Cellular cAMP Content Research Articles

Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells.

Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg2+ transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg(2+)-depleted cells. Intracellular free Mg2+ concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 +/- 0.01 mM) by culturing in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg2+]i/dt, of 164 +/- 5 nM/s. Both glucagon and AVP stimulated Mg2+ uptake into MDCT cells, 196 +/- 11 and 189 +/- 6 nM/s, respectively, at concentrations of 3 x 10(-7) M and 10(-7) M, respectively. Enhanced Mg2+ uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg2+ entry was not dependent on protein synthesis. 8-Bromo-cAMP, 10(-4) M, enhanced Mg2+ uptake (225 +/- 13 nM/s), whereas phorbol esters were without effect. Finally, protein kinase A inhibition prevented glucagon and AVP stimulation of Mg2+ uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg2+ uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.

A1 adenosine receptors in human neutrophils. Electron microscope localization.

Adenosine is an ubiquitous nucleoside that mediates several important physiological effects, trough the binding of three pharmacologically distinct receptor subtypes, named A1 A2 and A 3 .1 Adenosine receptor subtypes has been distinguished on the basis of the effect of adenosine on the cellular content of cAMP: occupancy of A2 adenosine receptors induces an increase in cAMP level in several cells types, by contrast stimulation of A 1 receptors causes the opposite effect. These effects are mediated by a coupling of these receptor subtypes with Gs and Gi transduction-proteins, respectively.2

Enhancement of the vanadate-stimulated release of lipoprotein lipase activity by astilbin from the leaves of Engelhardtia chrysolepis.

Astilbin, a dihydroflavonol rhamnoside isolated from the leaves of Engelhardtia chrysolepis, enhanced the vanadate-stimulated release of lipoprotein lipase (LPL) activity from rat isolated fat pads. N-[2-(Methyl-amino)ethyl]-5-isoquinolinesulfonamide (H-8), a potent inhibitor of cAMP-dependent protein kinase (PKA), markedly inhibited the enhancement by astilbin. Lipolysis in the fat pads was stimulated by astilbin alone in a dose-dependent manner and this stimulation was suppressed in the presence of vanadate, probably due to its antilipolytic action. A significant enhancement by astilbin was observed with increasing effects of vanadate on cAMP content in the fat pads and on cAMP phosphodiesterase (PDE) activity in the particulate fraction although astilbin alone showed only a slight increase in the cellular cAMP content and PDE activity. Astilbin may enhance the vanadate-stimulated release of LPL activity through a synergistic effect on an increase in the cellular cAMP content produced by vanadate accompanied by more potent activation of PKA.

Ocular hypotensive and vasodilative effects of two β-adrenergic blockers with intrinsic sympathomimetic activity

Purpose. The ocular hypotensive and vasodilative effects of vaninolol and eugenolol, two β-adrenergic blockers with intrinsic sympathomimetic activity, were tested in rabbits and their pharmacologic mechanisms were also studied in vitro.Methods. Intraocular pressure was measured in ocular hyper-tonic rabbits which were induced by infusing 20% NaCl or 5% glucose solution. The rabbit's ocular blood flow was determined using the colored microsphere technique. The concentrations of cAMP were evaluated in porcine ciliary body and cultured A7r5 smooth muscle cells by radioimmunoassay. Ca+2 concentration was measured in A7r5 cells by spectrofluorometry after loading cells with Fura-2-AM.Results. It was found that 0.5% eugenolol and vaninolol could suppress the intraocular pressure in glucose-induced ocular hypertensive rabbits and delay the intraocular pressure recovery in NaCl-induced ocular hypertensive rabbits. In addition, both agents improved the ocular blood flow in the iris, ciliary body, retina and choroid. Vaninolol and eugenolol of 10 μM inhibited the basal cAMP accumulation from 23.9 ± 2.0 of control to 8.7 ± 0.4 and 2.4 ± 0.1 respectively and inhibited the isoproterenol-induced cAMP accumulation from 154.3 ± 13.6 to 120.6 ± 8.3 and 74.2 ± 6.1 respectively in the porcine ciliary body. The cellular cAMP concentration was significantly increased from 10 ± 1 of control to 96 ± 5 (vaninolol) and 38 ± 3 (eugenolol) in cultured A7r5 smooth muscle cells. Both agents also increased the intracellular calcium concentration in A7r5 cells.Conclusions. These results indicate that the lowering of intraocular pressure by vaninolol and eugenolol may be due to cAMP suppression in the ciliary body by β-antagonist and/or α2-agonist activities. Both agents cause vasodilation via β2-agonist action that increase the smooth muscle cellular cAMP level more than vasoconstriction via α-agonist activity by increasing an influx of extracellular Ca+2. Curr. Eye Res. 17: 700–707, 1998.

Ocular hypotensive and vasodilative effects of two β-adrenergic blockers with intrinsic sympathomimetic activity

Purpose. The ocular hypotensive and vasodilative effects of vaninolol and eugenolol, two β-adrenergic blockers with intrinsic sympathomimetic activity, were tested in rabbits and their pharmacologic mechanisms were also studied in vitro.Methods. Intraocular pressure was measured in ocular hyper-tonic rabbits which were induced by infusing 20% NaCl or 5% glucose solution. The rabbit's ocular blood flow was determined using the colored microsphere technique. The concentrations of cAMP were evaluated in porcine ciliary body and cultured A7r5 smooth muscle cells by radioimmunoassay. Ca+2 concentration was measured in A7r5 cells by spectrofluorometry after loading cells with Fura-2-AM.Results. It was found that 0.5% eugenolol and vaninolol could suppress the intraocular pressure in glucose-induced ocular hypertensive rabbits and delay the intraocular pressure recovery in NaCl-induced ocular hypertensive rabbits. In addition, both agents improved the ocular blood flow in the iris, ciliary body, retina and choroid. Vaninolol and eugenolol of 10 μM inhibited the basal cAMP accumulation from 23.9 ± 2.0 of control to 8.7 ± 0.4 and 2.4 ± 0.1 respectively and inhibited the isoproterenol-induced cAMP accumulation from 154.3 ± 13.6 to 120.6 ± 8.3 and 74.2 ± 6.1 respectively in the porcine ciliary body. The cellular cAMP concentration was significantly increased from 10 ± 1 of control to 96 ± 5 (vaninolol) and 38 ± 3 (eugenolol) in cultured A7r5 smooth muscle cells. Both agents also increased the intracellular calcium concentration in A7r5 cells.Conclusions. These results indicate that the lowering of intraocular pressure by vaninolol and eugenolol may be due to cAMP suppression in the ciliary body by β-antagonist and/or α2-agonist activities. Both agents cause vasodilation via β2-agonist action that increase the smooth muscle cellular cAMP level more than vasoconstriction via α-agonist activity by increasing an influx of extracellular Ca+2. Curr. Eye Res. 17: 700–707, 1998.

Alteration of glucose uptake in cultured human corneal endothelial cells grown in high glucose media via cAMP-dependent pathway.

In this study, cultured human corneal endothelial cells were incubated in media containing various concentrations of glucose at 5 mM, 10 mM, and 25 mM for 2 days. Then, the cellular 2-deoxyglucose uptake and cAMP concentration of cultured human corneal endothelial cells were measured. The results indicated that the activity of cellular glucose uptake of nmole/min/mg protein was decreased gradually from 0.18 (5 mM), 0.10 (10 mM), 0.07 (20 mM) to 0.06 (25 mM) after 2 days incubation with a high concentration of glucose. The glucose uptake in insulin-treated human corneal endothelial cells also exhibited a similar declining effect in high glucose media from 0.30 (5 mM), 0.11 (10 mM), 0.08 (20 mM) to 0.05 (25 mM). The cAMP concentration in human corneal endothelial cells was measured in the presence of high glucose media. It was indicated that the cAMP concentrations of pmole/well in both insulin-treated and non-insulin treated cells were also decreased after increasing the glucose concentration in the media from 73 (5 mM) to 20 (25 mM) and 101 (5 mM) respectively. The cAMP concentration in insulin-treated cells was less than in non-insulin treated cells. This decreasing effect was significantly reversed by the addition of 1 mM dibutyryl-cAMP to the cells for 1 hour in both groups. These results suggest that the diabetic state may decrease the 2-deoxyglucose uptake in human corneal endothelial cells via cAMP-dependent pathway.

Effects of Ethanol on Basal and Prostaglandin E1‐Induced Increases in β‐Endorphin Release and Intracellular cAMP Levels in Hypothalamic Cells

Our recent studies determining the effect of cAMP-elevating agents forskolin and dibutyryl cAMP on ethanol-induced immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic cells in primary cultures suggested the possibility that both stimulatory and adaptive secretory responses of beta-EP neurons after ethanol exposure may involve the cAMP system. To determine further the role of cAMP, the effects of prostaglandin E1 (PGE1) on basal and ethanol-regulated IR-beta-EP secretion and cAMP productions were determined in primary cultures of hypothalamic cells. The results presented in this study show that a 50 mM dose of ethanol, which is within the EC50 dose of ethanol required to elevate IR-beta-EP release from hypothalamic cells, increased cellular levels of cAMP and elevated IR-beta-EP release simultaneously from the cultured neurons for a period of 6 hr. The cAMP and IR-beta-EP secretory responses developed desensitization to ethanol challenge after 24 hr of constant ethanol incubation. The cAMP-elevating agent PGE1 increased the cellular content of cAMP and IR-beta-EP release in a dose-dependent manner. The EC50 dose of PGE1 for elevation of IR-beta-EP and cAMP was approximately 0.5 microM. As with ethanol, chronic treatment with PGE1 desensitized the cAMP and IR-beta-EP responses of hypothalamic neurons to PGE1. Acute exposure to ethanol increased the PGE1-stimulated levels of cAMP and IR-beta-EP, whereas chronic exposure to ethanol resulted in diminished cAMP responses to PGE1. These data provide evidence that the cAMP system may be involved in controlling hypothalamic beta-EP secretion, as well in regulating the stimulatory and adaptive responses of beta-EP neurons to ethanol.

Stimulation of cyclic AMP, 17beta-oestradiol and protein synthesis by human chorionic gonadotrophin in human endometrial cells.

Human endometrial tissue was collected from 30-37 year old ovulatory women and enzymatically dispersed and processed to obtain endometrial stromal cells. Cells (2 x 10(4)/well) were incubated in vitro in the absence (control) or presence of human chorionic gonadotrophin (HCG) (10 IU/well) either for 1 h (to determine cAMP) or for 4 h (to estimate protein synthesis, 17beta-oestradiol and aromatase activity). HCG significantly stimulated cellular cAMP content and release in comparison to control. Both cellular accumulation and release of cAMP reached the peak at 10 min and then sharply declined in the absence of phosphodiesterase (PDE) inhibitor or remained steady for 1 h in the presence of PDE inhibitor. Content and release of 17beta-oestradiol from stromal cells steeply increased from 30 min to 1 h and then slowly increased for 4 h in response to HCG. Incubation of cells with HCG significantly stimulated (P < 0.01) cellular aromatase activity in comparison to control. HCG stimulated protein synthesis of the cells in two phases, initially at 15 min which remained steady for 1 h and then significantly increased (P < 0.01) between 1 and 2 h. The initial phase of protein synthesis appears to be the direct effect of HCG while the later phase was due to the higher concentration of oestradiol caused by HCG. The initial phase of protein synthesis was inhibited by cycloheximide but not by actinomycin D, whereas the later phase of protein synthesis was inhibited both by cycloheximide and actinomycin D. Results therefore indicate a probable mechanism of HCG action on stromal cells and demonstrate the physiological relevance of gonadotrophin receptor in human endometrial cells reported earlier from this laboratory.

Modulation of epinephrine-stimulated gluconeogenesis by insulin in hepatocytes isolated from genetically obese (fa/fa) Zucker rats.

Genetically obese (fa/fa) Zucker rats present an impaired response of hepatic glucose production to the inhibition by insulin. In this work, we have investigated the modulation by this hormone of epinephrine-stimulated gluconeogenesis, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine (1 microM) caused a maximal stimulation of [14C]lactate conversion to [14C]glucose in hepatocytes isolated from either obese or lean animals. The stimulation of gluconeogenesis by epinephrine was accompanied by a significant reduction of fructose 2,6-bisphosphate levels, an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, and by a 2-fold increase in the cellular concentrations of cAMP. The presence of insulin in the incubation medium antagonized, in a concentration-dependent manner, the effects of epinephrine. In hepatocytes isolated from lean rats, the reversion caused by insulin was complete, the concentration required for half-maximal insulin action ranging from 0.22 to 0.56 nM. In contrast, in obese rat hepatocytes, insulin only partially blocked epinephrine-mediated effects, and the sensitivity to insulin was 2- to 4-fold lower, as indicated by the corresponding half-maximal insulin action values. Furthermore, insulin (10 nM) almost completely blocked the increase in cAMP levels induced by epinephrine in lean rat hepatocytes, whereas it only provoked a small and nonsignificant reduction of epinephrine-stimulated levels of the cyclic nucleotide in hepatocytes obtained from obese rats.

Endothelial permeability for macromolecules. Mechanistic aspects of pathophysiological modulation.

The endothelium separates blood from tissue and actively regulates the exchange between these compartments. Throughout the body, this one-cell-thick lining of blood vessels extends over several thousand square meters. It harbors many receptors and functions that actively regulate the extravasation of nutrients, solutes, hormones, macromolecules, and leukocytes. If the barrier function of the endothelium does not perform appropriately, leakage of macromolecules occurs, causing edema formation and exposure of the interstitium to high concentrations of plasma constituents. In severe cases of vascular leakage, platelets will adhere to the exposed subendothelium and red blood cells may even accumulate in the interstitium. Vascular leakage may be desirable for recruiting plasma proteins, such as complement factors during infection, but it can become life-threatening, particularly when it occurs in the lungs or pericardium or when it causes hypovolemic shock. Furthermore, frequent or prolonged leakage may impair proper functioning of the affected tissue. In recent years, considerable progress has been made in understanding the molecular processes that contribute to the regulation of endothelial permeability. With the exception of the liver, adrenal, and bone marrow sinusoids, in which the endothelium has rather large pores, the endothelium constitutes a selective barrier between blood and tissue. In principle, this barrier consists of the EC monolayer and its matrix. Macromolecules can cross the endothelial barrier in three ways: (1) between the cells, through cell junctions (paracellular); (2) through the EC, via pores (diaphragms or fused vesicles); and (3) transcellularly, via shuttling vesicles and specific receptors. It is generally believed that the charge and compactness of the endothelial matrix (both glycocalyx and basal membrane) contribute additionally to the selectivity of the endothelial barrier toward molecules of different size and charge. Electron microscopic evidence strongly suggests that in tissue capillaries, hormones and macromolecules are shuttled across the endothelial barrier via vesicles, in …

Gastric GATA-6 DNA-binding protein: proteolysis induced by cAMP

The rat gastric GATA DNA-binding protein, GATA-6 (GATA-GT1), was stably expressed in CHO-K1 cells. The GATA-6 protein was localized in the nucleus but not in the cytoplasm. Interestingly, when cells were treated with dibutyryl cAMP, the GATA-6 protein was specifically degraded. Such a phenomenon was not observed in the presence of 5′-AMP or dibutyryl cGMP. The cellular level of the GATA-6 protein was restored upon removal of dibutyryl cAMP. Degradation was also induced by cholera toxin, which increased the cellular cAMP concentration, and was inhibited by a protein kinase A inhibitor. However, activators of protein kinase C did not have any effect. The degradation was inhibited by proteasome inhibitors (PSI (benzyloxycarbonyl-Ile-Glu( O- t-Bu)-Ala-leucinal) and MG115 (benzyloxycarbonyl-Leu-Leu-norvalinal)) but not by those of lysosomes and serine proteases. These results suggest that a kinase-mediated protein phosphorylation is the cellular signal for degradation of the GATA-6 protein. This finding constitutes a novel aspect of regulation by GATA DNA-binding proteins, which are essential for developmental processes and tissue-specific transcription.

Defects of beta 2-adrenergic signal transduction in chronic lymphocytic leukaemia: relationship to disease progression.

The second messenger 3':5'-cyclic adenosine monophosphate (cAMP) inhibits the proliferation of human B lymphocytes. In lymphoid malignancies, cAMP levels or the number of beta 2-adrenergic receptors seem to be decreased. In order to explore this phenomenon further, the function of the beta 2-adrenergic receptor complex was examined in mononuclear leucocytes (MNLs) from patients with B-cell chronic lymphocytic leukaemia (CLL). Peripheral blood MNLs from 25 CLL patients (16 male, nine female: aged 62 +/- 9 years) and 10 healthy volunteers (seven male, three female; aged 47 +/- 19 years) were used. The binding characteristics of beta 2-adrenergic receptors (beta 2-AR) on MNLs were determined by radioligand binding assays with [125I]-cyanopindolol ([125I]-CYP). The number of high-affinity binding sites for [125I]-CYP was significantly lower in CLL patients (313 +/- 300 sites per cell; mean +/- SD) than in control subjects (1479 +/- 1268 sites per cell). Moreover, the density of beta 2-AR decreased with disease progression, from Binet stage A (371 +/- 236, n = 13) to B (236 +/- 136, n = 7) and C (141 +/- 59, n = 5) (P < 0.05; Kruskal-Wallis analysis). Functional analyses of the beta 2-AR complex were performed by measuring the cellular cAMP content of MNLs in response to different stimulators. The cAMP production of MNLs upon isoprenaline stimulation (ISO; 10 min, 10(-4) mol L-1) was slightly lower in CLL patients (12.5 +/- 7.04 pmol 10(-6) cells) than in control subjects (15.91 +/- 10.08 pmol 10(-6) cells), and decreased with CLL progression (stage A 14 +/- 7; stage B 13.66 +/- 3.91; stage C 3.07 +/- 0.79 pmol 10(-6) cells). In contrast, cAMP accumulation in response to cholera toxin (CHO; 10(-4) gml-1, 120 min) was not different in control subjects (70.07 +/- 31.30 pmol 10(-6) cells) and CLL patients (stage A 95.24 +/- 123.07, stage B 70.76 +/- 57.37, stage C 33.21 +/- 33.73 pmol 10(-6) cells). When stimulated with forskolin (100 mumol L-1, 15 min), control MNLs produced about ten-fold more cAMP than CLL MNLs (188.56 +/- 92.53 vs. 17.88 +/- 10.32 pmol 10(-6) cells); this response was not stage dependent. Taken together, the results show that the beta 2-AR transmembrane signalling is impaired in CLL patients. The correlation of some beta 2-AR signalling defects with disease progression suggests that they may contribute to the disease progression of CLL patients.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

von Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10(-6) M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled beta-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]i as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

Prostaglandin E2 inhibits antidiuretic hormone induced transepithelial sodium transport and cAMP production in frog skin epithelium (Rana esculenta).

The stimulation of transepithelial sodium transport by arginine vasotocin (AVT) and the ability of prostaglandin E2 (PGE2) to inhibit the AVT dependent sodium transport was examined on pieces of frog skin taken from different regions of the frog. The basal sodium transport was of the same magnitude in the abdominal, thoracal and upper and lower dorsal region. In all regions the sodium transport could be stimulated by AVT, though the stimulation was highest in the abdomen. Subsequent addition of PGE2 caused a significant inhibition of the AVT dependent sodium transport in the abdomen but had only minor effects on the other parts of the skin. Therefore, pieces of skin from the abdomen were selected for the remaining experiments. Addition of PGE2 to skin pieces in the presence of AVT resulted in a hyperpolarization of the cellular potential and a decrease in the transepithelial sodium transport, indicating a decreased apical sodium permeability. This was correlated with a decrease in cellular cAMP contents. We conclude that frog skin exhibits large regional differences in sensitivity to AVT and that PGE2 only modulates the natriferic effect of AVT in the abdomen.

Vasopressin-stimulated Ca2+ reabsorption in rabbit cortical collecting system: effects on cAMP and cytosolic Ca2+.

The effect of arginine vasopressin (AVP) on transepithelial Ca2+ transport in primary cultures of rabbit cortical collecting system cells was examined. Addition of AVP to the basolateral side of the monolayer dose-dependently (EC50 = 0.7 nM) increased active Ca2+ reabsorption from a basal value of 85 +/- 2 nmol.h-1.cm-2 to a maximum value of 124 +/- 3 nmol.h-1.cm-2. This was paralleled by a dose-dependent (EC50 = 1.1 nM) increase in cellular adenosine 3', 5'-cyclic monophosphate (cAMP) content. Both effects of AVP were mimicked by the V2 agonist deamino-Cys,D-Arg8-vasopressin (dDAVP) and forskolin. Addition of either AVP or dDAVP to the basolateral side evoked a sustained increase in cytosolic free Ca2+ concentration, which resulted from both Ca2+ entry and release from internal stores. Only the effect on Ca2+ entry was mimicked by forskolin, demonstrating that cAMP acts by activating a Ca2+ influx pathway. The present findings demonstrate that AVP stimulates transcellular Ca2+ transport in the cortical collecting system through activation of basolateral V2 receptors coupled to adenylyl cyclase to increase the cellular cAMP content.

Neuropeptide Y reduces macromolecule permeability of coronary endothelial monolayers

The effect of neuropeptide Y (NPY) on cellular adenosine 3',5'-cyclic monophosphate (cAMP) contents and macromolecule permeability was studied in cultured monolayers of microvascular coronary endothelial cells from rat. Macromolecule permeability was continuously determined as passage of albumin across the monolayers. NPY (10(-10)-10(-7) M) decreased albumin flux and cellular cAMP content in a dose-dependent manner, with a half-maximal effect on albumin flux at 1.4 x 10(-9) M and on cAMP contents at 0.7 x 10(-9) M. A maximum effect of NPY was observed at 10(-7) M, decreasing albumin flux by 71 +/- 8% and cellular cAMP contents by 80 +/- 9% (mean +/- SD, n = 6, P < 0.05) compared with control. The effect of NPY on albumin flux was not altered in the presence of 10(-5) M indomethacin (an inhibitor of cyclooxygenase) and 10(-5) M NG-nitro-L-arginine (an inhibitor of nitric oxide synthase). NPY (10(-7) M) also antagonized the increase of albumin flux and cAMP content induced by 10(-6) M isoproterenol. Pretreatment of endothelial monolayers with pertussis toxin (1 microgram/ml for 2 h) abolished the effect of NPY on albumin flux and cAMP contents. This study shows that NPY can modulate macromolecule permeability of endothelial monolayers by reducing the cellular cAMP contents. Together with the effect of pertussis toxin, the data suggest that NPY exerts its antiadrenergic effect on cAMP metabolism and endothelial barrier function by receptors linked to adenylyl cyclase via an inhibitory guanosine-binding protein in coronary endothelial cells.

Adrenergic responses of R. ridibunda red cells.

We studied the effects of beta-adrenergic stimulation on the formation of adenosine 3', 5'-cyclic monophosphate (cAMP) and on the cell volume in Rana ridibunda red blood cells under O2 (high oxygen atmosphere) and under N2 (hypoxia) gases. Adrenergic stimulation caused the accumulation of cAMP and the swelling of R. ridibunda erythrocytes. 10(-5) M adrenaline and 10(-5) M isoproterenol seem to be the more efficient concentration in causing greater cell swelling. The amount of cAMP required for adrenergic swelling was minimal; swelling was observed at cAMP concentrations higher than 20 nM. Maximal swelling was seen when the cellular cAMP concentration reached 40 nM at high oxygen atmosphere. Isoproterenol caused a greater increase in water content than adrenaline. At hypoxic conditions the water content and cAMP concentrations obtained after stimulation with adrenaline and isoproterenol were greater than at high oxygen atmosphere. Since the adrenergic response seems to be blocked both by prazosin and propranolole the adrenoreceptors of R. ridibunda red cells may be classified as alpha 1- and beta-receptors. The enhancement of cAMP and cell swelling may be either due to an increase in the number of adrenoreceptors, or to increased responsiveness of the erythrocyte Na(+)-H+ antiporter to catecholamines during hypoxia. These responses are believed to enhance blood oxygen transport during periods of increased oxygen demands.

Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance

This study describes the establishment of a rat kidney cortical collecting duct (CCD) clonal cell line (RCCD1 cells) that maintains high transepithelial resistance and specific hormonal sensitivities. Immortalized cells were obtained by infection of primary cultured CCD cells with the wild-type simian virus 40. Grown on Petri dishes, RCCD1 cells are organized as monolayers of cuboid cells separated by tight junctions and form domes. Grown on permeable filters, confluent RCCD1 cells exhibit high transepithelial resistance (Rt: 2390 +/- 140 omega. cm2), transepithelial potential difference (PD) of -10.5 +/- 1.2 mV lumen negative, an associated short-circuit current (Isc) of 4.3 +/- 0.5 microA/cm2, and generated significant Na+, K+, H+ and HCO3- gradients, reflecting Na+ and H+ reabsorption and K+ and HCO3- secretion. RCCD1 cells exhibit features of both principal (PC) and intercalated (IC) cells. Consistent with PC phenotype, about 50% of the cells were positively stained by a PC-specific agglutinin. In situ hybridization studies revealed the presence of alpha, beta and gamma subunit mRNAs of the amiloride-sensitive epithelial Na+ channel and alpha 1 and beta 1 subunits of Na(+)-K(+)-ATPase. Moreover, Na(+)-K(+)-ATPase was immunolocalized at the basolateral side of the cells. Arginine vasopressin (AVP) induced a significant increase in both cellular cAMP content and Isc. Amiloride decreased in a dose-dependent manner Isc from untreated and AVP-treated RCCD1 cells. In addition, a barium-sensitive K+ conductance was evidenced in the apical side of the cells. Consistent with IC phenotype, isoproterenol (ISO) provoked a large increase in cellular cAMP and stimulated Isc. The effect of ISO on Isc was blocked by 5 x 10(-3) M DPC, a chloride channel blocker. Finally, AVP plus ISO had additive effect on Isc. Taken together, these results provide evidence that the RCCD1 cell line has maintained many of the original properties of rat CCD from which they were derived.

Signaling mechanisms underlying the insulinotropic effect of pituitary adenylate cyclase-activating polypeptide in HIT-T15 cells.

The signaling mechanisms underlying the insulinotropic effect of the pancreatic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) were studied in insulin-producing HIT-T15 cells. It was found that unlabeled PACAP38 dose-dependently displaced [125I]PACAP27 from HIT-T15 cell membranes, showing binding of the peptide to these cells. Furthermore, at levels above 0.1 nM, PACAP38 stimulated insulin secretion in a Ca2+ -dependent manner, requiring 0.5 mM glucose. The peptide also markedly increased the cellular cAMP content and slightly stimulated the formation of inositol phosphates. Moreover, PACAP38 elevated the cytoplasmic Ca2+ concentration ([Ca2+]cyt) in fura-2-AM-loaded cell suspensions. The PACAP38-induced increase in cellular cAMP content was also seen at zero glucose, whereas the increases in insulin secretion and [Ca2+]cyt were abolished by removal of glucose or extracellular Ca2+. We conclude that PACAP38 binds to HIT-T15 cell membranes and acts in a glucose-dependent manner on adenylate cyclase to form cAMP, which both directly and indirectly, through increased [Ca2+]cyt, stimulates exocytosis.

15-lipoxygenase metabolite(s) of arachidonic acid mediates adrenocorticotropin action in bovine adrenal steroidogenesis.

The acute activation of adrenal glucocorticoid synthesis by ACTH has long been believed to be mediated by cAMP as the major second messenger, although increases in cellular cAMP concentration have not been observed at low concentrations of ACTH. We found that steroidogenesis in bovine adrenal fasciculata-reticularis cells was activated by the addition of arachidonic acid or its 15-lipoxygenase metabolite, 15-hydroperoxyeicosatetraenoic acid. The cellular 15-lipoxygenase pathway was significantly activated by 1 pM ACTH, at which concentration no increase in cellular cAMP synthesis was observed. The 1 pM ACTH-induced stimulation of steroidogenesis was completely suppressed by a lipoxygenase inhibitor, AA-861. The stimulation was independent of the increase in cellular cAMP. These results show that the action of 1 pM ACTH on steroidogenesis may be mediated by the 15-lipoxygenase metabolite(s) as a solo second messenger. The addition of ACTH at concentrations higher than 10 pM increased both the 15-lipoxygenase activity and cellular cAMP synthesis. Under these conditions, the 15-lipoxygenase metabolite(s) and cAMP were shown to mediate the activation of steroidogenesis synergistically. The presence of a dual second messenger system could explain the stimulation of steroidogenesis by ACTH at physiological concentrations.