The structural and functional interrelationships of muscarinic acetylcholine receptor subtypes

Molecular cloning of the genes encoding the muscarinic acetylcholine receptors has shown that receptor subtypes classified on the basis of pharmacological properties are related polypeptides encoded by distinct genes. These studies have also revealed the existence of novel muscarinic receptor subtypes. Functional analysis of each of the subtypes expressed in mammalian cells indicates that the different subtypes activate distinct biochemical pathways, a finding that explains the tissue-specific physiological response elicited by the neurotransmitter, acetylcholine.     

The renal Na+/Ca2+ exchange system is located exclusively in the distal tubule.

The movement of Ca2+ across the basolateral plasma membrane was determined in purified preparations of this membrane isolated from rabbit proximal and distal convoluted tubules. The ATP-dependent Ca2+ uptake was present in basolateral membranes from both these tubular segments, but the activity was higher in the distal tubules. A very active Na+/Ca2+ exchange system was also demonstrated in the distal-tubular membranes, but in proximal-tubular membranes this exchange system was not demonstrable. The presence of Na+ outside the vesicles gradually inhibited the ATP-dependent Ca2+ uptake in the distal-tubular-membrane preparations, but remained without effect in those from the proximal tubules. The activity of the Na+/Ca2+ exchange system in the distal-tubular membranes was a function of the imposed Na+ gradient. These results suggest that the major differences in the characteristics of Ca2+ transport in the proximal and in the distal tubules are due to the high activity of a Na+/Ca2+ exchange system in the distal tubule and its virtual absence in the proximal tubule.     

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

Specificity of tyrosine protein kinases of the structurally related receptors for insulin and insulin-like growth factor I: Tyr-containing synthetic polymers as specific inhibitors or substrates

The receptors for insulin and insulin-like growth factor (IGF) I are structurally similar transmembrane proteins. Ligand binding to the extracellular domain of the receptor stimulates its cytoplasmic tyrosine protein kinase which phosphorylates its own beta subunit as well as exogenous substrates. It is believed, from several lines of evidence, that tyrosine-specific protein kinases are mediating some or all of the actions of insulin (or IGF-I). In order to gain insights into the substrate specificity of the structurally related insulin and IGF-I receptor kinases, we have studied the action of highly purified receptors isolated from human placental membranes. Present studies using selected tyrosine-containing polymers have revealed: (i) Polymers such as (Y,A,E)n and (Y-A-E)n inhibit beta subunit autophosphorylation and exogenous substrate phosphorylation by autophosphorylated receptors. (ii) Insulin receptor kinase is at least 10 times more sensitive to these inhibitors than IGF-I receptor kinase. (iii) (Y-A-E)n is approximately 8 times more potent an inhibitor than (Y,A,E)n toward both receptors. (iv) While (E4,Y1)n and (E6,A3,Y1)n are good substrates for both receptor kinases, the ratio of phosphate incorporation into the former to the latter is characteristically high (approximately 4) for the IGF-I receptor and low (approximately 1) for the insulin receptor. These results imply that the substrate specificity and enzymatic action of these two receptor kinases are distinct.     

Specific intracellular hyaluronic acid binding to isolated rat hepatocytes is membrane-associated

Intact isolated rat hepatocytes show a small amount of specific 125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of 125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for 125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the 125I-HA binding activity could be pelleted by centrifugation at 100,000 x g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release 125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for 125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the 125I-HA binding activity, since (1) purified rat fibrinogen did not bind to 125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the 125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.     

Studies on the role of transferrin and endocytosis in the uptake of Fe3+ from Fe-nitrilotriacetate by mouse duodenum

Addition of iron-binding proteins (human serum transferrin, mouse serum transferrin, human lactoferrin) to the luminal fluid in tied-off segments of mouse intestine in vivo led to reduced 59Fe3+ absorption from 59Fe3+-nitrilotriacetate when compared to 59Fe3+-nitrilotriacetate alone. Assay of transferrin in luminal fluid from tied segments revealed only trace amounts of immunoreactivity. The levels of luminal transferrin are unaltered in chronic hypoxia where iron absorption is significantly enhanced. Studies in vitro revealed that NH4Cl, dansylcadavarine, para-chloromercuribenzoate and trinitrobenzenesulphonate have no effect on initial 59Fe3+ uptake rates from 59Fe3+-nitrilotriacetate, while N-ethylmaleimide (1 mM) caused a 40% inhibition. In vivo 59Fe3+ uptake was unaffected by preincubation of tied-off segments with colchicine (5 mM) for up to 2 h. These results suggest that receptor-mediated endocytosis of transferrin is not a significant mechanism in the uptake of luminal Fe3+ by mouse duodenum.