Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.     

Behavioral,physiological, and morphological components of dominance and mate attraction in male green iguanas

This study investigated the morphological, physiological, and behavioral components of social dominance important for mate attraction in male green iguanas (Iguana iguana). A group of 9 male and 11 female adult green iguanas was studied in a large semi-natural enclosure during one reproductive season (October–January). Four of the nine males never initiated aggressive encounters; the other five were observed to display aggressively toward each other and were ranked in a linear dominance hierarchy. Head size was the most important factor influencing fighting success. Head size and display frequency were positively correlated with plasma testosterone levels. Dominance rank directly influenced ability to monopolize areas containing resources used by females. The quality of a male's home range, measured as his access to a large basking rock in the enclosure, was related to the proportion of potential mates found within his home range. One male greatly surpassed the others in his ability to defend a home range of high quality and attract potential mates. These data suggest that physiological and morphological factors, through their influence on social behavior, may ultimately affect male reproductive fitness. © 1992 Wiley-Liss, Inc.     

Surface heterogeneity of bovine sperm revealed by aqueous two-phase partition

Ejaculated, bovine sperm have been subjected to multiple partition in aqueous two-phase systems. This partition, carried out in a countercurrent fashion, reveals heterogeneity of the sperm population with respect to surface properties. The sperm, when partitioned in phase systems that detect non-change associated surface properties (change-insensitive) are largely distributed as two distinct populations. In charge-sensitive phase systems (which principally detect cell surface molecules carrying charge) the sperm do not show any obvious surface heterogeneity. Considerable heterogeneity is revealed in affinity-ligand phase systems containing palmitic acid coupled to one of the phase components-poly(ethylene glycol). There is a difference in surface heterogeneity between sperm which have been washed in buffer or left unwashed, direct from the ejaculate. This is indicative of weak adsorption of proteins to the sperm surface in seminal fluid. These results show that bovine ejaculated sperm is a heterogeneous cell population having unequal distributions of a number of different surface molecules.     

Cloning, sequencing and distribution of the Salmonella typhimurlum LT2 siaiidase gene, nanH, provides evidence for interspecies gene transfer

The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli. Sialidase expression was regulated positively by cAMP. In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity. A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S. typhimurium LT2. The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi. Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S. typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event. At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases. The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase. From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S. typhimurium obtained its nanH copy most recently from Salmonella arizonae. S. typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH. These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids.     

Inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoyl[3H]inosine is accompanied by modification of tyrosine 345 in a single beta subunit

The inactivation of the bovine heart mitochondrial F1-ATPase by 5'-p-fluorosulfonylbenzoylinosine (FSBI) proceeds with pseudo-first order kinetics. The rate of inactivation increased from pH 7 to 9 revealing a pKa of about 8.2. When a tryptic digest of the enzyme which had been inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine ([3H]FSBI) was submitted to reversed phase high pressure liquid chromatography, a single major peak of radioactivity, T1, was resolved. Amino acid sequence analysis of purified peptide fragments derived from T1 showed that the modification of beta-Tyr-345 is responsible for inactivation of the enzyme. Complete inactivation of the enzyme by [3H]FSBI is estimated to proceed with modification of 0.8 mol of beta-Tyr-345/mol of enzyme. Another notable observation is that inosine triphosphatase (ITPase) activity catalyzed by F1 from bovine heart mitochondria is much more sensitive to inactivation by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) than is ATPase activity. Whereas complete inactivation of ATPase activity by FSBA has been shown to proceed with the mutually exclusive modification of Tyr-368 or His-427 in all three copies of the beta subunit (Bullough, D. A., and Allison, W. S. (1986) J. Biol. Chem. 261, 5722-5730), it is shown here that complete inactivation of ITPase activity by FSBA is accompanied by modification of these residues in only one copy of the beta subunit. Inactivation of both the ATPase and ITPase activities of the enzyme by FSBI proceeds with modification of Tyr-345 in a single copy of the beta subunit.     

Hepatotoxic doses of dioxin do not damage mouse bone marrow chromosomes

We report on a study of the cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mice of the C57B1/6J (with high-affinity TCDD receptor) or DBA/2J (with low-affinity TCDD receptor) strains were given single intraperitoneal injections of 50, 100 or 150 micrograms of TCDD/kg body weight. At various times (8-48 h) after injection, we examined bone marrow cells for cytogenetic effects by performing structural aberration, sister-chromatid exchange, and micronucleus tests. 1 month after exposure, liver sections were studied for hepatotoxic effects. We found no evidence of chromosome damage by TCDD given in doses that cause liver damage in both strains of mice.     

Proton-translocating ATPase and lysosomal cystine transport

A proton-translocating ATPase was identified in highly purified lysosomes from Epstein-Barr virus-transformed human lymphoblasts. Activity of this ATPase caused acidification of highly purified, fluorescein isothiocyanate dextran-loaded lysosomes and correlated with the ATP-dependent efflux of lysosomal cystine. The lysosomal ATPase was distinct from mitochondrial F1-ATPase in its responses to a variety of inhibitors. Although ATP-dependent lysosomal cystine efflux is not demonstrable in cultured lymphoblasts from individuals with nephropathic cystinosis, ATPase activity and acidification in lysosomes from these cells is comparable to that in noncystinotic lysosomes. ATPase activity in lymphoblasts from normal individuals was 543 +/- 79 nmol/mg/min while in lymphoblasts from cystinotic individuals this activity was 541 +/- 25 nmol/mg/min. ATP-dependent acidification of lysosomes from normals was -0.5 +/- 0.1 pH units compared to -0.5 +/- 0.1 pH units in cystinotic lysosomes. Activity of the lysosomal proton-translocating ATPase is a necessary, but not sufficient, condition for lysosomal cystine efflux.     

High Dosage Metolazone in Chronic Renal Failure

Metolazone is a modified quinazolinesulphonamide and in a dose of between 4 and 7·5 mg is an effective diuretic in man with normal renal function. Fourteen patients with non-oedematous stable chronic renal failure (creatinine clearance ranging from 1·2 to 12 ml/min) were given metolazone in doses ranging from 20-150 mg. A noticeable increase in urine flow and sodium excretion occurred, free water clearance increased, and there was a small but significant increase in potassium excretion. No side effects were noted.