Transplantation of human renal carcinomas into athymic mice.

Thirty-one primary human renal carcinomas were transplanted into athymic mice of which ten produced tumors in the mouse host. Only tumors with a nuclear grade of 3 or 4 were successfully transplanted. The nuclear grades of the human tumor and transplant were similar; however, the cellular histology often varied. Patient prognosis appeared to be inversely related to successful tumor transplantation. In the transplant group, the 1-year survival was 30% in contrast to a 1-year survival of 83% among patients with renal cancers of similar stage and grade which did not produce tumors in the mice.

Characterization of two human cell lines (TK-10, TK-164) of renal cell cancer.

Two previously unreported cell lines of human renal cell carcinoma are presented. TK-10 and TK-164 have each been in culture for over 4 years. The epithelial nature of both cell lines has been documented by light and electron microscopy. The cells in each line contain a Y chromosome, have specific marker chromosomes, and a distinct flow cytometric histogram. Both lines grow in agar, albeit not in athymic mice.

Increased number of beta-adrenergic receptors in the hypertrophied myocardium.

Development of cardiac hypertrophy is associated with depletion of endogenous catecholamine stores and increased inotropic response to exogenous catecholamines. A biochemical basis for these changes is provided by the observation that the number of cardiac beta-adrenergic receptors - as reflected in specific [3H]dihydroalprenolol binding - is increased in hypertrophy without a change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. This change in beta-receptor numbers may be an important adaptive mechanism for preserving the contractile performance of the hypertrophied myocardium.

Increased phospholipid methylation in the myocardium of hyperthyroid rats.

Administration of L-thyroxine (1 mg/kg) to adult rats results in cardiac hypertrophy and enhanced contractility. Phosphatidylcholine synthesis through methylation of phosphatidylethanolamine by S-adenosylmetionine in enhanced in the myocardium of hyperthyroid rats. Increased microsomal methylation occurs pari passu with the development of cardiac hypertrophy and may be partly responsible for preservation of cardiac performance.

Enhanced myocardial RNA synthesis in spontaneously hypertensive rats possible role of high-mobility group non-histone proteins.

Cardiac hypertrophy in spontaneously hypertensive rats is associated with increased nuclear RNA polymerase activity. In order to explore mechanisms facilitating the interaction of the enzyme with its endogenous template, we compared the structure of nuclear chromatin from myocytes of 20-week-old spontaneously hypertensive rats and normotensive Wistar-Kyoto controls. Enhanced RNA synthesis in hypertensive rats was accompanied by increased susceptibility to digestion by deoxyribonuclease I. Nick translation of nuclei also resulted in higher nucleotide incorporation in hypertensive rats. Salt-extraction abolished the differences in deoxyribonuclease I sensitivity between the two animal groups. Reconstitution with either 0.35 M NaCl-extract or high mobility group (HMG) non-histone proteins restored digestion susceptibility but did not equalize SHR and WKY cells. SDS-polyacrylamide gel electrophoresis of 0.35 M NaCl-extracts and supernatants from deoxyribonuclease I digestion revealed the presence of HMG proteins which were preferentially released in hypertensive rats. There was a small but statistically significant increase in nuclear HMG protein content in hypertensive rats (0.12 +/- 0.02 mg/mg DNA vs. 0.09 +/- 0.02 mg/mg DNA in Wistar-Kyotos, P less than 0.05) but no difference in their electrophoretic appearance. These results indicate that chromatin structure is altered in the hypertrophied myocardium with resultant increase in deoxyribonuclease I susceptibility. This increase appears to be partly dependent on the high-mobility group non-histone proteins.

Selective stimulation of venous prostaglandin E 9-ketoreductase by bradykinin.

The effects of bradykin on prostaglandin metabolism in canine mesenteric vessels were examined. Bradykinin stimulated microsomal prostaglandin synthesis in both artery and vein; this stimulation was more pronounced when [14C] hosphatidylcholine rather than [14C] arachidonate was used as the substrate for prostaglandin synthetase. This suggested that bradykinin enhanced a membrane phospholipase. In addition, bradykinin selectively stimulated prostaglandin E 9-ketoreductase activity from veins but not arteries. This may explain the finding that bradykinin induces the release of prostaglandin E compounds from arteries but prostaglandin F compounds from veins.

Phospholipid metabolism in the rat renal inner medulla.

In view of the importance of phospholipids as a source of precursor fatty acids for the high prostaglandin synthesis in the renal inner medulla, we studied pathways of phospholipid esterification and degradation in the rat inner medulla. De novo acylation of [14C]arachidonate occurred predominantly in position 2 of phosphatidylcholine in the microsomal fraction. This newly esterified [14C]arachidonate was accessible to deacylation by a microsomal phospholipase A2 (EC 3.1.1.4) with alkaline optimum which was Ca2+-dependent and resistant to 0.1% deoxycholate. No phospholipase A1 (EC 3.1.1.32) activity against endogenous labeled phosphatidylcholine could be demonstrated in the microsomal fraction. When exogenous phosphatidylcholine labeled at position 2 was deacylated by renomedullary homogenates, labeled free fatty acid but no labeled lysophosphatidylcholine was recovered in the reaction products. This could be attributed to further degradation of generated lysophosphatidylcholine by a cytosolic lysophospholipase (EC 3.1.1.5). Sodium deoxycholate at a concentration of 0.1% or higher inhibited the lysophospholipase and allowed the demonstration of both A2 and A1 alkaline phospholipase activities in the homogenate. The major in vitro pathway of lysophosphatidylcholine disposition is further degradation by a cytosolic lysophospholipase, while reutilization for phosphatidylcholine synthesis through the action of a predominantly microsomal acyltransferase appears to be a minor pathway. In the presence of several acyl-CoAs, reutilization of lysophosphatidylcholine is significantly increased by an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) but there is no preferential transfer of arachidonyl-CoA compared to other acyl-CoAs.

Involvement of microtubules in the isoproterenol-induced 'down'-regulation of myocardial beta-adrenergic receptors.

The number of cardiac beta-adrenergic receptors identified by [3H]dihydroalprenolol binding decreases in a concentration-dependent manner during prolonged administration of isoproterenol. Loss of membrane beta-receptors is paralleled by the appearance of [3H]dihydroalprenolol binding activity in the cytosol. This redistribution of receptors is prevented by colchicine and vinblastine but not lumicolchicine. Cardiac receptor desensitization is, therefore, dependent on microtubules and may be influenced by agents interfering with tubulin polymerization.

Decreased number of beta-adrenergic receptors in hypertensive vessels.

Responsiveness to inotropic agents is altered in hypertension and may contribute to its initiation and maintenance. A biochemical basis for this change was provided by the observation that the number of beta-adrenergic receptors, as reflected in specific [3H]dihydroalprenolol binding, was diminished in both arteries and veins of spontaneously hypertensive rats. There was no change in the affinity of dihydroalprenolol for the binding sites or in the capacity of isoproterenol to displace dihydroalprenolol. The decline in beta-adrenergic receptor numbers is not secondary to blood pressure elevation but may, instead, contribute to the pathogenesis of hypertension.

Age-dependency of vascular phospholipid deacylation-reacylation in spontaneously hypertensive rats.

We have studied the temporal relation of phospholipid turnover and prostaglandin synthesis to the evolution of hypertensive vascular disease in the spontaneously hypertensive rat. The incorporation of arachidonate into aortic phospholipids, its release by phospholipase A2 and its utilization for prostaglandin synthesis were compared in spontaneously hypertensive and Wistar-Kyoto rats aged 7, 20 and 42 weeks. When expressed per mg of protein in the assay medium, arachidonate incorporation into aortic phospholipids decreased, while prostaglandin synthesis increased, with age in both rat strains. No significant differences were noted between hypertensive and normotensive animals at 7 weeks of age whereas both enhanced phospholipid turnover and prostaglandin synthesis was demonstrated in hypertensive rats at 20 and 42 weeks of age. The higher phospholipase activity in hypertensive aortas was associated with a significant increase in the capacity for exogenous lysophosphatide hydrolysis. Transacylation and reacylation of lysolecithin, however, were not significantly enhanced in hypertensive aortas. These biochemical changes accompany, and may be related to, structural modifications of the aortic wall in the course of hypertension.

Nuclear chromatin changes during post-natal myocardial development.

The proliferative capacity of rat myocardium declines rapidly during the first few weeks of post-natal life. In order to gain insight into the mechanisms involved in this decline, we studied the structure and function of nuclear chromatin from isolated rat myocardial cells during post-natal growth. Chromatin template activity decreased progressively (7.5 +/- 0.3 pmol [3H]UTP/microgram DNA per min at age 5 days compared to 2.2 +/- 0.1 pmol [3H]UTP/microgram DNA per min at age 6 months) and was associated with a decrease in the number of transcription initiation sites. This decline was accompanied by changes in chromatin structure as evidenced by: (a) decreased susceptibility to DNAase I digestion with advancing age, (b) decreased poly-L-lysine binding (60% decrease between day 5 and six months of age), (c) progressive decline in positive ellipticity of circular dichroism spectra between 250--300 nm, and (d) derivative melting profiles showing a decrease in DNA regions bound by non-histone proteins and concomitant increase in histone-bound regions. The protein composition of myocardial chromatin also changed during post-natal development, chiefly due to a progressive increase in the histone/DNA ratio. These results indicate substantial changes in the organization and functional capacity of myocardial chromatin during early post-natal growth. These changes accompany, and may contribute to, the restriction in the proliferative capacity of myocardial cells.

Adenosine 3',5'-monophosphate-dependent protein kinases of myocardial non-histone nuclear proteins.

Myocardial acidic non-histone nuclear proteins (NHPs) contain endogenous protein kinase activity. Phosphocellulose chromatography of purified NHPs identifies nine separate peaks of protein kinases which can phosphorylate both endogenous and exogenous substrates to a variable degree; endogenous NHPs are the best substrates. Cyclic AMP-stimulated protein kinase induced phosphorylation of endogenous and exogenous substrates; the extent of this stimulation varied according to the protein kinase fraction and substrate used. Cyclic AMP also enhanced NHP-induced stimulation of RNA polymerase activity. This enhancement was dependent on protein kinase-induced phosphorylation of NHPs since it was prevented by alkaline phosphatase pretreatment. It is concluded that nuclear protein kinases regulate myocardial RNA synthesis by enhancing phosphorylation of NHPs and that this regulation is under cyclic AMP control.

Comparison of the handling of norepinephrine in the myocardium of adult and old rats.

The handling of norepinephrine was compared in the myocardium of adult and old rats. Endogenous norepinephrine levels were significantly lower in the hearts of old animals. Following intravenous injection of 3H-norepinephrine [3H-NE] the initial uptake was slightly higher in the old animals; subsequently, however, there was a more pronounced and prolonged fall in 3H-NE specific activity in the myocardium of the old rats. The increased turnover rate was associated with normal catecholamine synthesis. Studies of the subcellular distribution of 3H-NE showed an inability of the myocardium from ageing rats to retain norepinephrine in the storage granules with subsequent loss of the soluble fraction.

Effect of antihypertensive therapy on calcium transport by cardiac sarcoplasmic reticulum of SHRs.

Cardiac hypertrophy develops during the course of blood pressure elevation in spontaneously hypertensive rats (SHRs) and is associated with defective calcium transport by cardiac sarcoplasmic reticulum (SR). AT 20 weeks of age, calcium uptake is reduced in SHRs (42 +/- 1.3 vs 64 +/- 1.6 nmol X mg-1 X min-1 in age-matched normotensive Wistar-Kyoto rats, P less than 0.01), while Ca2+ ATPase activity is enhanced (44 +/- 1.1 vs 35 +/- 0.7 nmol X mg-1 X min-1 in WKYs, P = 0.02); this results in low stoichiometry between calcium uptake and ATP hydrolysis in SHRs. The steady-state levels of the phosphoprotein intermediate [EP] of the transport ATPase are higher in normotensive rats (0.97 +/- 0.1 vs 0.67 +/- 0.08 nmol X mg-1 in SHRs, P less than 0.01) but the Ca2+- and ATP-dependency are similar in the two groups. In order to study the relative roles of hypertension and cardiac hypertrophy in the depression of SHR function, 20-week old SHRs and normotensive rats were treated for 10 weeks with either hydralazine (100 mg X litre-1) or alpha-methyldopa (8 g X litre-1). Both therapeutic regimens resulted in near normalisation of blood pressure of SHRs (hydralazine: 18.1 +/- 0.5 kPa [136 +/- 4 mmHg]; alpha-methyldopa 17.6 +/- kPa [132 +/- 3 mmHg]). Regression of cardiac hypertrophy, however, was seen only in the alpha-methyldopa-treated group, as judged by changes in left ventricular weight, RNA/DNA ratio, and hydroxyproline content. Furthermore, improvement in calcium transport capacity by the SHR, as reflected in higher calcium uptake and stoichiometric ratio between uptake and ATP hydrolysis, was found after alpha-methyldopa, but not hydralazine treatment. These results indicate that reversal of cardiac hypertrophy is required for improvement in calcium transport by cardiac SR after antihypertensive therapy of SHRs.

Nulceoprotein changes in the hearts of cardiomyopathic turkeys.

The possible involvement of nuclear proteins in the pathogenesis of a spontaneously occurring model of congestive cardiomyopathy in turkeys was examined. This model is characterised by cardiac hypertrophy and dilatation, reduced cardiac output and depressed contractility. The protein composition of myocardial nuclei was compared in normal (n = 9) and cardiomyopathic (n = 18) turkeys, 70 to 140 days old. Myopathic hearts as a group have a higher histone content (1.75 +/- 0.09 (SD) mg . mg DNA-1 vs 1.65 +/- 0.07 in controls, P less than 0.01) and histone/nonhistone protein (NHP) ratio (1.07 +/- 0.07 vs 0.95 +/- 0.02 in controls, P less than 0.01). The latter was independent of age and correlated well with the degree of cardiac dilatation. The electrophoretic patterns of chromatin proteins was decreased in myopathic hearts. This decrease was primarily accounted for by lower NHP phosphorylation (5.78 +/- 1.38 pmol 32P . mg prot-1 . 15 min-1 vs 8.33 +/- 0.81 in controls, P less than 0.01). DEAE-Sephacel chromatography separated cyclic AMP-dependent and -independent nuclear protein kinases with similar substrate specificities but lower specific activities in myopathic hearts. SDS-polyacrylamide similar substrate specificities but lower specific activities in myopathic hearts. SDS-polyacrylamide gel electrophoresis of phosphorylated nucleoproteins revealed differences in the lower molecular species of NHPs between control and myopathic hearts. There was a significant correlation between NHP phosphorylation and degree of cardiac dilatation (r = -0.78) or contractility as reflected by left ventricular systolic time intervals (r = -0.57). These results suggest that development of this model of spontaneous cardiomyopathy is associated with, and may, in part, be secondary to changes in the composition and function of myocardial nucleoproteins.

Contrasting effects of spontaneous and induced cardiomyopathy on the nucleoproteins of turkey hearts.

The possible involvement of nuclear proteins in the pathogenesis of cardiomyopathy was studied in a spontaneously occurring and a furazolidone-induced model of turkey cardiomyopathy. Both models are characterised by cardiac hypertrophy and dilatation, systemic hypotension and depressed contractility. The protein composition of myocardial nuclei was compared in normal (n = 9) and cardiomyopathic (spontaneous n = 6, furazolidone-induced n = 21) turkeys. Cardiac nuclei from spontaneously myopathic animals had a higher histone content (1.827 +/- 0.058 (mean +/- SD) mg . mg DNA-1 vs 1.688 +/- 0.187 in controls, P less than 0.05) and histone/nonhistone protein ratio (1.122 +/- 0.020 vs 0.882 +/- 0.128 in controls, P less than 0.01). Nuclear protein phosphorylation was lower in spontaneously cardiomyopathic turkeys primarily because of decreased nonhistone protein phosphorylation (5.100 +/- 0.759 pmol 32 P . mg prot-1 .15 min-1 vs 8.456 +/- 0.886 in controls, P less than 0.01). In contrast, furazolidone-induced cardiomyopathy of similar severity to the spontaneously occurring model was not associated with changes in nucleoprotein composition or degree of phosphorylation. These results indicate that development of spontaneous cardiomyopathy in turkeys may be related to the composition and function of nuclear nonhistone proteins. These changes are not secondary to the cardiac hypertrophy/dilatation accompanying the myopathic process.

Calcium uptake by cardiac sarcoplasmic reticulum in human dilated cardiomyopathy.

To determine the biochemical basis of abnormal diastolic properties in human dilated cardiomyopathy calcium uptake by the sarcoplasmic reticulum in ventricular homogenates of biopsy specimens from 21 patients with dilated cardiomyopathy was compared with that in nine normal controls. As a group, patients with cardiomyopathy had considerably lower calcium uptake rates (3.3(0.6) nmol.mg-1.min-1 vs 6.5(0.5) nmol.mg-1.min-1, p less than 0.01). Calcium uptake rates correlated modestly with resting haemodynamic values and significantly with plasma noradrenaline concentrations but not with plasma renin activity. These results show that sarcoplasmic reticulum function is impaired in human dilated cardiomyopathy and that this impairment is related both to the severity of haemodynamic dysfunction and to the extent of sympathetic nervous system activation.

Up-regulation of renal prostaglandin receptors in genetic salt-dependent hypertension.

Development of hypertension in Dahl salt-sensitive rats (DS) is accompanied by reduced renomedullary prostaglandin synthesis, which may be responsible for their lower natriuretic capacity. To examine the changes in renomedullary prostaglandin E2 synthesis, the effects of high (8.0%) and normal (0.6%) NaCl diets were examined in DS and in Dahl salt-resistant rats (DR). In response to an 8.0% NaCl diet, the number of prostaglandin E2 receptors in the renal outer medulla of DR increased (2.97 +/- 0.2 vs 2.18 +/- 0.2 pmol/mg on 0.6% NaCl diet) while no change was noted in their affinities (Kd, 9.5 +/- 0.2 vs 9.4 +/- 0.3 nM). Receptor number and affinity in the renal cortex, inner medulla, and liver of DR were not affected. In contrast, renomedullary receptors of DS had a lower affinity than those of age-matched DR (Kd, 13.9 +/- 0.2 nM on 0.6% NaCl diet and 14.0 +/- 0.3 nM on 8.0% NaCl diet) and did not increase in number after a high salt diet. This apparent inability of DS to modulate prostaglandin receptors may contribute to their susceptibility to salt-induced hypertension.

Decreased isoproterenol-induced "down"-regulation of beta-adrenergic receptors in the myocardium of SHR.

The number of cardiac beta-adrenergic receptors and the activity of isoproterenol-stimulated adenylate cyclase are lower in spontaneously hypertensive rats (SHRs) and may account for diminished inotropic responsiveness to beta-agonists. Differences in isoproterenol-induced desensitization of the cardiac adenylate cyclase system between 14- to 16-week-old SHRs and Wistar-Kyoto (WKY) controls were studied using cardiac membranes, isolated cardiac myocytes, and intact animals. In both animal strains, the isoproterenol-induced "loss" of beta-adrenergic receptors in the cardiac membranes required adenosine triphosphate (ATP), magnesium (Mg2+), and guanosine 5'-triphosphate (GTP). The ATP and Mg2+ requirement may reflect a crucial role for a phosphorylation step, since desensitization was prevented by cordycepin, a nonspecific phosphorylation inhibitor. In both isolated myocytes and intact animals, isoproterenol induced a redistribution of the beta-adrenergic receptors (but not of adenylate cyclase) from the cell membrane to the cytosol. Internalization of the receptors was not secondary to the loss of membrane fragments to the cytosol, since the receptors sequestered in a fraction devoid of cell surface markers. In all three preparations, the extent of isoproterenol-induced "loss" of beta-receptors and isoproterenol-stimulated adenylate cyclase was considerably lower in SHRs than WKYs. This difference appeared to be due to a reduced capacity of isoproterenol to induce translocation of beta-receptors from the membranes to the cytosol of SHRs, because of a change in the physical properties of either the beta-receptors themselves or the membranes into which they were embedded.

Cardiac beta-adrenergic receptors in salt-dependent genetic hypertension.

The pattern of cardiac beta-adrenergic receptor changes in different hypertrophy models varies according to the pathophysiology. In salt-sensitive Dahl rats, high dietary salt intake leads to a moderate degree of cardiac hypertrophy associated with increased numbers of cardiac beta-adrenergic receptors but unchanged affinity for agonists. Isoproterenol-stimulated cardiac adenylate cyclase is also higher in salt-loaded hypertensive rats without any change in basal or NaF-stimulated activities. In contrast, neither beta-adrenergic receptors nor adenylate cyclase activities are affected by variations in dietary salt in salt-resistant Dahl rats. The extent of isoproterenol-induced down regulation of beta-adrenergic receptors on isolated cardiac myocytes as well as the recovery from this down regulation is not significantly different in either strain of Dahl rats and is not influenced by dietary salt. The enhancement of beta-adrenergic pathways in salt-dependent genetic hypertension may be involved both in the initiation of cardiac hypertrophy and the preservation of contractile function.