Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit

Purified acetylcholine receptor is rapidly and specifically phosphorylated by partially purified protein kinase C, the Ca2+/phospholipid-dependent enzyme. The receptor delta subunit is the major target for phosphorylation and is phosphorylated on serine residues to a final stoichiometry of 0.4 mol of phosphate/mol of subunit. Phosphorylation is dose-dependent with a Km value of 0.2 microM. Proteolytic digestion of the delta subunit phosphorylated by either protein kinase C or the cAMP-dependent protein kinase yielded a similar pattern of phosphorylated fragments. The amino acids phosphorylated by either kinase co-localized within a 15-kDa proteolytic fragment of the delta subunit. This fragment was visualized by immunoblotting with antibodies against a synthetic peptide corresponding to residues 354-367 of the receptor delta subunit. This sequence, which contains 3 consecutive serine residues, was recently shown to include the cAMP-dependent protein kinase phosphorylation site (Souroujon, M. C., Neumann, D., Pizzighella, S., Fridkin, M., and Fuchs, S. (1986) EMBO J. 5, 543-546). Concomitantly, the synthetic peptide 354-367 was specifically phosphorylated in a Ca2+- and phospholipid-dependent manner by protein kinase C. Furthermore, antibodies directed against this peptide inhibited phosphorylation of the intact receptor by protein kinase C. We thus conclude that both the cAMP-dependent protein kinase and protein kinase C phosphorylation sites reside in very close proximity within the 3 adjacent serine residues at positions 360, 361, and 362 of the delta subunit of the acetylcholine receptor.     

Role of peripheral and central opioid activity in analgesia induced by restraint stress

Rats subjected to prolonged restraint showed an increase in tail flick latency which outlasted the period of restraint by 15 min. This restraint could be blocked but not reversed by 1 mg/kg of naltrexone hydrochloride given subcutaneously. Naltrexone methobromide, administered subcutaneously in doses of 10 or 25 mg/kg, did not block the analgesia indicating that peripheral opioid receptors were probably not involved. Naltrexone hydrochloride was shown to have no effect on brain tryptophan uptake in restrained rats, a neurochemical event which had previously been shown to be critical to restraint-induced analgesia.     

Molecular cloning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain B13.

The genes specifying the utilization of 3-chlorobenzoate by Pseudomonas sp. strain B13 WR1 have been cloned by using a broad-host-range cosmid cloning system. Analysis of the catabolic products of the enzymatic reactions encoded by two hybrid cosmids, pMW65 and pMW90, by thin-layer and high-performance liquid chromatography demonstrated that both encoded the genes for the complete catabolism of 3-chlorobenzoate. Physical analysis of one of the cosmid derivatives, pMW65, by restriction endonuclease mapping and subcloning demonstrated that the pathway genes are encoded on a fragment no larger than 11 kilobases.     

Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization

When human epidermal cells were seeded on floating rafts of collagen and fibroblasts, they stratified at the air-liquid interface. The suprabasal cells synthesized the large type II (K1) and type I (K10/K11) keratins characteristic of terminal differentiation in skin. At earlier times in culture, expression of the large type II keratins appeared to precede the expression of their type I partners. At later times, all suprabasal cells expressed both types, suggesting that the accumulation of a critical level of K1 keratin may be a necessary stimulus for K10 and K11 expression. Expression of the terminal differentiation-specific keratins was completely suppressed by adding retinoic acid to the culture medium, or by submerging the cultures in normal medium. In submerged cultures, removal of vitamin A by delipidization of the serum restored the keratinization process. In contrast, calcium and transforming growth factor-beta did not influence the expression of the large keratins in keratinocytes grown in the presence of retinoids, even though they are known to induce certain morphological features of terminal differentiation. Retinoic acid in the raft medium not only suppressed the expression of the large keratins, but, in addition, induced the synthesis of two new keratins not normally expressed in epidermis in vivo. Immunofluorescence localized one of these keratins, K19, to a few isolated cells of the stratifying culture. In contrast, the other keratin, K13, appeared uniformly in a few outer layers of the culture. Interestingly, K13 expression correlated well with the gradient of retinoid-mediated disruptions of intercellular interactions in the culture. These data suggest that K13 induction may in some way relate to the reduction in either the number or the strength of desmosomal contacts between suprabasal cells of stratified squamous epithelial tissues.     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

Summary ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102–126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.     

Species specificity of anti-acetylcholine receptor antibodies elicited by synthetic peptides

Two peptides corresponding to amino acid residues 351-368 of the alpha-subunits of Torpedo and human acetylcholine receptor (AChR) were synthesized. These peptides contain a segment (residues 355-364) which displays the greatest variability in amino acid sequence between the two species. Antibodies elicited against the two peptides cross-reacted with the respective native AChRs and were shown to be species specific by radioimmunoassay, immunoblotting, and immunofluorescence microscopy. Thus, antibodies against the Torpedo peptide cross-reacted with Torpedo AChR but did not bind to mammalian or chicken AChR. Antibodies against the human peptide proved to be specific probes for mammalian muscle AChR. They cross-reacted with mammalian AChR (human, calf, mouse, and rat) but not with Torpedo or chicken AChR. These antibodies were also shown to react preferentially with the extrajunctional form of muscle AChR, as compared to their reactivity with junctional muscle AChR. In immunofluorescence experiments, the anti-human peptide antibody stained AChR aggregates in sectioned or ethanol-permeabilized rat and mouse myotubes grown in culture but did not stain living myotubes. This indicates that the sequence 351-368 of the alpha-subunit of mammalian AChR is on the cytoplasmic face of muscle cell membranes, as predicted theoretically.     

Activation of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate

Despite their physicochemical and mechanistic differences platelet activating factor (or acetylglycerylether phosphorylcholine; AGEPC) and thrombin, both platelet stimulatory agents, induce phosphoinositide turnover in platelets. We therefore investigated the stimulation of the phosphoinositide phosphodiesterase by these agents and questioned whether they evoked hydrolysis of the same or different pools of phosphoinositides. [3H]Inositol-labelled rabbit platelets were challenged with thrombin and/or AGEPC under a variety of protocols, and the phospholipase C mediated production of radioactive inositol monophosphate (IP); inositol bisphosphate (IP2) and inositol trisphosphate (IP3) was used as the parameter. AGEPC (1 X 10(-9) M) caused a transient maximum (5 to 6-fold) increase in [3H]IP3 at 5 s followed by a decrease. Thrombin (2 U/ml) elicited an increase in [3H]IP3 at a much slower rate than AGEPC; 2 fold at 5 s, 5 fold at 30 s and a maximum 6 to 8-fold at 2-5 min. Compared to AGEPC, thrombin stimulated generation of [3H]IP2 and [3H]IP were severalfold higher. When thrombin and AGEPC were added together to platelets there was no evidence for an additive increase in inositol polyphosphate levels except at earlier time points where increases were submaximal. When AGEPC was added at various time intervals after thrombin pretreatment, no additional increases in [3H]IP3 were observed over that maximally seen with thrombin or AGEPC alone. In another set of experiments, submaximal increases (about 1/4 and 1/2 of maximum) in [3H]IP3 were achieved by using selected concentrations of thrombin (0.1 U and 0.3 U, respectively) and then AGEPC (1 X 10(-9) M) was added for 5 s. Once again the increase in [3H]IP3 was close to the maximal level seen with thrombin or AGEPC individually. It is concluded that thrombin and AGEPC differentially activated phosphoinositide phosphodiesterase (phospholipase C) in rabbit platelets and that the stimulation of the phospholipase C by these two stimuli causes IP3 production via hydrolysis of a common pool of phosphatidylinositol 4,5-bisphosphate.