Regulation of cyclic AMP metabolism in bovine adrenal medullary cells.

The capacity of cultured bovine adrenal medullary cells to metabolize and export cyclic AMP has been studied. Basal cellular cyclic AMP levels were increased 50% by 100 microM 3-isobutyl-1-methylxanthine (IBMX) and rolipram, a class IV (cyclic AMP-specific) phosphodiesterase (PDE) inhibitor. They were not affected by inhibition of class I (Ca2+/calmodulin-dependent), class III (cyclic GMP-inhibited) or class V PDE (cyclic GMP-specific) with vinpocetine or 3-isobutyl-8-methoxymethyl-1-methylxanthine (8-methoxymethyl-IBMX), SK&F 94120, or MB 22,948, respectively, all at 100 microM. Furthermore, only IBMX and rolipram enhanced the cyclic AMP response to 0.3 microM forskolin. Rolipram had an EC50 of < or = 1 microM and was equally effective at 100 microM and 1 mM. IBMX enhanced cyclic AMP levels significantly more at 1 mM than at 100 microM. Neither vinpocetine nor 8-methoxymethyl-IBMX (100 microM) enhanced the Ca(2+)-dependent cyclic AMP response to K+ depolarization. Elevation of cyclic GMP levels with sodium nitroprusside (10 or 100 microM), to activate any cyclic GMP-stimulated class II PDE and to inhibit any cyclic GMP-inhibited class III PDE, also had no effect on basal or forskolin-stimulated cyclic AMP levels. In the presence of IBMX (1 mM), forskolin (5 microM) caused a rapid and large increase in cellular cyclic AMP levels which was maximal after about 5 min and declined slightly over 3 hr. Over this period, extracellular cyclic AMP levels rose almost linearly reaching levels 2-3 times those in the cells. The results indicate bovine adrenal medullary cells have a high capacity for sustained cyclic AMP export. Furthermore, two PDE isozymes appear to degrade cyclic AMP in these cells, a rolipram-sensitive, cyclic AMP-specific, class IV isozyme and a rolipram-insensitive isoform.     

Histamine-induced increases in cyclic AMP levels in bovine adrenal medullary cells.

1. The effect of histamine on cellular cyclic AMP levels in cultured bovine adrenal medullary cells has been studied. 2. Histamine (0.3-30 microM) increased cyclic AMP levels transiently, with a maximal response after 5 min, a smaller response after 20 min, and no increase seen after 80 or 180 min. The EC50 at 5 min was approximately 2 microM. Histamine had no effect on cyclic AMP release from the cells over 5 min, but increased it after 90 min. 3. The cyclic AMP response to 5 microM histamine was reduced by 45% by 1 microM mepyramine and by almost 30% by 1 microM cimetidine, and was abolished by the combination of both antagonists. Cimetidine at 100 microM did not inhibit the response to histamine more than 1 microM cimetidine. The H3-receptor antagonist, thioperamide (1 microM), had no effect on the response to histamine. 4. The H1-receptor agonist, 2-thiazolyethylamine (5-100 microM) and the H2-receptor agonist, dimaprit (5-100 microM), each induced a cyclic AMP response, and gave more-than-additive responses when combined. The H3 agonist (R) alpha-methylhistamine (100 microM) had no effect either on its own or in combination with either the H1 or the H2 agonist. The response to 100 microM 2-thiazolylethylamine was unaffected by cimetidine (100 microM). 5. The cyclic AMP responses to 5 microM histamine, 100 microM thiazolylethylamine and 100 microM dimaprit were each weakly enhanced in the presence of 1 mM 3-isobutyl-1-methylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholinoceptor regulation of cyclic AMP levels in bovine adrenal medullary cells.

1. The regulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels by cholinoceptors has been studied in cultured bovine adrenal medullary cells. 2. Acetylcholine (100 microM), nicotine (10 microM) and dimethylphenylpiperazinium (20 microM) each increased cellular cyclic AMP levels 2 to 4 fold over 5 min in the absence of phosphodiesterase inhibitors. The muscarinic agonist acetyl-beta-methylcholine (100 microM) had no effect either on its own or on the response to nicotine. The responses to acetylcholine and nicotine were unaffected by atropine (1 microM) but were abolished by mecamylamine (5 microM). 3. Cellular cyclic AMP increased transiently during continuous exposure to nicotine (1-20 microM), with the largest response seen after 5 min, a smaller response after 20 min, and no change in cyclic AMP levels seen after 90 or 180 min. The maximal response after 5 min stimulation was seen with 5-10 microM nicotine and the EC50 was about 2 microM. In contrast, extracellular cyclic AMP levels did not change after 5 or 20 min stimulation with nicotine, but increased slightly after 90 min and further after 180 min. 4. The cellular cyclic AMP response to nicotine (10 microM) was unchanged or weakly enhanced in the presence of the unselective phosphodiesterase inhibitor, isobutylmethylxanthine, and was unchanged in the presence of rolipram. Nicotine did not interact synergistically with low concentrations of forskolin. The response was however completely abolished in the absence of extracellular Ca2+.     

Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP.

1. In bovine aortic endothelial cells (BAEC), thrombin (1 mu ml-1), bradykinin (1-10 nM) and adenosine triphosphate (ATP) (0.3 microM-100 microM) each induced a biphasic elevation of cytosolic calcium ([Ca2+]i), consisting of an initial transient followed by a sustained plateau phase. 2. Pretreatment of BAEC with 4 beta-phorbol 12-myristate 13-acetate (PMA; 100 nM) reduced the magnitude of the initial transient elevation of [Ca2+]i, induced by thrombin (1 mu ml-1), low concentrations of bradykinin (1 nM) or ATP (0.3 microM, 3 microM), but not by higher concentrations of the latter two agonists. Addition of PMA (100 nM) during the plateau phase of the increase in [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (10 nM) or ATP (30 microM) resulted in a fall in [Ca2+]i. 3. The inhibitory effects of PMA (100 nM) were inhibited by staurosporine (100 nM) but not mimicked by the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD; 100 nM). Furthermore, staurosporine (100 nM) increased [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by thrombin or bradykinin. In contrast, staurosporine (100 nM) reduced [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by ATP (30 microM). 4. Pretreatment with forskolin (10 microM) had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 mu ml-1), bradykinin (1 nM and 10 nM) or ATP (30 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Diazepam potentiates the corticoidogenic response of bovine adrenal fasciculata cells to dibutyryl cyclic AMP

To provide a possible role of peripheral type benzodiazepine receptors in the regulation of glucocorticoid biosynthesis. We have examined the effect of diazepam on the corticoidogenic response to dibutyryl cyclic AMP in isolated bovine adrenal fasciculata cells. Diazepam alone (up to 100 microM) had no effect on the corticoidogenesis. Diazepam caused a dose-dependent potentiation of dibutyryl cyclic AMP-induced corticoidogenesis. However, diazepam had no effect on the corticoidogenic response to ACTH and a high concentration of KCl. The potentiating effect by diazepam was clearly detected after 90 min-incubation, and it was blocked by YM-684 (diazepam antagonist) and ML-236B (cholesterol de novo synthesis inhibitor). Diazepam caused no significant decrease of intracellular content of cholesteryl esters during the corticoidogenic response to dibutyryl cyclic AMP. When the cells were incubated in the presence of (+)-PN200-110, a potent voltage-dependent Ca channel inhibitor, the potentiating effect by diazepam was not affected in spite of a significant inhibition of dibutyryl cyclic AMP-induced corticoidogenesis. These results indicate that the potentiating effect of diazepam on dibutyryl cyclic AMP-induced corticoidogenesis is due in part to the activation of the intracellular cholesterol supply system (cholesterol de novo synthesis) without any change of voltage-dependent Ca channels.     

Pertussis toxin inhibits noradrenaline accumulation by bovine adrenal medullary chromaffin cells

Summary Bovine adrenal medullary chromaffin cells maintained in tissue culture accumulated [³H]-noradrenaline by a high affinity, Na⁺-dependent, desipramine-sensitive process. The accumulation was linear with time (1–90min) and had an apparent K_m of 0.52±0.24 mol/l and V_max of 1.70±0.48 pmol/(10⁵ cells · 15 min). Pretreatment of the cells with the ADP-ribosylating agent pertussis toxin resulted in a reduction in the V_max, [0.81±0.39 pmol/(10⁵cells · 15 min)] but no significant change in the apparent affinity (K_m = 0.42±0.07 mol/l). This inhibition of [³H]noradrenaline accumulation was distinct from that produced by the vesicular transport inhibitor reserpine. Pertussis toxin inhibition probably did not arise through an indirect action on the Na⁺-gradient because while, as expected, Na⁺, K⁺-ATPase inhibition reduced [³H]noradrenaline accumulation, pertussis toxin pretreatment always caused a further significant reduction even in the presence of maximally effective concentrations of ouabain. Stimulation of the CAMP-protein kinase A system by forskolin or 8-bromocyclic AMP also caused a reduction in [³H] noradrenaline accumulation but again pertussis toxin pretreatment always resulted in a further reduction. Thus, the data provide evidence for a pertussis toxin-sensitive element in the catecholamine accumulation process and are consistent with an action at a site directly associated with the transporter itself rather than with an indirect action via secondary processes.Correspondence to S. J. Bunn at the above address     

Regulation of calcium current by low-Km cyclic AMP phosphodiesterases in cardiac cells.

The voltage-gated Ca2+ current (ICa) in cardiac myocytes is regulated by cAMP-dependent phosphorylation. Although the regulation of ICa via mechanisms involving modulation of cAMP synthesis is well understood, the regulation of cAMP degradation has been less thoroughly investigated. The goal of the present study was to investigate the participation of different subclasses of cAMP phosphodiesterase (PDE) in regulating cAMP-dependent phosphorylation of Ca2+ channels in frog ventricular myocytes. Cardiomyocytes were isolated enzymatically and mechanically and were patch-clamped using the whole-cell configuration of the patch-clamp technique. The effects of various low-Km cAMP PDE inhibitors on ICa were examined. None of the inhibitors tested [milrinone, indolidan, 1-methyl 3-isobutyl xanthine (MIX), rolipram, or Ro 20-1724] were able to elevate ICa in the absence of elevated cAMP, although they all increased ICa in the presence of submaximal levels of cAMP. This result suggests that these compounds do not act directly on Ca2+ channels but rather modulate cAMP degradation. Half-maximal effects were observed with 1.4 microM milrinone and 3.4 microM MIX. Milrinone was effective when applied from either the extracellular or intracellular surface, whereas MIX was effective only when applied from the extracellular solution. In the presence of internal cGMP, which stimulates the cGMP-stimulated PDE, the low-Km cAMP PDE inhibitors had no effect on ICa, whereas high concentrations of MIX, which inhibit the cGMP-stimulated PDE, increased ICa. This would support the hypothesis that cGMP-stimulated PDE either has a much stronger capacity to hydrolyze cAMP or is more efficiently coupled to Ca2+ channels than the low-Km cAMP PDEs.     

Atherogenic lipoproteins inhibit catecholamine secretion in cultured bovine adrenal medullary cells

The effects of lipoproteins on ion channel-mediated catecholamine secretion were investigated in cultured bovine adrenal medullary cells. Low density lipoprotein (LDL; 20–80 mg/dl) and lipoprotein(a) [Lp(a); 10–80 mg/dl] inhibited catecholamine secretion induced by carbachol, an activator of nicotinic acetylcholine receptor-ion channels. LDL and Lp(a) suppressed carbachol-induced ²²Na⁺ influx as well as ⁴⁵Ca²⁺ influx in a concentration-dependent manner similar to that of catecholamine secretion. The inhibition of catecholamine secretion by Lp(a) was not overcome by increasing the concentration of carbachol. On the other hand, high density lipoprotein (HDL; <150 mg/dl) had no effect on ²²Na⁺ influx, ⁴⁵Ca²⁺ influx, and catecholamine secretion. Like LDL and Lp(a), a synthetic peptide homologous to human plasma apolipoprotein B (apoB), apoB fragment_3358–3372-amide (3–60 μM), attenuated ²²Na⁺ influx, ⁴⁵Ca²⁺ influx, and catecholamine secretion caused by carbachol. The apoB fragment also suppressed ²²Na⁺ influx induced by veratridine (an activator of voltage-dependent Na⁺ channels) and ⁴⁵Ca²⁺ influx induced by 56 mM K⁺ (an indirect activator of voltage-dependent Ca²⁺ channels). These findings suggest that atherogenic lipoproteins such as LDL and Lp(a) suppress catecholamine secretion by interfering with Na⁺ influx through nicotinic acetylcholine receptor-ion channels, in which apoB, a structural component common to both LDL and Lp(a), plays an important role. The inhibition by atherogenic lipoproteins of catecholamine secretion may influence the progression of atherosclerosis induced by these lipoproteins. Received: 5 December 1997 / Accepted: 9 June 1998     

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells

Summary In the human T -cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E₂ receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects.Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as G_i2 and G_i3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment.In conclusion, activation of protein kinase C by phorbol esters reveals a G_i-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A₂ receptor stimulated CAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive G_i-protein. Send offprint requests to I. van der Ploeg at the above address     

Inhibition of the norepinephrine transporter function in cultured bovine adrenal medullary cells by bisphenol A

We report here the effects of an environmental estrogen, bisphenol A, on norepinephrine (NE) transporter function in cultured bovine adrenal medullary cells. The effects of bisphenol A were compared to those of 17beta-estradiol. Bisphenol A significantly inhibited [3H]NE uptake by the cells in a concentration-dependent manner (1-100 microM). Kinetic analysis revealed that bisphenol A, as well as 17beta-estradiol, noncompetitively inhibited [3H]NE uptake. Bisphenol A and 17beta-estradiol inhibited the specific binding of [3H]desipramine to plasma membranes isolated from bovine adrenal medulla. As shown by Scatchard analysis of [3H]desipramine binding, bisphenol A increased the dissociation constant (K(d)) and decreased the maximal binding (B(max)), indicating a mixed type of inhibition. 17beta-Estradiol increased the K(d) without altering the B(max), thereby indicating competitive inhibition. The present findings suggest that bisphenol A inhibits the function of the NE transporter by acting on a site different from that of 17beta-estradiol in the adrenal medulla and probably in the brain noradrenergic neurons.     

Inhibition by carbamazepine of various ion channels-mediated catecholamine secretion in cultured bovine adrenal medullary cells

The effects of carbamazepine (CBZ) on ²²Na⁺ influx, ⁴⁵Ca²⁺ influx, catecholamine secretion and cyclic GMP production were examined in cultured bovine adrenal medullary cells. 1 CBZ (40–120 mol/l) inhibited ²²Na⁺ influx evoked by carbachol in a concentration-dependent manner. CBZ inhibited carbachol-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion at concentrations similar to those which suppressed ²²Na⁺ influx. 2 CBZ (4–120 mol/l) inhibited veratridine-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion. 3 CBZ (12 or 40–120 mol/l) suppressed 56 mmol/1 K⁺-evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, respectively. 4 Combination of CBZ with nitrendipine or -agatoxin-IVA produced further inhibition of 56 mmol/l K⁺ - evoked ⁴⁵Ca²⁺ influx and catecholamine secretion, compared to the effect of CBZ alone, whereas CBZ plus -conotoxin-GVIA did not produce any further inhibition. 5 CBZ (40 mol/1) attenuated the production of cyclic GMP caused by muscarine. These results suggest that CBZ at therapeutic concentrations (16–48 mol/l: 4–12 g/ml) inhibits catecholamine secretion by interfering with nicotinic acetylcholine receptor-associated ion channels, voltage-dependent Na⁺ channels and N-type voltage-dependent Ca²⁺ channels, and may have an antimuscarinic effect in adrenal medullary cells.     

Ionic and metabolic requirements for stimulation of secretion by ouabain in bovine adrenal medullary cells

The effect of ouabain on the secretion of catecholamines from isolated bovine adrenal medullary cells was investigated. Ouabain enhances the basal rate of secretion approximately 2-fold, with half-maximal stimulation occurring at a glycoside concentration of around 5 X 10(-7) M. Parallel measurements of the release of dopamine beta-hydroxylase (EC 1.14.17.1) (an enzyme associated with chromaffin granules) and lactate dehydrogenase (EC 1.1.1.27) (which is confined to the cytosolic compartment) suggest that this increase in secretion occurs as a result of an enhanced rate of exocytosis rather than by any other route. The stimulatory effect of ouabain is dependent on extracellular sodium but is maintained in the nominal absence of calcium and is unaffected by changes in the major external anion. Neither tetrodotoxin nor phenoxybenzamine alters the response to glycoside treatment, but the calcium channel blocker methoxyverapamil reduces the catecholamine secretion evoked by ouabain in a dose-dependent fashion. This study serves to characterize the secretory action of ouabain in isolated chromaffin cells and to provide a foundation for the ion flux studies reported in the following paper.