Adenylyl Cyclase Signaling Pathway Research Articles

Crosstalk between adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism under red blood cell microrheological changes

There are evidences that red blood cell (RBC) deformation and aggregation change under their incubation with catecholamines and it is connected with activation of intracellular signaling pathways. The present study was designed to explore the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism of RBCs together with their microrheological changes. The washed RBCs were resuspended in PBS. In each of the three research sessions RBC suspensions were divided into two aliquots: 1) control (without drug) and 2) with an appropriate drug. After cell incubation RBC deformability (RBCD) and aggregation (RBCA) were estimated. RBC incubation with catecholamines resulted in RBCD changes by 18-30%. RBCs incubation with forskolin facilitated an increase of RBCD by 17% (p < 0.05). A significant deformability rise under dB-AMP incubation was found by 27% (p < 0.01). Ca2+ cell influx, stimulated by A23187, was accompanied by an increase of RBCA; whereas red cell deformability was changed only slightly. On the other hand, Ca2+ entry blocking into the cells by verapamil has led to significant RBCA decrease and RBCD rise. The obtained results make us believe that RBCD change was closely associated with Ca2+ control mechanisms. An effect of Ca2+ concentration increase on RBC microrheology was removed, if it was preliminary added to incubation medium EGTA as Ca2+ chelator. It was found that all four PDE inhibitors: IBMX, vinpocetine, rolipram, pentoxifylline decreased RBCA significantly and, quite the contrary, they increased red cell deformability. Our data have shown that Ca2+ entry increase was accompanied by red cell aggregation rise, while adenylyl cyclase-cAMP system stimulation led to red cell deformability increase and its aggregation lowered. The crosstalk between two intracellular signaling systems is probably connected with phosphodiesterase activity.

Alterations in corticostriatal synaptic plasticity in mice overexpressing human α-synuclein

Most forms of Parkinson's disease (PD) are sporadic in nature, but some have genetic causes as first described for the α-synuclein gene. The α-synuclein protein also accumulates as insoluble aggregates in Lewy bodies in sporadic PD as well as in most inherited forms of PD. The focus of the present study is the modulation of synaptic plasticity in the corticostriatal pathway of transgenic (Tg) mice that overexpress the human α-synuclein protein throughout the brain (ASOTg). Paired-pulse facilitation was detected in vitro by activation of corticostriatal afferents in ASOTg mice, consistent with a presynaptic effect of elevated human α-synuclein. However basal synaptic transmission was unchanged in ASOTg, suggesting that human α-synuclein could impact paired-pulse facilitation via a presynaptic mechanism not directly related to the probability of neurotransmitter release. Mice lacking α-synuclein or those expressing normal and A53T human α-synuclein in tyrosine hydroxylase-containing neurons showed, instead, paired-pulse depression. High-frequency stimulation induced a presynaptic form of long-term depression solely in ASOTg striatum. A presynaptic, N-methyl- d-aspartate receptor-independent form of chemical long-term potentiation induced by forskolin (FSK) was enhanced in ASOTg striatum, while FSK-induced cAMP levels were reduced in ASOTg synaptoneurosome fractions. Overall the results suggest that elevated human α-synuclein alters presynaptic plasticity in the corticostriatal pathway, possibly reflecting a reduction in glutamate at corticostriatal synapses by modulation of adenylyl cyclase signaling pathways. ASOTg mice may recapitulate an early stage in PD during which overexpressed α-synuclein dampens corticostriatal synaptic transmission and reduces movement.

Role of muscarinic signal transduction and CREB phosphorylation in dichlorvos-induced memory deficits in rats: An acetylcholine independent mechanism

The present study was designed to explore the alternative mechanism (other than AChE inhibition) for chronic, low-level exposure to dichlorvos, an organophosphate, in vivo. Dichlorvos, at a dose of 1.0 and 6.0 mg/kg body weight (b.wt.) for 12 weeks, showed impairment in neurobehavioral indices viz. rota rod, passive avoidance and water maze tests. Though high dose of dichlorvos had a detrimental effect on acetylcholinesterase activity, no significant inhibition was seen with low dose of dichlorvos. Western blot analysis and immunofluorescence studies showed a significant reduction in the expression of M 1, M 2 and M 3 muscarinic receptor subtypes in high dose group animals, whereas in low dose group animals only the M 2 receptor subtype was reduced significantly. Further, the signal transduction cascade linked to these receptor subtypes was affected in high dose group animals whereas in low dose group only adenylyl cyclase-linked signaling pathway was impaired. Finally, the phosphorylation of CREB, a memory enhancing transcription factor, was significantly reduced in both low dose and high dose group animals. Thus, the present study reveals the significance of M 2 muscarinic receptor linked adenylyl cyclase signaling pathway and phosphorylation of CREB in the development of neurobehavioral impairments after chronic low-level exposure to dichlorvos.

A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC).

We previously identified a sperm-specific Na(+)/H(+) exchanger (sNHE) principally localized to the flagellum. Disruption of the sNHE gene in mice resulted in absolute male infertility associated with a complete loss of sperm motility. Here, we show that the sNHE-null spermatozoa fail to develop the cAMP-dependent protein tyrosine phosphorylation that coincides with the functional maturation occurring upon incubation in capacitating conditions in vitro. Both the sperm motility defect and the lack of induced protein tyrosine phosphorylation are rescued by the addition of cell-permeable cAMP analogs, suggesting that cAMP metabolism is impaired in spermatozoa lacking sNHE. Our analyses of the bicarbonate-dependent soluble adenylyl cyclase (sAC) signaling pathway in sNHE-null sperm cells reveal that sNHE is required for the expression of full-length sAC, and that it is important for the bicarbonate stimulation of sAC activity in spermatozoa. Furthermore, both codependent expression and coimmunoprecipitation experiments indicate that sNHE and sAC associate with each other. Thus, these two proteins appear to be components of a signaling complex at the sperm flagellar plasma membrane. We propose that the formation of this complex efficiently modulates intracellular pH and bicarbonate levels through the rapid and effective control of sAC and sNHE activities to facilitate sperm motility regulation.

Cloning and Functional Identification of Novel Endothelin Receptor Type A Isoforms in Pituitary

Mammalian endothelin (ET) receptors, termed ET(A)R and ET(B)R, are derived from two intron-containing genes and the functional splice variants of ET(B)R but not ET(A)R have been identified. Here, we report about the isolation of cDNAs of ET(A)R transcripts from rat anterior pituitary, which are generated by alternative RNA splicing. Deletion of exon 2 and insertion of fragments from intron 1 and 2 accounted for formation of three misplaced proteins, whereas the insertion of a fragment from intron 6 resulted in generation of a functional plasma membrane receptor, termed ET(A)R-C13. In this splice variant, the C-terminal 382S-426N sequence of ET(A)R was substituted with a shorter 382A-399L sequence, resulting in alteration of the putative domains responsible for coupling to G(q/11) and G(s) proteins and the endocytotic recycling, as well as in deletion of the predicted protein kinase C/casein kinase 2 phosphorylation sites. The mRNA transcripts for ET(A)R-C13 were identified in normal and immortalized pituitary cells and several other tissues. The pharmacological profiles of recombinant ET(A)R and ET(A)R-C13 were highly comparable, but the coupling of ET(A)R-C13 to the calcium-mobilizing signaling pathway was attenuated, causing a rightward shift in the potency for agonist. Furthermore, the efficacy of ET(A)R-C13 to stimulate adenylyl cyclase signaling pathway and to internalize was significantly reduced. These results indicate for the first time the presence of a novel ET(A) splice receptor, which could contribute to the functional heterogeneity among secretory pituitary cell types.

Suppression of Thyrotropin Receptor Constitutive Activity by a Monoclonal Antibody with Inverse Agonist Activity

TSH binding to the TSH receptor (TSHR) induces thyrocyte growth and proliferation primarily by activating the adenylyl cyclase signaling pathway. Relative to the other glycoprotein hormone receptors, the TSHR has considerable ligand-independent (constitutive) activity. We describe a TSHR monoclonal antibody (CS-17) with the previously unrecognized property of being an inverse agonist for TSHR constitutive activity. This property is retained, even when constitutive activity is extremely high consequent to diverse TSHR extracellular region mutations. A similar effect on an activating mutation at the base of the sixth transmembrane helix (not accessible to direct CS-17 contact) indicates that CS-17 is acting allosterically. Administered to mice in vivo, CS-17 reduces serum T(4) levels. The CS-17 epitope is conformational and a significant portion lies in the C-terminal region of the TSHR leucine-rich domain (residues 260-289). By interacting with the large TSHR extracellular domain, CS-17 is, to our knowledge, the first antibody reported to be an inverse agonist for a member of the G protein receptor superfamily. After humanization of its murine constant region, CS-17 has the potential to be an adjunctive therapeutic agent in athyreotic patients with residual well-differentiated thyroid carcinoma as well as pending definitive treatment in some selected hyperthyroidism states.

Functional Interaction between Gonadotropin-Releasing Hormone and PACAP in Gonadotropes and αT3-1 Cells

Gonadotropes, like other cells, receive informational input from multiple receptor types, acting through multiple intracellular signaling pathways, and are therefore faced with the task of integrating this input in order to respond appropriately to their environment. In recent years an increasing number of examples of functional interactions occurring between the PIC and adenylyl cyclase signaling pathways in gonadotropes have been described, and the discovery that these cells are targets for PACAP has provided a physiological context for earlier work on gonadotrope regulation by cyclic AMP. The development of the alpha T3-1 cell line has greatly facilitated investigation of the interaction between these signaling systems. In these cells we have obtained no evidence for interaction between the GnRH and PACAP receptor-effector systems at the level of receptor occupancy or expression, but these systems clearly do have reciprocal modulatory effects on second messenger generation and/or mobilization. We are now faced with the challenge of determining the physiological and/or pathophysiological relevance of such interactions.

Dependence of Electrical Activity and Calcium Influx-Controlled Prolactin Release on Adenylyl Cyclase Signaling Pathway in Pituitary Lactotrophs

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.

Atrial natriuretic peptide-C receptor-induced attenuation of adenylyl cyclase signaling activates phosphatidylinositol turnover in A10 vascular smooth muscle cells.

Atrial natriuretic peptide (ANP)-C receptor activation has been shown to inhibit adenylyl cyclase (AC) activity as well as to stimulate phospholipase C (PLC) signaling pathways. The present studies were undertaken to investigate whether ANP-C receptor-mediated decreased cAMP levels contribute to the activation of PLC signaling. C-ANP(4-23) [des(Gln(18),Ser(19), Glu(20),Leu(21),Gly(22))ANP(4-23)-NH(2)], a ring-deleted peptide of ANP that interacts specifically with ANP-C receptor, stimulated inositol 1,4,5-tris-phosphate (IP(3)) production (PLC activity) in A10 vascular smooth muscle cells in a concentration- and time-dependent manner. The maximal stimulation observed was about 75% at 2 h of treatment, with an apparent EC(50) of about 20 to 30 nM. Pertussis toxin treatment of the cells completely abolished the C-ANP(4-23)-mediated stimulation of IP(3) production. Forskolin (FSK), a stimulator of adenylyl cyclase, dibutyryl cAMP (db cAMP), and isoproterenol (ISO), a beta-adrenergic agonist that stimulates adenylyl cyclase activity and cAMP levels, inhibited IP(3) production by about 35, 30, and 50%, respectively, whereas dideoxyadenosine (DDA), an inhibitor of adenylyl cyclase activity, and oxotremorine stimulated IP(3) production by about 90 and 80%, respectively, in these cells, suggesting a functional interaction between these two signaling pathways. Treatment of the cells with antisense oligonucleotide of ANP-C receptor that attenuated ANP-C receptor-mediated inhibition of adenylyl cyclase resulted in a complete attenuation of C-ANP(4-23)-induced stimulation of IP(3) formation, whereas FSK, db cAMP, and ISO-mediated decrease and oxotremorine and endothelin-1 (ET-1)-induced increase in IP(3) production was not affected by this treatment. Furthermore, C-ANP(4-23)-induced increase in IP(3) formation was significantly potentiated by DDA and inhibited by FSK and db cAMP, whereas ET-1-induced increase in IP(3) production was not affected by FSK. In addition, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A, completely abolished C-ANP(4-23) and not ET-1-induced stimulation of IP(3) production. These results indicate that ANP-C receptor activation by C-ANP(4-23) and resulting decrease in cAMP levels may be responsible for the activation of phosphatidylinositol (PI) turnover signaling, suggesting a cross-talk between ANP-C receptor-mediated adenylyl cyclase and PLC signaling pathways.

Inhibitory G-proteins and their role in desensitization of the adenylyl cyclase pathway in heart failure.

Heart failure is accompanied by stereotypic alterations in cardiac gene expression. These changes are most likely secondary in the pathogenesis and can be viewed as protective, e.g. as energy-saving mechanisms, but at the same time, they aggravate contractile dysfunction and the deficit of failing cardiac myocytes to respond to altered hemodynamic needs. One of the best-studied, paradigmatic examples of this dichotomy is heterologous desensitization of the cardiac adenylyl cyclase (AC) signaling pathway. It protects against detrimental consequences of the excessive adrenergic drive, but it also blunts the most powerful inotropic support of the heart. Desensitization is associated with downregulation of beta-adrenergic receptors, increased beta-adrenoceptor kinases and increased inhibitory G protein alpha-subunits, G(alphai). Whereas a causative role of the former is generally accepted, the role of the increase in G(alphai) has remained controversial for many years. The present article summarizes early and novel findings that, in the view of the authors, provide solid evidence for G(alphai) to play an important role in the adaptation of cardiac AC to various pathophysiological conditions.

Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Beta-adrenoceptor-mediated cell signaling in the neonatal heart and liver: responses to terbutaline.

Terbutaline, a beta(2)-adrenoceptor (beta(2)-AR) agonist, is a widely used tocolytic that also crosses the placenta to stimulate fetal beta-ARs. The current study examines the effects of terbutaline administered to neonatal rats. Terbutaline (10 mg/kg sc) given on postnatal day (PN) 2-5 or PN 11-14 elicited significant downregulation of both cardiac and hepatic beta-ARs, with a much greater effect in the liver. Despite the reduction in cardiac beta-ARs, receptor desensitization was absent as evidenced by the maintained ability of isoproterenol to stimulate adenylyl cyclase (AC) in membrane preparations. The underlying mechanism was dissected by using stimulants that operate at different points in the AC signaling pathway, NaF, forskolin, and Mn(2+). When administered in the early neonatal period, terbutaline failed to evoke any changes in cardiac AC activity; however, treatment on PN 11-14 evoked heterologous sensitization downstream from the receptor, evidenced by increases in the response to NaF and forskolin. In the liver, neonatal terbutaline administration elicited a small (approximately equal to 10%) decrease in the AC response to isoproterenol, an effect much smaller than the downregulation of beta-ARs (>40%). In this tissue, desensitization was again offset by heterologous sensitization of AC signaling. These results indicate that, in the developing organism, beta-AR-mediated cell signaling responses are maintained in the face of receptor downregulation through heterologous induction of downstream signaling elements. These unique responses serve to sustain beta-AR signaling in the perinatal period.

Constitutive Activation of A3 Adenosine Receptors by Site-Directed Mutagenesis

The objective of this study was to create constitutively active mutant human A3 adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A3 ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A3 agonist Cl-IB-MECA (1-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC50 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F, and Y282F and mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.

CREM, a master-switch of the transcriptional cascade in male germ cells

In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signalling pathway is mediated by a family of cAMP-responsive nuclear factors. The CREB and CREM transcription factors are activated by phosphorylation of a key serine residue by kinase stimulated by cyclic AMP, calcium, growth factors and stress signals. Phosphorylation allows recruitment of CBP (CREB Binding Protein), a large co-activator that contacts the general transcriptional machinery. The CREM gene plays a key physiological and developmental role within the hypothalamic-pituitary-gonadal axis. CREM is highly expressed in postmeiotic cells upon a striking developmental switch regulated by the pituitary hormone FSH. CREM-mutant mice generated by homologous recombination reveal that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent while there is a significant increase in apoptotic germ cells. Mutant male mice completely lack spermatozoa, a phenotype reminiscent of cases of human infertility. Interestingly, in male germ cells, CREM is not phosphorylated but associates with ACT, a member of the LIM-only class of proteins that has intrinsic transcriptional activity. Thus, in some circumstance, CREM can bypass the classical requirement for phosphorylation and association with CBP.

Pertussis toxin inhibits cholecystokinin- and epidermal growth factor-induced mitogen-activated protein kinase activation by disinhibition of the cAMP signaling pathway and inhibition of c-Raf-1.

Pertussis toxin (PTx), which inactivates G(i/o) type G proteins, is widely used to investigate the involvement of G(i/o) proteins in signal transduction. Activation of extracellular-regulated kinases 1 and 2 (ERK1/2) by G protein-coupled receptors has been described to occur either through a PTx-insensitive pathway involving activation of phospholipase C and protein kinase C (PKC), or through a PTx-sensitive pathway involving G(i)betagamma-mediated activation of Src. Cholecystokinin (CCK) activates ERK1/2 by a PKC-dependent, and thus presumably PTx-insensitive, pathway. However, CCK has recently been shown to induce activation of G(i) proteins in addition to G(q/11). In the present study, PTx partially inhibited CCK-induced ERK1/2 activation in pancreatic AR42J cells, although activation of phospholipase C was not reduced. PTx also inhibited ERK1/2 activation in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) as well as activation of c-Raf-1 by EGF and CCK. In contrast, PTx, CCK, and EGF had only minor effects on A-Raf and B-Raf activity. Forskolin, a direct activator of adenylyl cyclase, inhibited CCK- and EGF-induced activation of c-Raf-1 and ERK1/2 in a manner similar to that of PTx. In PTx-treated cells, the cAMP content was increased and forskolin did not further inhibit CCK- and EGF-induced activation of c-Raf-1 or ERK1/2. In conclusion, the present study shows that PTx-sensitivity of receptor-induced ERK1/2 activation could be a consequence of disinhibition of the adenylyl cyclase signaling pathway, which in turn causes inhibition of c-Raf-1 activation rather than indicating involvement of a PTx-sensitive G protein in this signaling pathway.

Signal Transduction Pathways in Entamoeba histolytica: PKA Activity and Translocation During the Interaction of Trophozoites with FN

Signal transduction mechanisms have been demonstrated to operate in Entamoeba histolytica trophozoites after diverse external stimuli (1). Interaction with FN transduces information into the amebas that activates the phosphoinositide as well as the adenylyl cyclase signaling pathways (2,3). cAMP has been shown to have a modulatory effect on the organization of the actin cytoskeleton to form specialized adhesive structures, and also in the regulation of actin mRNA levels (4). In both processes, the participation of proteins phosphorylated by a cAMP-dependent kinase (PKA) has been inferred by comparison with similar phenomena observed in other eukaryotic cells (5). Here, we present evidence for the presence and cAMP-dependent activation of PKA in trophozoites and the nuclear translocation of its catalytic subunit after stimulation of the cells with FN.

Pituitary Adenylyl Cyclase-activating Peptide Activates Multiple Intracellular Signaling Pathways to Regulate Ion Channels in PC12 Cells

Pituitary adenylyl cyclase-activating peptide (PACAP) stimulates calcium transients and catecholamine secretion in adrenal chromaffin and PC12 cells. The PACAP type 1 receptor in these cells couples to both adenylyl cyclase and phospolipase C pathways, but although phospolipase C has been implicated in the response to PACAP, the role of adenylyl cyclase is unclear. In this study, we show that PACAP38 stimulates Ca(2+) influx in PC12 cells by activating a cation current that depends upon the dual activation of both the PLC and adenylyl cyclase signaling pathways but does not involve protein kinase C. In activating the current, PACAP38 has to overcome an inhibitory effect of Ras. Thus, in cells expressing a dominant negative form of Ras (PC12asn17-W7), PACAP38 induced larger, more rapidly activating currents. This effect of Ras could be overidden by intracellular guanosine-5'-O-3-(thio)triphosphate (GTPgammaS), suggesting that it was mediated by inhibition of downstream G proteins. Ras may also inhibit the current through a G protein-independent mechanism, because cAMP analogues activated the current in PC12asn17-W7 cells, provided GTPgammaS was present, but not in PC12 cells expressing wild type Ras. We conclude that coupling of PACAP to both adenylyl cyclase and phospholipase C is required to activate Ca(2+) influx in PC12 cells and that tonic inhibition by Ras delays and limits the response.

CAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway

: Cellular proliferation is a key factor in the enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). We determined the extent to which adenosine 3':5'-cyclic monophosphate (cAMP) may regulate the in vitro proliferation of cyst epithelial cells derived from human ADPKD cysts. : Epithelial cells from cysts of individuals with ADPKD and from normal human kidney cortex (HKC) of individuals without ADPKD were cultured. The effects of agonists and inhibitors on the rate of cellular proliferation and the activation of extracellular signal-regulated kinase (ERK1/2) were determined. : 8-Br-cAMP (100 micromol/L) stimulated the proliferation of cells from eight different ADPKD subjects to 99.0% above baseline; proliferation was inhibited by protein kinase A (PKA) antagonists H-89 (97%) and Rp-cAMP (90%). Forskolin (10 micromol/L), which activates adenylyl cyclase, increased proliferation 124%, and receptor-mediated agonists arginine vasopressin, desmopressin, secretin, vasoactive intestinal polypeptide, and prostaglandin E2 stimulated proliferation 54.2, 56.3, 46.7, 37.1, and 48.3%, respectively. The mitogen extracellular kinase (MEK) inhibitor PD98059 completely inhibited ADPKD cell proliferation in response to cAMP agonists, but genistein, a receptor tyrosine kinase inhibitor, did not block cAMP-dependent proliferation. cAMP agonists increased the activity of ERK above control levels within five minutes. In contrast to ADPKD, proliferation and ERK activity of cells derived from normal HKC were not stimulated by cAMP agonists, although electrogenic Cl- secretion was increased by these agonists in both ADPKD and HKC cell monolayers. : We conclude that cAMP agonists stimulate the proliferation of ADPKD but not HKC epithelial cells through PKA activation of the ERK pathway at a locus distal to receptor tyrosine kinase. We suggest that the adenylyl cyclase signaling pathway may have a unique role in determining the rate of cyst enlargement in ADPKD through its actions to stimulate cellular proliferation and transepithelial solute and fluid secretion.

Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance.

Pituitary gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone stimulate the gonads by regulating germ cell proliferation and differentiation. FSH receptors (FSH-Rs) are localized to testicular Sertoli cells and ovarian granulosa cells and are coupled to activation of the adenylyl cyclase and other signaling pathways. Activation of FSH-Rs is considered essential for folliculogenesis in the female and spermatogenesis in the male. We have generated mice lacking FSH-R by homologous recombination. FSH-R-deficient males are fertile but display small testes and partial spermatogenic failure. Thus, although FSH signaling is not essential for initiating spermatogenesis, it appears to be required for adequate viability and motility of the sperms. FSH-R-deficient females display thin uteri and small ovaries and are sterile because of a block in folliculogenesis before antral follicle formation. Although the expression of marker genes is only moderately altered in FSH-R -/- mice, drastic sex-specific changes are observed in the levels of various hormones. The anterior lobe of the pituitary gland in females is enlarged and reveals a larger number of FSH- and thyroid-stimulating hormone (TSH)-positive cells. The phenotype of FSH-R -/- mice is reminiscent of human hypergonadotropic ovarian dysgenesis and infertility.

Regulating the balance between differentiation and apoptosis: role of CREM in the male germ cells.

Various endocrine and neuronal functions are governed by the cAMP-dependent signaling pathway. In eukaryotes, transcriptional regulation upon stimulation of the adenylyl cyclase signaling pathway is mediated by a family of cAMP-responsive nuclear factors. This family consists of a large number of members which may act as activators or repressors. These factors contain the basic domain/leucine zipper motifs and bind as dimers to cAMP-response elements (CRE). The function of CRE-binding proteins (CREBs) is modulated by phosphorylation by several kinases. Direct activation of gene expression by CREBs requires phosphorylation by the cAMP-dependent protein kinase A to the serine-133 residue. The gene CREM encodes various transcription factors which play key physiological and developmental roles within the hypothalamic-pituitary-gonadal axis. We have previously shown that the transcriptional activator CREMtau is highly expressed in postmeiotic cells. Spermiogenesis is a complex process by which postmeiotic male germ cells differentiate into mature spermatozoa. This process involves remarkable structural and biochemical changes which are under the hormonal control of the hypothalamic-pituitary axis. We have addressed the specific role of CREM in spermiogenesis using CREM-mutant mice generated by homologous recombination. Analysis of the seminiferous epithelium from mutant male mice reveals that spermatogenesis stops at the first step of spermiogenesis. Late spermatids are completely absent while there is a significant increase in apoptotic germ cells. A series of postmeiotic germ cell-specific genes are not expressed. Mutant male mice completely lack spermatozoa. This phenotype is reminiscent of cases of human infertility.