical experiments, glutamate and gamma-amino-butyric acid (GABA) have been proposed in both mammalian and non-mammalian species as afferent transmitters between the sensory cell and the afferent dendrite. GABA is also suspected to act as an efferent neurotransmitter in the cochlea. We describe in this study the immunocytochemical localization of GABA within the vestibular end organs in the guinea pig. GABA immunoreactivity was found in the calyceal nerve endings surrounding type I hair cells of the vestibular epithelia. The most significant labelings were obtained in the crista ampullaris. Labeling was more difficult to observe in the utricular and saccular macula. These results contribute to the recent proposal that the calyx has a secretory function, and suggest that GABA may have a modulatory influence upon the type I hair cells.     

Intravesical BCG administration in the guinea pig

Intravesical BCG administration is used as an adjuvant therapy after transurethral resection for superficial bladder cancer in man. The mechanisms of its antitumor activity are not known. The aim of this study was to characterize the histomorphological changes in various organs of the guinea pig after intravesical BCG administration. The BCG preparation used was BCG-RIVM, a Dutch BCG preparation. Instillations were performed in previously undamaged bladders weekly for 6 consecutive weeks and lasted 30 min or 1 h. Different doses were used ranging from 10³ culturable particles (c.p.) to 5 × 10⁷ c.p. of BCG. After 6 weeks, the animals were killed and postmortem examination was performed. The bladder wall, retroperitoneal lymph nodes, spleen, liver, lungs and distant lymph nodes were examined histologically. The BCG therapy, with a dose of 10₆ culturable particles and higher, induced an inflammatory reaction consisting of mononuclear infiltrates in the subepithelial tissue of the bladder wall. In approximately 50% of the animals investigated, the infiltrates were accompanied by noncaseating granulomatous lesions indicated by the presence of epithelioid cells. In general, the epithelial layer of the bladder showed no visible alterations. Similarly, a granulomatous inflammatory reaction was observed in the first retroperitoneal (iliac) lymph nodes draining the bladder. Granulomatous lesions were occasionally also present in liver and lung. In three of the 29 animals investigated, lesions were present both in liver and lungs, and in two of these three animals a granulomatous reaction was observed in the spleen and distant lymph nodes indicating a generalized inflammatory response induced by BCG. No microorganisms were detected by Ziehl-Neelsen (ZN) staining or culture in L?wenstein-Jensen medium in the first draining (iliac) lymph nodes of the bladder or in the spleen. In this study we found that BCG could induce inflammatory reactions in the bladder wall after its introduction into the previously undamaged bladder. Ulceration of the epithelium covering the mononuclear infiltrates was not observed. Occasionally a generalized inflammatory response to BCG was present in the animals investigated.     

Forms and activity of high molecular weight adrenocorticotropin in guinea pig

High molecular weight forms of adrenocorticotropin (ACTH) and endorphin were identified in extracts of guinea pig anterior and intermediate/posterior pituitary. Extracts of anterior pituitary contained ACTH immunoactive material with apparent molecular weights of 36,000, 24,000 and 4,500 daltons. The highest molecular weight form the ACTH co-migrated with a peak of endorphin immunoactive material. No material the size of glycosylated ACTH(1--39) was detected. Separated forms of high molecular weight ACTH prepared from mouse tumor cell culture medium stimulated the same maximal production of steroid as ACTH(1--39) in the guinea pig adrenal cell bioassay. Pro-ACTH/endorphin and ACTH biosynthetic intermediate were two orders of magnitude less potent than synthetic human ACTH(1--39); glycosylated ACTH(1--39) was equipotent to ACTH(1--39) although no similar material was detected in guinea pig pituitary extracts. Isolated guinea pig adrenal cortical cells were incubated with the various separated form of mouse tumor cell ACTH and products synthesized from (3H)pregnenolone were analyzed by two-dimensional thin-layer chromatography. The ratio of cortisol-related to corticosterone-related products was the same in response in glycosylated and nonglycosylated ACTH.     

A cytofluorometric study of the myenteric plexus in the guinea pig

Summary The myenteric plexus of the guinea pig ileum was studied in stretch preparations of the longitudinal muscle layer with adherent plexuses, and in freeze-dried transverse sections from the small intestinal wall. Catecholamines and serotonin (5-HT) were visualized according to the Falck-Hillarp technique. Emission spectra from the resulting fluorophores and recordings of their rates of photodecomposition were analysed. Adrenergic nerve terminals showed a slow fluorescence fading rate and a fluorescence spectrum compatible with their known contents of noradrenaline (NA), while the enterochromaffin cells showed a rapid exponential fading and a fluorescence spectrum compatible with their known contents of 5-HT. In order to unmask any low amounts of 5-HT in the neurons of the plexus, analysis of fluorescence parameters at various time intervals after pretreatment with reserpine followed by MAO-inhibition was performed. With the methods used no evidence of the presence of 5-HT in the myenteric plexus of the guinea pig could be found.We thank Iréne Svensson and Uno Johansson for skilful technical assistance. We are also indebted to Ciba, Pfizer and Draco for generous supplies of Reserpine, Nialamide and Pheniprazine. —This work was supported by grants from the Swedish Medical Research Council (Project 14 X-2235) and Göteborgs Läkaresällskap.