Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.     

The thermodynamics of bovine and porcine insulin and proinsulin association determined by concentration difference spectroscopy

Difference spectroscopy was used to determine the equilibrium constants and thermodynamic parameters for the monomer-dimer association of bovine and porcine insulin and bovine proinsulin at pH 2.0 and 7.0. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -6.6 kcal/mol, -18 cal/mol-deg, and -12 kcal/mol, respectively. Porcine insulin behaved similarly to bovine insulin in its dimerization properties in that delta G degree 25, delta S degree, and delta H degree were found to be -6.8 kcal/mol, -14 cal/mol-deg, and -11 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -7.2 kcal/mol, -16 cal/mol/deg, and -12 kcal/mol, respectively. At pH 7.0 delta G degree 25, delta S degree, and delta H degree for dimerization of porcine insulin were -6.7 kcal/mol, -11.6 cal/mol-deg, and -10 kcal/mol, respectively. The similarity in the thermodynamic parameters of both insulin species at the different pH's suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range. The dimerization of both insulin species is under enthalpic control. This may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine proinsulin were found to be -5.3 kcal/mol, -26 cal/mol-deg, and -13 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of proinsulin were -5.9 kcal/mol, -4.2 cal/mol-deg, and -7.2 kcal/mol, respectively. Although the presence of the C-peptide on proinsulin does not drastically affect the overall free energy change of dimer formation (as compared to insulin), the other thermodynamic parameters are rather drastically altered. This may be because of electrostatic interactions of groups on the C-peptide with groups on the B-chain which are near the subunit contact site in the insulin dimer.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

Evidence for two distinct intracellular pools of inorganic sulfate in Penicillium notatum.

A strain of Penicillium notatum unable to metabolize inorganic sulfate can accumulate sulfate internally to an apparent equilibrium concentration 10(5) greater than that remaining in the medium. The apparent Keq is near constant at all initial external sulfate concentrations below that which would eventually exceed the internal capacity of the cells. Under equilibrium conditions of zero net flux, external 35SO42- exchanges with internal, unlabeled SO42- at a rate consistent with the kinetic constants with the sulfate transport system. Efflux experiments demonstrated that sulfate occupies two distinct intracellular pools. Pool 1 is characterized by the rapid release of 35SO42- when the suspension of preloaded cells is adjusted to 10 mM azide at pH 8.4 (t 1/2, 0.38 min). 35SO42- in pool 1 also rapidly exchanges with unlabeled medium sulfate. Pool 2 is characterized by the slow release of 35SO42- induced by azide at pH 8.4 or unlabeled sulfate (t 1/2, 32 to 49 min). Early in the 35SO42- accumulation process, up to 78% of the total transported substrate is found in pool 1. At equilibrium, pool 1 accounts for only about 2% of the total accumulated 35SO42-. The kinetics of 35SO42- accumulation is consistent with the following sequential process: medium----pool 1----pool 2. Monensin (33 microns) accelerates the transfer of 35SO42- from pool 1 to pool 2. Valinomycin (0.2 microM) and tetraphenylboron- (1 mM) retard the transfer of 35SO42- from pool 1 to pool 2. At the concentrations used, neither of the ionophores nor tetraphenylboron- affect total 35SO42- uptake. Pool 2 may reside in a vacuole or other intracellular organelle. A model for the transfer of sulfate from pool 1 to pool 2 is presented.     

Histologic assessment of nerve regeneration in the rat

This study reports the degree of spontaneous regeneration that will occur in the sciatic nerve of a rat 5 months after complete resection of the nerve. In 30 animals, the sciatic nerve was excised. Histological assessment at 5 months revealed evidence of regeneration for a variable distance (mean 23.7 mm +/- 6.4 mm). Histological sections were studied at 1-cm intervals along the length of the nerve. Evidence of compartmentation with "minifascicle" formation was noted. The orientation of the nerve fibers was parallel to the long axis of the nerve. This study assessing spontaneous regeneration is meant to serve as a control for other studies evaluating the effect of factors that may influence nerve regeneration in the rat model.     

Which component of sulphasalazine is active in rheumatoid arthritis?

Sulphasalazine is known to be effective as a second line agent in the treatment of rheumatoid arthritis. The two chemical constituents of sulphasalazine (sulphapyridine and 5-aminosalicylic acid) were assessed separately in the treatment of rheumatoid arthritis. Over 24 weeks sulphapyridine showed a pronounced second line effect comparable with sulphasalazine and with a similar toxicity profile, whereas 5-aminosalicylic acid showed only a weak first line effect. Thus sulphapyridine appears to be the active moiety responsible for the second line effect of sulphasalazine in rheumatoid arthritis. The efficacy of the antibacterial component of sulphasalazine yet again permits speculation about the role of a bacterial pathogen in the aetiopathogenesis of rheumatoid disease.     

C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity

The Ca2+- and phospholipid-dependent protein kinase (C-kinase) binds tightly in the presence of Ca2+ to purified membranes of A431 human epidermoid carcinoma cells. The major membrane substrate for C-kinase is the epidermal growth factor (EGF) receptor. Phosphorylation of the EGF receptor is Ca2+-dependent and occurs at threonine and serine residues. After tryptic digestion of the receptor, three major phosphothreonine-containing peptides were identified. These are identical with three new phosphopeptides present in the EGF receptor isolated from A431 cells treated with either of the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. C-kinase catalyzes phosphorylation at these same sites in purified EGF receptor protein. These results indicate that, in A431 cells exposed to tumor promoters, C-kinase catalyzes phosphorylation of a significant population of EGF receptor molecules. This phosphorylation of EGF receptors results in decreased self-phosphorylation of the EGF receptor at tyrosine residues both in vivo and in vitro and in decreased EGF-stimulated tyrosine kinase activity in vivo.     

The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats

We have isolated and sequenced a full-length cDNA clone for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain). This sequence predicts a 339 amino acid (Mr 38,493) protein containing an N-terminal region of 20 amino acids, known to interact with a 10 kd protein (light chain), and a C-terminal region, found to contain two Ca2+/phospholipid-binding sites, that can be aligned as four 70 amino acid repeats. A single p36 gene was detected in the mouse genome, and a major p36 mRNA of 1.6 kb was found to be expressed in different mouse tissues. Unexpectedly, p36 and the phospholipase A2 inhibitor lipocortin I were found to be 50% identical in sequence over the C-terminal 300 residues. The function of p36 and its relation to other proteins are discussed.     

The protein-tyrosine kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo.

p36, a major in vivo substrate of protein-tyrosine kinases, is shown to be phosphorylated at serine 25, a site very close to the major site of tyrosine phosphorylation by pp60v-src, tyrosine 23 (J. R. Glenney, Jr., and B. F. Tack, Proc. Natl. Acad. Sci. USA 82:7884-7888, 1985). We present evidence suggesting that protein kinase C mediates phosphorylation of serine 25.     

Structural characterisation of the bromouracil.guanine base pair mismatch in a Z-DNA fragment.

The deoxyoligonucleotide d(BrU-G-C-G-C-G) was crystallised at pH 8.2 and its structure analysed by X-ray diffraction. The unit cell, of dimensions a = 17.94, b = 30.85, c = 49.94A contains four DNA duplexes in space group P2(1)2(1)2(1). The duplexes are in the Z conformation, with four Watson-Crick G.C base pairs and two BrU.G base pairs. The structure was refined to an R factor of 0.16 at a resolution of 2.2A with 64 solvent molecules located. The BrU.G base pair mismatch is of the wobble type, with both bases in the major tautomer form and hydrogen bonds linking 0-2 of BrU with N-1 of G and N3 of BrU with 0-6 of G. There is no indication of the presence of ionised base pairs, in spite of the high pH of crystallisation. The results are discussed in terms of the mutagenic properties of 5- bromouracil.