Increased cAMP Formation Research Articles

PACAP Stimulates Biosynthesis and Release of Endozepines from Rat Astrocytes

Astrocytes synthesize and release endozepines, a family of neuropeptides related to diazepam-binding inhibitor (DBI). Astroglial cells also express the receptors of pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP). In the present article, we show that PACAP dose dependently increases DBI gene expression and stimulates endozepine release through activation of PAC1-R. PACAP increases cAMP formation, enhances polyphosphoinositide turnover, and evokes calcium mobilization from intracellular Ca2+ pools. The effect of PACAP on endozepine release is mediated through the adenylyl cyclase/PKA pathway while the downregulation of astrocyte response to PACAP can be ascribed to activation of the PLC/PKC pathway.

Compared pharmacology of human histamine H3 and H4 receptors: structure–activity relationships of histamine derivatives

Various histamine derivatives were investigated at the human H3 receptor (H3R) and H4 receptor (H4R) stably expressed in human embryonic kidney (HEK)-293 cells using [125I]iodoproxyfan and [3H]histamine binding, respectively. In Tris buffer, [3H]histamine binding to membranes of HEK(hH4R) cells was monophasic (K(D) of 3.8+/-0.8 nM). In phosphate buffer, the Hill coefficient was decreased (n(H) = 0.5+/-0.1) and a large fraction of the binding was converted into a low-affinity component (K(D) = 67+/-27 nM). The inhibition of [3H]histamine binding by two agonists, a protean agonist and five antagonists/inverse agonists confirms that the potency of many H3R ligands is retained or only slightly reduced at the H4R. Histamine derivatives substituted with methyl groups in alpha, beta or N(alpha) position of the side chain retained a nanomolar potency at the H3R, but their affinity was dramatically decreased at the H4R. With relative potencies to histamine of 282 and 0.13% at the H3R and H4R, respectively, (+/-)-alpha,beta-dimethylhistamine is a potent and selective H3R agonist. Chiral alpha-branched analogues exhibited a marked stereoselectivity at the H3R and H4R, the enantiomers with a configuration equivalent to L-histidine being preferred at both receptors. The methylsubstitution of the imidazole ring was also studied. The relative potency to histamine of 4-methylhistamine (4-MeHA) at the H4R (67%) was similar to that reported at H2 receptors but, owing to its high affinity at the H4R (Ki = 7.0+/-1.2 nM) and very low potency at H1- and H3-receptors, it can be considered as a potent and selective H4R agonist. On inhibition of forskolin-induced cAMP formation, all the compounds tested, including 4-MeHA, behaved as full agonists at both receptors. However, the maximal inhibition achieved at the H4R (approximately -30%) was much lower than at the H3R (approximately -80%). Thioperamide behaved as an inverse agonist at both receptors and increased cAMP formation with the same maximal effect (approximately +25%). In conclusion, although the pharmacological profiles of the human H3R and H4R overlap, the structure-activity relationships of histamine derivatives at both receptors strongly differ and lead to the identification of selective compounds.

ATP stimulates Na+-glucose cotransporter activity via cAMP and p38 MAPK in renal proximal tubule cells

Extracellular ATP plays an important role in the regulation of renal function. However, the effect of ATP on the Na(+)-glucose cotransporters (SGLTs) has not been elucidated in proximal tubule cells (PTCs). Therefore, this study was performed to examine the action of ATP on SGLTs and their related signal pathways in primary cultured rabbit renal PTCs. ATP increased [(14)C]-alpha-methyl-d-glucopyranoside (alpha-MG) uptake in a time-dependent (>1 h) and dose-dependent (>10(-6) M) manner. ATP stimulated alpha-MG uptake by increasing in V(max) without affecting K(m). ATP-induced increase of alpha-MG uptake was correlated with the increase in both SGLT1 and SGLT2 protein expression levels. ATP-induced stimulation of alpha-MG uptake was blocked by suramin (nonspecific P2 receptor antagonist), RB-2 (P2Y receptor antagonist), and MRS-2179 (P2Y(1) receptor antagonist), suggesting a role for the P2Y receptor. ATP-induced stimulation of alpha-MG uptake was blocked by pertussis toxin (PTX, a G(i) protein inhibitor), SQ-22536 (an adenylate cyclase inhibitor), and PKA inhibitor amide 14-22 (PKI). ATP also increased cAMP formation, which was blocked by PTX and RB-2. However, pretreatment of adenosine deaminase did not block ATP-induced cAMP formation. In addition, ATP-induced stimulation of alpha-MG uptake was blocked by SB-203580 (p38 MAPK inhibitor), but not by PD-98059 (p44/42 MAPK inhibitor) or SP-600125 (JNK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK. In conclusion, ATP increases alpha-MG uptake via cAMP and p38 MAPK in renal PTCs.

Angiotensin II AT 1 receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT 1 receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2 + influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation.

The NK1 Receptor Localizes to the Plasma Membrane Microdomains, and Its Activation Is Dependent on Lipid Raft Integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.

PHCCC, a Specific Enhancer of Type 4 Metabotropic Glutamate Receptors, Reduces Proliferation and Promotes Differentiation of Cerebellar Granule Cell Neuroprecursors

Exposure of immature rat cerebellar granule cell cultures to the type 4 metabotropic glutamate (mGlu4) receptor enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC) reduced [3H]thymidine incorporation. Its action was sensitive to the growth conditions and was attenuated by two mGlu4 receptor antagonists. An antiproliferative action of PHCCC was also seen in cultures from wild-type, but not mGlu4, knock-out mice. At least in rat cultures, PHCCC was not neurotoxic and enhanced neuritogenesis. Although PHCCC reduced the increase in cAMP formation and phospho-AKT levels induced by forskolin, none of these transduction pathways significantly contributed to the reduction of [3H]thymidine incorporation. Interestingly, PHCCC reduced the expression of Gli-1, a transcription factor that mediates the mitogenic action of Sonic hedgehog. Finally, we treated newborn rats with PHCCC either intracerebrally (infusion of 5 nmol/2 microl in the cerebellar region once every other day) or systemically (5 mg/kg, i.p., once daily) from postnatal days 3-9. Local infusion of PHCCC induced substantial changes in the morphology of the developing cerebellum. In contrast, systemic injection of PHCCC induced only morphological abnormalities of the cerebellar lobule V, which became visible 11 d after the end of the treatment. These data suggest that mGlu4 receptors are involved in the regulation of cerebellar development.

Heterologous desensitization of the sphingosine-1-phosphate receptors by purinoceptor activation in renal mesangial cells.

1 Sphingosine-1-phosphate (S1P) is considered a potent mitogen for mesangial cells and activates the classical mitogen-activated protein kinase (MAPK) cascade via S1P receptors. In this study, we show that S1P signalling is rapidly desensitized upon S1P receptor activation. A complete loss of S1P sensitivity occurs after 10 min of S1P pretreatment and remains for at least 8 h. A similar desensitization is also seen with the S1P mimetic FTY720-phosphate, but not with the nonphosphorylated FTY720, nor with sphingosine or ceramide. 2 Prestimulating the cells with extracellular ATP or UTP, which bind to and activate P2Y receptors on mesangial cells, a similar rapid desensitization of the S1P receptor occurs, suggesting a heterologous desensitization of S1P receptors by P2Y receptor activation. Furthermore, adenosine binding to P1 receptors triggers a similar desensitization. In contrast, two other growth factors, PDGF-BB and TGFbeta2, have no significant effect on S1P-induced MAPK activation. 3 S1P also triggers increased inositol trisphosphate (IP3) formation, which is completely abolished by S1P pretreatment but only partially by ATP pretreatment, suggesting that IP3 formation and MAPK activation stimulated by S1P involve different receptor subtypes. 4 Increasing intracellular cAMP levels by forskolin pretreatment has a similar effect on desensitization as adenosine. Moreover, a selective A3 adenosine receptor agonist, which couples to phospholipase C and increases IP3 formation, exerted a similar effect. 5 Pretreatment of cells with various protein kinase C (PKC) inhibitors prior to ATP prestimulation and subsequent S1P stimulation leads to a differential reversal of the ATP effect. Whereas the broad-spectrum protein kinase inhibitor staurosporine potently reverses the effect, the PKC-alpha inhibitor CGP41251, the PKC-delta inhibitor rottlerin and calphostin C show only a partial reversal at maximal concentrations. 6 Suramin, which is reported as a selective S1P3 receptor antagonist compared to the other S1P receptor subtypes, has no effect on the S1P-induced MAPK activation, thus excluding the involvement of S1P3 in this response. 7 In summary, these data document a rapid homologous and also heterologous desensitization of S1P signalling in mesangial cells, which is mechanistically triggered by PKC activation and eventually another staurosporine-sensitive protein kinase, as well as by increased cAMP formation.

Irsogladine, an anti-ulcer drug, suppresses superoxide production by inhibiting phosphodiesterase type 4 in human neutrophils

Neutrophil superoxide production is implicated in the pathogenesis of gastric mucosal damage induced by various ulcerative agents and Helicobacter pylori infection. We investigated here the effects of an anti-ulcer drug irsogladine [2, 4-diamino-6-(2, 5-dichlorophenyl)-s-triazine maleate] on cAMP formation in isolated human neutrophils. The cAMP level in human neutrophils was elevated by a phosphodiesterase (PDE) type 4 selective inhibitor rolipram, but not by any inhibitors of PDE1, PDE2 and PDE3. Irsogladine also increased cAMP formation in a concentration-dependent manner in neutrophils. A non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) alone significantly increased cAMP level, whereas irsogladine was unable to further increase cAMP level in the presence of IBMX. Irsogladine inhibited concentration-dependently the superoxide (O2−) production induced by various stimuli including formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, guanosine 5′-[gamma-thio] triphosphate, A23187 and phorbol 12-myristate 13-acetate. These effects of irsogladine were mimicked by rolipram, IBMX and dibutyryl cAMP. The inhibitory effects of irsogladine and rolipram on the O2− production were reversed by a protein kinase A inhibitor H-89. These results indicate that irsogladine inhibits the superoxide production in human neutrophils by the increase of cAMP content by PDE 4 inhibition, which in turn contributing to the anti-ulcer effects of irsogladine on gastric mucosal lesions associated with oxidative stress.

Melanocortin receptor type 3 as a potential target for anti-inflammatory therapy.

Alpha-melanocyte stimulating hormone (alpha-MSH) and other melanocortin peptides are potent anti-inflammatory agents exhibiting efficacy in many animal models of acute and chronic inflammation. They are derived from a larger precursor molecule known as the POMC prohormone and are produced both centrally and peripherally. They exert their effect by binding to melanocortin receptors, of which five have been cloned and partially characterised. Agonism at these receptors leads to adenylate cyclase activation and subsequent increases in cAMP formation. Two receptors to date have been proposed to mediate the actions of the melanocortin peptides in an inflammatory scenario, the MC1 and 3-R, and here we discuss our findings proposing the MC3-R as a novel therapeutic target. The potential anti-inflammatory role for MC3-R is in its infancy, however, recent studies have shown that melanocortin peptides are effective in mice bearing a non-functional MC1-R (recessive yellow e/e mice). This ability to inhibit cell migration appears to be via inhibition in cytokine and adhesion molecule expression and is due to their abilities to interfere with cell signalling pathways. Identification of endogenous mediators of anti-inflammation, their receptors and the pathologies they are effective in, is of benefit to the medical community, and will hopefully have reduced side effects. We believe that specific small molecule agonists directed at MC3-R could be potential novel therapeutics for inflammatory conditions.

Possible Role of a Neuropeptide PACAP (Pituitary Adenylate Cyclase‐Activating Polypeptide) on Stimulus‐Secretion Coupling in Catecholamine Neuron

AbstractFor Abstract see ChemInform Abstract in Full Text.

Antagonism of VIP-stimulated cyclic AMP formation in chick brain.

Of eight peptides tested (0.01-5 microM), only two, that is, pituitary adenylate cyclase-activating polypeptide (PACAP27) and chicken vasoactive intestinal peptide (cVIP), potently stimulated cyclic AMP (cAMP) production in cerebral cortical slices of the chick. Mammalian VIP (mVIP) showed some activity only at the highest dose tested, whereas truncated forms of PACAP or VIP, that is, PACAP6-27, cVIP6-28, and mVIP6-28, or hybrid compounds, that is, neurotensin6-11-cVIP7-28 (NT-cVIP) and neurotensin6-11-mVIP7-28 (NT-mVIP), were inactive. Thirty-minute preincubation of chick cortical slices with 5 microM PACAP6-27, NT-cVIP, or NT-mVIP competitively antagonized the cAMP effects of cVIP (0.03-1 microM), with the truncated form of PACAP being the best antagonist. Preincubation of slices with 5 microM mVIP6-28 also produced a significant inhibition of the cVIP (0.1-1 microM)-induced increase in cAMP production; however its action was independent of the concentration of cVIP. In contrast to mVIP6-28, cVIP6-28 showed no antagonistic activity against the full-length peptide. In parallel experiments, 30-min pretreatment of cortical slices with 5 microM PACAP6-27 significantly antagonized the PACAP38-evoked increase in cAMP formation, whereas mVIP6-28 or the NT-mVIP hybrid was ineffective. It has been concluded that in the chick brain, PACAP and cVIP stimulate cAMP biosynthesis via PAC1 and VPAC-type receptors, respectively, and PACAP6-27 seems to be the most potent, yet PACAP/VIP receptor-nonselective antagonist. Unlike truncated PACAP, the NT-VIP hybrid peptides tested may represent VPACtype receptor-selective blocking activity.

Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y 1 receptor

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y 1 receptor, while other P2Y subtypes, except perhaps P2Y 4, are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y 1 and P2Y 11 receptors, while P2Y 2 and P2Y 4 receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y 1 receptors and Gα q/11 subunits. Various nucleotides induced intracellular Ca 2+ mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPγS, a profile resembling that of human P2Y 1 receptors. Furthermore, P2Y 1 receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca 2+ responses in a concentration-dependent manner, consistent with expression of a P2Y 1 receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPγS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y 11 receptors were expressed. P2Y 1 receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y 1 receptor.

Localization of Adenylyl Cyclase Isoforms and G Protein-Coupled Receptors in Vascular Smooth Muscle Cells: Expression in Caveolin-Rich and Noncaveolin Domains

A number of different agonists activate G protein-coupled receptors to stimulate adenylyl cyclase (AC), increase cAMP formation, and promote relaxation in vascular smooth muscle. To more fully understand this stimulation of AC, we assessed the expression, regulation, and compartmentation of AC isoforms in rat aortic smooth muscle cells (RASMC). Reverse transcription-polymerase chain reaction detected expression of AC3, AC5, and AC6 mRNA, whereas immunoblot analysis indicated expression of AC3 and AC5/6 protein primarily in caveolin-rich membrane (cav) fractions relative to noncaveolin (noncav) fractions. Beta(1)-adrenergic receptors (AR), beta(2)AR, and G(s) were detected in both cav and noncav fractions, whereas the prostanoid receptors EP(2)R and EP(4)R were excluded from cav fractions. We used an adenoviral construct to increase AC6 expression. Overexpressed AC6 localized only in noncav fractions. Two-fold overexpression of AC6 caused enhancement of forskolin-, isoproterenol- and prostaglandin E(2)-stimulated cAMP formation but no changes in basal levels of cAMP. At higher levels of AC6 overexpression, basal and adenosine receptor-stimulated cAMP levels were increased. Stimulation of cAMP levels by agents that increase Ca(2+) in native cells was consistent with the expression of AC3, but overexpression of AC6, which is inhibited by Ca(2+), blunted the Ca(2+)-stimulable cAMP response. These data indicate that: 1) RASMC express multiple AC isoforms that localize in both caveolin-rich and noncaveolin domains, 2) expression of AC6 in non-caveolin-rich membranes can increase basal levels of cAMP and response to several stimulatory agonists, and 3) Ca(2+)-mediated regulation of cAMP formation depends upon expression of different AC isoforms in RASMC. Compartmentation of GPCRs and AC is different in cardiomyocytes than in RASMC, indicating that targeting of these components to caveolin-rich membranes can be cell-specific. Moreover, our results imply that the colocalization of GPCRs and the AC isoforms they activate need not occur in caveolin-rich fractions.

Possible role of a neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) on stimulus-secretion coupling in catecholamine neuron

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide first isolated from ovine hypothalamic tissue. This peptide stimulates adenylate cyclase activation. However, few details were known of the function of this peptide on stimulus-secretion coupling in neuronal cells. The authors have investigated the role of PACAP on catecholamine biosynthesis and secretion using cultured bovine adrenal chromaffin cells as a model for catecholamine-containing neurons. PACAP38, the 38-amino acid form of PACAP, increased cAMP formation in bovine adrenal chromaffin cells. In addition, PACAP38 increased [Ca2+]i associated with PI turnover and Ca2+ influx into the cells. The synthesis of catecholamine and the phosphorylation of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, stimulated by the maximal effective concentration of dibutyryl cAMP or a high concentration (56 mM) of K+ were further enhanced by PACAP38. Thus PACAP38 stimulated the pathway of catecholamine biosynthesis mainly by both activation of cAMP- and Ca2(+)-dependent protein kinases. On catecholamine secretion from the cells, the effect of PACAP38 was markedly potentiated by addition of ouabain, an inhibitor of Na+/K+ ATPase. This markedly potentiated secretion was greatly reduced with Na+ omitted-sucrose medium. PACAP38 increased 22Na+ influx into the cells treated with ouabain. Thus PACAP38 with ouabain stimulated catecholamine secretion by accumulation of intracellular Na+, resulting in an increase in Ca2+ influx. These results indicate that the neuropeptide PACAP has an important role in stimulus-secretion coupling in adrenal chromaffin cells.

Involvement of Raf-1 in chronic δ-opioid receptor agonist-mediated adenylyl cyclase superactivation

Chronic δ-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic δ-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 μM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% ( n=6, P<0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.

Mechanisms of melatonin action in the pituitary and SCN.

We have compared melatonin effects in two different cell types in order to determine general intracellular mechanisms of its action. In neonatal rat pituitary, melatonin acts via the specific membrane receptors to inhibit GnRH-induced LH release. The melatonin effect disappears in adulthood due to the disappearance of the receptors. The mechanism of the melatonin action involves inhibition of the GnRH induced increase of intracellular calcium ([Ca2+])i. Our observations indicate that melatonin has dual inhibitory effect on GnRH-induced [Ca2+]i: it inhibits mobilisation of Ca2+ from endoplasmic reticulum as well as Ca2+ influx through voltage sensitive channels. Besides, melatonin also decreases basal and GnRH- or forskolin-induced increase of cAMP concentration in the pituitary. Although cAMP is not of primary importance for regulation of LH release, the cAMP decrease may participate in the mechanism of inhibitory melatonin action on LH release. Rat suprachiasmatic nuclei (SCN) have a high density of the melatonin receptors throughout the postnatal life. Cultures of dispersed SCN cells show circadian rhythm of vasopressin (AVP) release, with several fold increase in the middle of the day and decrease during night. Melatonin inhibits the spontaneous AVP release. Melatonin also inhibits the AVP release induced by vasoactive intestinal peptide (VIP). Intracellular mechanisms of the melatonin effect may involve cAMP, because melatonin inhibits the VIP-induced increase of cAMP and increase of cAMP formation by forskolin stimulates AVP release from the cultures. On the other hand, involvement of intracellular calcium in the regulation of AVP release may not be excluded. VIP induces [Ca2+]i increase in 14% of the SCN cells and AVP release is stimulated by Ca2+ ionophore ionomycin. Our observations indicate that some of the mechanisms of melatonin action are similar in the pituitary and SCN.

Adenosine modulates Mg(2+) uptake in distal convoluted tubule cells via A(1) and A(2) purinoceptors.

tk;1Adenosine plays a role in the control of water and electrolyte reabsorption in the distal tubule. As the distal convoluted tubule is important in the regulation of renal Mg(2+) balance, we determined the effects of adenosine on cellular Mg(2+) uptake in this segment. The effect of adenosine was studied on immortalized mouse distal convoluted tubule (MDCT) cells, a model of the intact distal convoluted tubule. The rate of Mg(2+) uptake was measured with fluorescence techniques using mag-fura 2. To assess Mg(2+) uptake, MDCT cells were first Mg(2+) depleted to 0.22 +/- 0.01 mM by being cultured in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl(2); next, changes in intracellular Mg(2+) concentration ([Mg(2+)](i)) were determined. [Mg(2+)](i) returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg(2+)](i))/dt, of 137 +/- 16 nM/s. Adenosine stimulates basal Mg(2+) uptake by 41 +/- 10%. The selective A(1) purinoceptor agonist N(6)-cyclopentyladenosine (CPA) increased intracellular Ca(2+) and decreased parathyroid hormone (PTH)-stimulated cAMP formation and PTH-mediated Mg(2+) uptake. On the other hand, the selective A(2) receptor agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS) stimulated Mg(2+) entry in a concentration-dependent fashion. CGS increased cAMP formation and the protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated Mg(2+) uptake. Selective inhibition of phospholipase C, protein kinase C, or mitogen-activated protein kinase enzyme cascades with U-73122, Ro-31-8220, and PD-98059, respectively, diminished A(2) agonist-mediated Mg(2+) entry. Aldosterone potentiated CGS-mediated Mg(2+) entry, and elevation of extracellular Ca(2+) diminished CGS-responsive cAMP formation and Mg(2+) uptake. Accordingly, MDCT cells possess both A(1) and A(2) purinoceptor subtypes with intracellular signaling typical of these respective receptors. We conclude that adenosine has dual effects on Mg(2+) uptake in MDCT cells through separate A(1) and A(2) purinoceptor pathways.

Modulation of glucagon receptor expression and response in transfected human embryonic kidney cells.

The modulation of glucagon receptor (GR) expression and biological response was investigated in human embryonic kidney cell (HEK-293) clones permanently expressing the GR with different densities. The GR mRNA expression level in these clones was upregulated by cellular cAMP accumulation and presented a good correlation with both the protein expression level and the maximum number of glucagon binding sites. However, the determination of glucagon-induced cAMP accumulation in these cell lines revealed that the enhancement of receptor expression did not lead to a proportional increase in cAMP formation. Under these conditions, the maximum cAMP production induced by NaF and forskolin was not significantly different among selected clones, regardless of the receptor expression level. High receptor-expressing clones showed the greatest susceptibility for agonist-induced desensitization compared with clones with lower GR expression levels. The results of the present study suggest that the GR can recruit non-GR-specific desensitization mechanism(s). Furthermore, the partial inhibition or alteration of the overall cAMP synthesis pathway at the receptor level may be a necessary adaptive step for a cell in response to a massive increase in membrane receptor expression level.

Chronic ethanol treatment reduces adenylyl cyclase activity in human erythroleukemia cells

Characteristic changes of platelet membrane adenylyl cyclase activity have been described in men with alcoholism. We studied the occurrence of these changes in human erythroleukemia (HEL) cells after chronic ethanol treatment. Chronic treatment of the HEL cell with ethanol (50 or 100 mM) for 48 h resulted in significant reduction of prostaglandin E 1-stimulated adenylyl cyclase activity. The acute ethanol (200 mM, 5 min) enhancement of adenylyl cyclase activity was significantly reduced after chronic ethanol treatment. We also observed a reduction in phorbol-12,13-dibutyrate (PDB) enhancement of prostaglandin E 1-stimulation after chronic ethanol treatment. Chronic ethanol treatment (50 or 100 mM) reduced the activity of adenylyl cyclase in response to stimulation by acute ethanol to a greater extent than that of after acute PDB. The increase in cAMP formation by ethanol and PDB was only evident when prostaglandin E 1 was present and under basal conditions (when no stimulatory agent was present) ethanol up to 200 mM, and PDB up to 1 M, had no significant effect on adenylyl cyclase activity. The reduced capacity of ethanol and/or PDB to stimulate adenylyl cyclase activity after chronic ethanol treatment suggests the involvement of a common denominator in the action of ethanol and PDB.

Zinc(II)-mediated enhancement of the agonist activity of histidine-substituted parathyroid hormone(1–14) analogues

Previous studies on parathyroid hormone (PTH)(1–14) revealed that residues (1–9) played a dominant role in stimulating PTH-1 receptor-mediated increases in cAMP formation. In the present study, we examined the effects of installing a metal-binding motif in the (10–14) region of rat PTH(1–14) on the peptide’s agonist activity. We found that substitution of histidine for the native asparagine at position 10 of PTH(1–14) provided a peptide that was approx. 8-fold more potent as an agonist in the presence of divalent zinc salts than it was in the absence of the metal. This enhancement in potency was dependent on the native histidine at position 14, the concentration of Zn(II) utilized, and did not occur with other divalent metal ions. The zinc-activated [His 10]-PTH(1–14) peptide was blocked by a classical PTH-1 receptor antagonist, PTHrP(7–36), and did not activate the PTH-2 receptor. The zinc-mediated enhancing effect did not require the large N-terminal extracellular domain of the PTH-1 receptor. Although we were able to demonstrate that [His 10]-PTH(1–14) binds Zn(II) using 1H-NMR, our spectroscopic studies (circular dichroism and nuclear magnetic resonance) were not consistent with the notion that zinc enhanced the activity of [His 10]-PTH(1–14) simply by inducing a helical structure in the 10–14 region. Rather, the data suggest that the enhancement in cAMP potency arises from the formation of a ternary complex between [His 10]-PTH(1–14), a zinc atom, and the extracellular loop/transmembrane domain region of the PTH-1 receptor.