Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle

1. 1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated by the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle.

2. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascualar (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There was no evidence of phosphate incorporatio into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation.

3. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.

Effect of thyroid status on α- and β-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased β-catecholamine responsiveness in hyperthyroid animals is due to a decrease in α-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the α₂-adrenergic inhibition of adenylate cyclase or the α₁-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamster. No effect of hyperthyroidism was found on the K_d of [³H]dihydroegocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to β-catecholamines was enhanced in adipocytes from hyperthyroid hamster, as was lipolysis. However, in adipocytes from hyperthyroid hamster the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on α-catecholamine action but enhances the activation of lipolysis by β-catecholamine agonists.     

Apparent absence of alpha-2 adrenergic receptors from hamster brown adipocytes

The possible presence of α adrenergic control of lipolysis and cyclic AMP production in brown adipocytes of hamsters was studied in adipocytes isolated from interscapular, subscapular, cervical and axillary regions of normal male hamsters maintained at 25°C. Lipolysis activated by either 3-isobutyl-1-methyl xanthine or isoproterenol was unaffected by the presence of the α adrenergic selective agonists clonidine and methoxamine. Similarly, accumulation of cyclic AMP in response to β-receptor stimulation, alone or in combination with a methyl xanthine, was unaffected by clonidine or methoxamine. In contrast, both lipolysis and cyclic AMP accumulation in brown fat cells were effectively suppressed in the presence of nicotinic acid, prostaglandin E₁ or N⁶-phenylisopropyl adenosine. Accumulation of cyclic AMP in response to the mixed agonist norepinephrine was not influenced when cells were exposed to the alpha adrenergic blocking drugs yohimbine or tolazoline. These observations suggest that alpha-2 adrenergic receptors which are present on hamster white fat cells and control production of cyclic AMP and lipolysis are absent from hamster brown adipocytes. On the other hand, brown fat cells of this species appear to respond to a number of other inhibitory compounds in a manner not markedly different from that of white adipocytes.     

The cyclic AMP receptor of Escherichia coli: Immunological studies in extracts of Escherichia coli and other organisms

An antiserum specific for the cyclic adenosine 3′,5′-monophosphate receptor from Escherichia coli has been employed to detect the presence of a similar protein in cellular extracts of a number of diverse organisms. In Ouchterlony double-diffusion experiments cellular extracts from Photobacterium fisheri, Aerobacter aerogenes, Proteus mirabilis, and Salmonella typhimurium all showed precipitin bands with E. coli cyclic AMP receptor-antiserum. The extract from Caulobacter crescentus exhibited slight cross-reactivity. Similar results were obtained with an immuno-precipitation assay used to quantitate the amount of cyclic AMP receptor-like protein present. Extracts from a variety of organisms were found to bind cyclic AMP when the usual (NH₄)₂SO₄ precipitation assay for cyclic AMP receptor was employed. Only the extract from Methanosarcina barkeri was inactive. Some extracts prepared from E. coli grown on Luria broth were observed to have no cyclic AMP binding activity. Antiserum was used to determine the presence of cyclic AMP receptor in these inactive extracts. These preparations usually regain binding activity on standing at 4°C for 2–3 days.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrrenal cells of vitamin E-deficient rats

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5′-triphosphate [¹⁴C]ATP to cyclic [¹⁴C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [¹⁴C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induce cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [¹⁴C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [¹⁴C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [¹⁴C]AMP continued to be formed. The in vitro addition of α-tocopherol reduced the inhibition of ACTH-induced cyclic [¹⁴C]AMP formation by ascorbate in Vitamin E-deficient rats.These studies suggest that α-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with membrane-bound enzyme adenylate cyclase.     

Evidence of multiple operational affinities for d-glucose inside the human erythrocyte membrane

1. 1. The Michaelis-Menten parameters of labelled d-glucose exit from human erythrocytes at 2°C into external solution containing 50 mM d-galactose were obtained. The K_m is 3.4 ± 0.4 mM, V 17.3 ± 1.4 mmol · 1⁻¹ cell water · min⁻¹ for this infinite-trans exit procedure.

2. 2. The kinetic parameters of equilibrium exchange of d-glucose at 2°C are K_m = 25 ± 3.4 mM, V 30 ± 4.1 mmol · 1⁻¹ cell water · min⁻¹.

3. 3. The K_m for net exit of d-glucose into solutions containing zero sugar is 15.8 ± 1.7 mM, V 9.3 ± 3.3 mol 9.3 ± 3.3 mol · 1⁻¹ cell water · min⁻¹.

4. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126–132] that there are sites with both high and low operational affinities for d-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.     

The effect of hormones on synthesis of the region linking chondroitin sulfate to protein

1. 1. Particulate fractions of costal cartilage from young rats are capable of catalyzing the formation of the first two monosaccharide units of the chondroitin sulfate-protein linkage region.

2. 2. Hormonal imbalance has been shown to influence the activity of the glycosyltransferases responsible for the sequential transfer of xylose and galactose from UDPxylose and UDPgalactose, respectively, in the formation of the linkage region.

3. 3. The activity of xylosyltransferase was found to be decreased in costal cartilage of diabetic, thyroidectomized and hypophysectomized rats, but not in rats injected with either testosterone or hydrocortisone. In the latter two treatment groups, galactosyltransferase activity was decreased only in the group receiving hydrocorsitone.

4. 4. The combined results of this and previous studies suggest that decreased levels of chondroitin sulfate in diabetic, thyroidectomized and hypophysectomized animals are due to interference in the synthesis of the linkage region of the proteoglycan at the xylosyltransferase level whereas hydrocortisone acts primarily at the level of the galactosyltransferase.

The effect of copper on frog skin. The role of sulphydryl groups

1. 1. Cu²⁺ at a concentration of 10⁻⁴ M, when applied to the external side of the frog skin produces an increase in the short-circuit current (I_sc).

2. 2. This effect was studied in skins of Rana temporaria adapted to cold (5°C) and room temperature (20°C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda.

3. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu²⁺ than skins with high I_sc.

4. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high I_sc give good responses to Cu²⁺.

5. 5. 5,5′-dithiobis(2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu²⁺, and dithiothreitol an SH-reducing agent, reverses the effect of this ion.

6. 6. Cu²⁺ also induces an increase in the unidirectional K⁺ fluxes and unmasks a net outward potassium flux.

7. 7. The outward K⁺ flux induced by Cu²⁺ is sensitive to ouabain.

8. 8. It is concluded that Cu²⁺ increases the permeability of the external barrier of the frog skin to Na⁺ and K⁺, probably by reacting with SH groups.