Alcohol potentiation of isoproterenol-stimulated cyclic AMP accumulation in rat parotid

The ability of beta-adrenergic agonists to elevate rat parotid cyclic AMP concentrations is potentiated greatly by certain organic solvents. Propanol was found to be more effective than other tested solvents. Propanol stimulated adenylate cyclase and inhibited low Km cyclic AMP phosphodiesterase activities however the magnitude of effect upon these enzyme activities probably does not account for the potentiation of cyclic AMP accumulation observed in intact cells.     

Calcium dependence of beta-adrenoceptor mediated cyclic AMP accumulation in human lymphocytes

In intact human lymphocytes, cyclic AMP accumulation in response to isoproterenol was inhibited by 5 mM EDTA, by deletion of calcium ions from the medium and by 1 mM lanthanum chloride, but not by 1 microM verapamil or by 10 microM nifedipine. A23187 caused a modest increase in cyclic AMP content. Exposure of lymphocytes to 5 microM 1-isoproterenol desensitized the cells to subsequent beta-adrenergic stimulation, reducing cyclic AMP accumulation. With higher concentrations of 1-isoproterenol (50 microM), receptor density was reduced as well. None of the above agents attenuated losses in agonist-stimulated cyclic AMP accumulation induced by treatment with 5 microM isoproterenol for 90 min. These data suggest that calcium ions, both those present in the extracellular medium and those bound to the plasma membrane, are required for isoproterenol-stimulation of adenylate cyclase. In addition, it appears that neither the presence of extracellular calcium ions nor full activation of adenylate cyclase are required for desensitization.     

Somatostatin inhibits VIP- and isoproterenol-stimulated cyclic AMP accumulation in rat prostatic epithelial cells

The dual regulation of cyclic AMP accumulation was studied in rat prostatic epithelial cells incubated with somatostatin, vasoactive intestinal peptide (VIP), and the beta-adrenergic agent isoproterenol. Somatostatin noncompetitively inhibited the stimulatory effect of VIP and isoproterenol, but it did not alter basal cyclic AMP levels. In addition to the multifactorial regulation of the cyclic AMP system in rat prostatic epithelium, these results suggest that somatostatin may play a physiological role at this level.     

Cyclic AMP does not alter taurine accumulation by rat renal brush border membrane vesicles

Secondary hyperparathyroidism has been attributed to be responsible for the generalized aminoaciduria and phosphaturia of vitamin D deficiency. Since PTH acts in the kidney to generate cAMP, we explored the possibility that its synthetic analog, dbcAMP, would alter the renal transport of taurine (an amino acid lost in the urine in vitamin D deficiency) and Pi. Exposure of renal BBMV prepared from normal and vitamin D-calcium-deficient rats to dbcAMP at concentrations ranging between 10(-4) and 10(-7) M did not alter taurine uptake by these vesicles. Higher dbcAMP concentrations blunted uptake, but these concentrations reduced intravesicular volume, thus representing an artifact of osmolarity. Preincubation of BBMV with dbcAMP for times between 0 and 60 min at 0 or 25 degrees C also did not alter taurine accumulation. Hypotonic lysis of BBMV, allowing entry of the cyclic nucleotide, followed by isotonic resealing did not influence taurine uptake. The addition of potassium fluoride (to inhibit phosphodiesterase activity) and ATP (as an energy source) did not alter taurine accumulation at 60 sec. The uptake of Pi, which is influenced by PTH, was decreased by 25% following exposure to dbcAMP on the internal surface of the vesicle. These data indicate that the taurinuria observed in vitamin D deficiency is unlikely to be related to a PTH-induced increase in intracellular cAMP, unlike the changes in Pi transport, which is sensitive to cyclic nucleotides.     

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. LYmphocytes from healthy subjects were incubated with isoproterenol and phosphodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.     

Hypoxanthine effects on cyclic AMP levels in human lymphocytes

We have measured hypoxanthine effect on cAMP levels in PBL in basal conditions (no agonist), and with the addition of 2-(p-[2-carboxyethyl] phenylethylamino)-5'-N-ethylcarboxamidoadenosine (CGS-21680, a specific A2 receptor agonist). We have found that hypoxanthine, at 25 microM and 50 microM concentrations, increases cAMP levels in PBL in basal and A2 agonist stimulated conditions.     

The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes

The effect of diamide (diazene dicarboxylic acid bis[N,N'-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 . 10(-3)-1 . 10(-4) M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 . 10(-4) M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 . 10(-4)-1 . 10(-5) M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 . 10(-6) M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3--2.5 mM for cyclic AMP and 3.3--10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.     

Chronic hormone replacement therapy does not alter resting or maximal skin blood flow

Postmenopausal women on estrogen replacementtherapy (ERT) regulate body core temperature at a lower baseline levelat rest in a thermoneutral environment. We conducted a series ofstudies to test whether, in a thermoneutral environment, chronic (2yr) oral ERT significantly alters baseline skin blood flow (SkBF) andcutaneous vascular conductance (CVC) and whether ERT alters maximal CVC(CVC_max) and SkBF inpostmenopausal women. In the first set of studies, forearm blood flow(FBF) was measured by venous-occlusion plethysmography in 24 postmenopausal women: 8 not taking exogenous hormone therapy (No HRTgroup), 8 on ERT, and 8 receiving combination of estrogen andprogesterone therapy, at rest and during prolonged (1 h) local heatingof the forearm at 42°C. Mean arterial pressure (MAP) was measuredby brachial auscultation before each set of FBF measurements tocalculate forearm vascular conductance (FVC = FBF/MAP). SkBF wasmeasured by laser-Doppler flowmetry (LDF), and CVC was calculated asLDF/MAP and standardized as%CVC_max. Baseline FVC,%CVC_max, and maximal FVC were notsignificantly different among the three groups of women. In the secondset of experiments, LDF in ERT and No HRT groups was measured at restin both thermoneutral and warm environments. %CVC_max was again notsignificantly different between ERT and No HRT groups at thermoneutralambient temperatures and increased similarly in the warm environment.Therefore, chronic exogenous ERT does not appear to influence eitherbaseline or maximal SkBF.

     

Native C3 does not bind to the C3b receptor (CR1) of human blood B lymphocytes or alter immunoglobulin synthesis

Complement receptors on lymphocytes were first described more than 12 yr ago (1-3) and have come to be used as a common marker for the identification of B cells (4). The function of these receptors on the lymphocyte and their possible role in induction and/or regulation of the immune response remain unclear. In particular, there continues to be controversy as to whether native C3 can bind to the C3b receptor of these cells without cleavage to C3b (5-10). The resolution of this question is critical in order to clarify the expected state of availability of the receptor in vivo, because in plasma, the C3 concentration is relatively high (1.1 to 1.5 mg/ml), whereas there is little or no circulating C3b due to efficient degradation by factor H and the C3-inactivator (11). With the recent development of an improved method for the isolation of C3 from human plasma, it has been possible to obtain biochemically and functionally pure C3 that has not undergone structural or conformational alteration during processing and fully retains the specific hemolytic activity of C3 in fresh serum (12). Berger et al. (13) were able to demonstrate that C3 prepared in this way failed to bind to the C3b receptor of human polymorphonuclear leukocytes or erythrocytes. Similar observations were made by Schreiber et al. (14), also with phagocytic cells and erythrocytes, and by Dixit et al. (15) with an isolated membrane receptor preparation from rabbit macrophages. In the present communication, we extend these observations to human peripheral blood B lymphocytes. Purified C3 in its native state fails to block B lymphocyte-EA (IgM) C4b3b rosettes, whereas C3b causes 50% inhibition at 5 to 6 micrograms/ml. Furthermore, C3 failed to alter polyclonal immunoglobulin (Ig) production by human B cells, whereas C3b inhibited this B cell function. These data suggest that native C3 does not bind to the C3b receptors of B lymphocytes, and thus they are not occupied under normal conditions in vivo.     

Neuropeptidergic control of cyclic AMP accumulation in human thyroid cell

Somatostatin (SRIF), cholecystokinin (CCK), gastrin and substance P, as single agents, do not influence baseline cellular cAMP levels in human thyroid cultures. SRIF inhibits TSH-induced cAMP accumulation in human thyroid cell, while CCK, gastrin and substance P do not modify cAMP response to TSH. Vasoactive intestinal peptide (VIP) increases cellular cAMP levels in human thyroid cultures and its effect is additive to increases produced by norepinephrine (NE) and isoproterenol (ISO). Neither SRIF nor the other tested peptides influence adrenergic and VIP-ergic cAMP stimulation.     

Pertussis toxin does not inhibit alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cyclic AMP accumulation in rat pinealocytes

The hypothesis that Gi might be involved in the alpha 1-adrenergic, protein kinase C (PKC)-mediated amplification of beta-adrenergic cyclic AMP stimulation in rat pinealocytes was investigated. Treatment of pinealocytes with a high concentration of pertussis toxin (500 ng/ml, 18 h) almost completely (approximately 95%) inactivated two cell membrane G-proteins (kDa 40.7 and 39.8) judged by back ADP-ribosylation of pinealocyte membrane proteins. However, this treatment failed to inhibit either the beta-adrenergic (isoprenaline, ISO 10(-6) M), alpha 1-plus beta-adrenergic (noradrenaline, NA 10(-5) M) or beta-adrenergic plus 12-O-tetradecanoylphorbol 13-acetate (TPA 10(-7) M) induced stimulation of cyclic AMP or cyclic GMP. These results suggest that alpha 1-adrenergic potentiation of beta-adrenergic stimulation of cyclic AMP and cyclic GMP does not involve a pertussis toxin-sensitive G-protein.     

Studies on the cyclic AMP response to prostaglandin in human lymphocytes

It is generally thought that cyclic AMP acts as the second messenger for prostaglandin E in human lymphocytes. We have recently found that the mitogen-induced proliferation of human lymphocytes is no longer inhibited by PGE₂ if the lymphocytes are preincubated overnight prior to the addition of mitogens and PGE₂. In this paper we report that lymphocytes also lose their cyclic AMP response to mitogens after preincubation. The loss of sensitivity to PGE with preincubation can be blocked by cyclohexamide (25 μg/ml). Indomethacin (1 μg/ml) partially blocked the loss of sensitivity, but removal of the glass-adherent cells did not. Since either manipulation effectively stops prostaglandin production in the preincubation cultures, it would appear that indomethacin prevented the loss of sensitivity to PGE₂ by a mechanism other than inhibition of PG synthetase. The addition of phytohemagglutinin to the preincubation cultures also blocked the loss of sensitivity to PGE₂.     

Light level does not alter ethylene sensitivity in radish or pea

Ethylene accumulation occurs in many plant growth environments. In some instances, low photosynthetic photon flux (PPF) is also a stress factor. Ethylene helps regulate the shade-avoidance mechanism and synthesis rates can be altered by light. We thus hypothesized that ethylene sensitivity in whole plants may be altered in low light. Radish (Raphanus sativus) and pea (Pisum sativum) plants were selected as models due to their rapid growth, use in previous studies and difference in growth habit. We first characterized radish and pea sensitivity to ethylene. Radish vegetation was less sensitive to ethylene than pea vegetation. Pea reproductive yield was highly sensitive. Plants grown under low light levels are typically etiolated and less robust than plants grown under higher light. In a second series of studies we examined the interaction of ethylene (50 ppb pea, 200 ppb radish) with PPFs from 50 to 400 μmol m^?2 s^?1. There was no statistically significant interaction between ethylene sensitivity and PPF, indicating that high PPF does not mitigate the detrimental effects of chronic low-level ethylene exposure. This also suggests there is no crosstalk between the shade avoidance pathway and the primary ethylene signaling pathway.     

Agonist- and mitogen-induced desensitization of isoproterenol-stimulated cyclic AMP formation in mouse epidermis in vivo

Injection of the beta-adrenergic agonist isoproterenol causes a rapid desensitization of cyclic AMP formation to subsequent beta-adrenergic challenge in mouse epidermis. Long-lasting catecholamine refractoriness is also observed after prolonged treatment of mouse skin with certain mitogens such as the phorbol ester TPA (tumor promoter), 12-retinoylphorbol-acetate, the TPA-analogue C14:4phorbol acetate or the divalent cation ionophore A 23187 but not after mitogenic stimulation by phorbol-12,13-dibenzoate and 4-O-methyl-TPA or by means of chemical depilation, removal of the horny layer or skin massage. Thus no clear-cut correlation exists between the desensitizing efficacy of a given treatment and its ability to provoke epidermal hyperplasia and to promote skin tumor formation. Both, agonist- and mitogen-induced desensitization are dependent on protein synthesis in epidermis, the mitogen-induced effect is in addition dependent on RNA synthesis. The putative desensitizing protein is not cyclic AMP phosphodiesterase but could be a feedback inhibitor of receptor-cyclase interaction ("refractoriness protein") which has recently been proposed to be responsible for catecholamine tachyphylaxis in certain in vitro systems. In contrast to epidermal hyperproliferation mitogen-induced tachyphylaxis is not mediated by prostaglandin synthesis and is apparently also independent of initial cyclic AMP formation. It can be prevented, however, by the antimitotic synthetic corticoid fluocinolone acetonide but not by colchicine, vincristine cytochalasin B or adrenergic blockers.     

Hypertonic aerosol inhalation does not alter central airway blood flow in dogs

Tracheobronchial blood flow in dogs increases with cold or dry air hyperventilation, possibly as a result of airway drying leading to increased osmolarity of airway surface fluid. This study was designed to examine whether administration of aerosols of various tonicity to alter airway surface fluid osmolarity would induce similar blood flow changes. Tracheobronchial blood flow was measured by the radioactive microsphere technique in six anesthetized dogs ventilated with warm humid air (100% relative humidity) for 15 min (period 1), air containing ultrasonically nebulized saline aerosol (1,711 mosmol/kg) for 3 min (period 2) and 12 min (period 3), and the same aerosol at a higher nebulizer output for a further 3 min (period 4). Between periods 3 and 4, the dogs were ventilated with warm humid air for 30 min to reestablish base-line conditions. In another five dogs, measurements were made after 30 min of ventilation with 1) warm humid air, 2) isotonic saline aerosol, 3) warm humid air, 4) distilled water aerosol (3 dogs), and hypertonic saline aerosol (2 dogs). After the last measurement was made, each dog was killed, the trachea and major bronchi were excised, and blood flow was calculated. No change in blood flow was found during any period of aerosol inhalation. The osmolar load imposed on the airways was estimated and was similar to that occurring during cold or dry air hyperventilation. These data suggest that increasing osmolarity of airway surface fluid does not explain the blood flow changes seen during hyperventilation of cold or dry air.     

Using a stick does not necessarily alter judged distances or reachability

Background

It has been reported that participants judge an object to be closer after a stick has been used to touch it than after touching it with the hand. In this study we try to find out why this is so.

Methodology

We showed six participants a cylindrical object on a table. On separate trials (randomly intermixed) participants either estimated verbally how far the object is from their body or they touched a remembered location. Touching was done either with the hand or with a stick (in separate blocks). In three different sessions, participants touched either the object location or the location halfway to the object location. Verbal judgments were given either in centimeters or in terms of whether the object would be reachable with the hand. No differences in verbal distance judgments or touching responses were found between the blocks in which the stick or the hand was used.

Conclusion

Instead of finding out why the judged distance changes when using a tool, we found that using a stick does not necessarily alter judged distances or judgments about the reachability of objects.     

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca_vs) are crucial for Ca_v activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca²⁺/CaM molecules bound to the Ca_v1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca²⁺/CaM bridged two PreIQ helixes to form a 4:2 Ca²⁺/CaM:Ca_v C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca_v1.2 inactivation or on the stoichiometry of full-length Ca_v1.2. These data provide further support for the monomeric Ca_v1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca²⁺/CaM:CaV C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.     

Calmodulin overexpression does not alter Cav1.2 function or oligomerization state

Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.