An RGD sequence in the P2Y(2) receptor interacts with alpha(V)beta(3) integrins and is required for G(o)-mediated signal transduction.

The P2Y(2) nucleotide receptor (P2Y(2)R) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the P2Y(2)R to K562 erythroleukemia cells was inhibited by antibodies against alpha(V)beta(3)/beta(5) integrins and the integrin-associated thrombospondin receptor, CD47. Immunofluorescence of cells transfected with epitope-tagged P2Y(2)Rs indicated that alpha(V) integrins colocalized 10-fold better with the wild-type P2Y(2)R than with a mutant P2Y(2)R in which the RGD sequence was replaced with RGE. Compared with the wild-type P2Y(2)R, the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular Ca(2+). Furthermore, an anti-alpha(V) integrin antibody partially inhibited these signaling events mediated by the wild-type P2Y(2)R. Pertussis toxin, an inhibitor of G(i/o) proteins, partially inhibited Ca(2+) mobilization mediated by the wild-type P2Y(2)R, but not by the RGE mutant, suggesting that the RGD sequence is required for P2Y(2)R-mediated activation of G(o), but not G(q). Since CD47 has been shown to associate directly with G(i/o) family proteins, these results suggest that interactions between P2Y(2)Rs, integrins, and CD47 may be important for coupling the P2Y(2)R to G(o).

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium.

The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO(3)(-) secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated P2Y(2) nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating Ca(2+)-activated Cl(-) secretion. However, the value of this treatment in facilitating transepithelial HCO(3)(-) secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the Ca(2+)-dependent anion conductance during activation of luminal P2Y(2) receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current (I(sc)) that declined to a stable plateau phase lasting 30-60 min. The plateau I(sc) after UTP was Cl(-) independent, HCO(3)(-) dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both I(sc) and serosal-to-mucosal HCO(3)(-) flux (J(s-->m)) during a 30-min period. Substitution of Cl(-) with gluconate in the luminal bath to inhibit Cl(-)/HCO(3)(-) exchange did not prevent the increase in J(s-->m) and I(sc) during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in J(s-->m) and I(sc). We conclude that P2Y(2) receptor activation results in a sustained (30-60 min) increase in electrogenic HCO(3)(-) secretion that is mediated via an intracellular Ca(2+)-dependent anion conductance in CF gallbladder.

Furosemide stimulates K transport in HCD57 erythroid cells.

We examined the influence of serum and furosemide on K movement and cell volume in HCD57 cells, a murine erythroleukemia cell line, which require erythropoietin (EPO) for survival. We found that maintenance of cell volume depends on the concentration of serum in the culture medium. In isotonic medium containing 20% serum, HCD57 cells maintain their steady-state volume. In contrast, the cells shrink progressively as medium serum content is reduced. In serum-free medium, raising external K to 75 mm prevents cell shrinkage and a further increase in K to 145 mm results in swelling, revealing a role for K permeability in the regulation of cell volume. Of particular interest has been a serendipitous finding with furosemide. Below an external K concentration of 2.1 +/- 0.3 mm in medium containing 2% serum, furosemide inhibits K uptake, probably stemming from its well known inhibitory action on KCl cotransport. However, above that K concentration, furosemide stimulates K uptake in a dose-dependent manner. Moreover, furosemide potentiates cell shrinkage induced by serum withdrawal. These findings suggest that the transport machinery mediating cellular shrinkage, once primed by serum depletion, becomes receptive to a second stimulus.

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor.

UTP activates P2Y, receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y, receptors with an EC50 of 0.2-1.0 microM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3-1.0 microM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50%, whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases-1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y, receptor agonists.

Identification of the adenylyl cyclase-activating 5-hydroxytryptamine receptor subtypes expressed in the rat submandibular gland.

1. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to increase cyclic AMP production in dispersed cell aggregates from the major salivary glands of the rat. The goal of the present study was to identify the 5-HT receptor subtypes that mediate these effects in rat submandibular glands (SMG). 2. Among the 5-HT receptor subtypes identified in the rat, 5-HT(4(a,b)), 5-HT(6) and 5-HT(7(a,b,c)) activate adenylyl cyclase (AC). We used subtype specific primers to screen rat SMG by reverse transcription-PCR. Results indicate the presence of mRNA for 5-HT(4(b)) and 5-HT(7(a)) but not for 5-HT(4(a)), 5-HT(6) and 5-HT(7(b,c)). 3. In dispersed SMG cells, 5-carboxyamidotryptamine (5-CT), a 5-HT(7) receptor agonist, stimulated cyclic AMP synthesis with higher potency (EC(50)=27+/-5 nM) but lower efficacy than 5-HT, suggesting a 5-HT(7) component and an additional component in the response to 5-HT. The 5-HT(7) contribution was further supported by antagonism of the 5-CT effect by metergoline, a 5-HT(7) antagonist, which exhibited an affinity (K(i)=50 nM) similar to that obtained at the cloned 5-HT(7) receptor. 4. In the presence of a maximally effective concentration of 5-CT, 5-HT produced an additional increase in cyclic AMP production that was inhibited by the 5-HT(4) receptor antagonist, GR113808, suggesting that the second component of cyclic AMP production is mediated by 5-HT(4) receptors. 5. These findings indicate the presence in rat SMG of both 5-HT(4(b)) and 5-HT(7(a)) receptors positively coupled to AC.

Differential regulation of vascular smooth muscle nuclear factor kappa-B by G alpha q-coupled and cytokine receptors.

NF kappaB has been implicated as a downstream effector of G alphaq-coupled receptor signaling, but whether these and cytokine receptors activate NF kappaB similarly remains unclear. Stimulation of rat vascular smooth muscle cell G alphaq-coupled P2Y nucleotide receptors with UTP induces luciferase transcription from a sensitive and specific NF kappaB dependent promoter. However, these responses are only;15% of that to the reference cytokine IL-1 beta. IL-1 beta is a powerful stimulator of I kappaB alpha degradation, RelA nuclear import, and isoform specific NF kappaB enhancer binding in vitro, responses that are not detectable after P2Y receptor stimulation. Expression of two trans -dominant NF kappaB polypeptides suppresses induction of the NF kappaB reporter and also IL-1 beta stimulated monocyte chemoattractant-1 mRNA, which is not induced by UTP. In contrast, UTP induces higher expression of the endogenous COX-2 and IL-6 mRNAs than does IL-1 beta, implying that G alphaq-coupled receptor evokes additional NF kappaB-independent transcription factors in regulating these two genes. P2Y receptors are as effective as the reference growth factor PDGF-BB at inducing CREB, AP-1, SRE and NFAT transcription, which are largely unaffected by IL-1 beta treatment. NF kappaB is less efficiently activated then several other transcriptional effectors of G alphaq-coupled receptor signaling in vascular smooth muscle cells, and is instead preferentially activated by inflammatory cytokines.

Desensitization of P2Y2 receptor-activated transepithelial anion secretion.

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current (Isc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the Isc with a potency profile of ATP = UTP > 2-methylthioATP >> alpha,beta-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2 receptor agonist UTP and increased in a concentration-dependent manner (IC50 approximately 10(-6) M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 x 10(-6) M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10(-5) M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.

Salivary gland P2 nucleotide receptors.

The effects of ATP on salivary glands have been recognized since 1982. Functional and pharmacological studies of the P2 nucleotide receptors that mediate the effects of ATP and other extracellular nucleotides have been supported by the cloning of receptor cDNAs, by the expression of the receptor proteins, and by the identification in salivary gland cells of multiple P2 receptor subtypes. Currently, there is evidence obtained from pharmacological and molecular biology approaches for the expression in salivary gland of two P2X ligand-gated ion channels, P2Z/P2X7 and P2X4, and two P2Y G protein-coupled receptors, P2Y1 and P2Y2. Activation of each of these receptor subtypes increases intracellular Ca2+, a second messenger with a key role in the regulation of salivary gland secretion. Through Ca2+ regulation and other mechanisms, P2 receptors appear to regulate salivary cell volume, ion and protein secretion, and increased permeability to small molecules that may be involved in cytotoxicity. Some localization of the various salivary P2 receptor subtypes to specific cells and membrane subdomains has been reported, along with evidence for the co-expression of multiple P2 receptor subtypes within specific salivary acinar or duct cells. However, additional studies in vivo and with intact organ preparations are required to define clearly the roles the various P2 receptor subtypes play in salivary gland physiology and pathology. Opportunities for eventual utilization of these receptors as pharmacotherapeutic targets in diseases involving salivary gland dysfunction appear promising.

Development and characterization of immortalized rat parotid and submandibular acinar cell lines.

The purpose of this investigation was to develop well-differentiated rat parotid and submandibular acinar cell lines. Acinar cells dissociated from rat parotid and submandibular glands were grown on Mitomycin C-treated 3T3 fibroblasts or Matrigel in primary culture and transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Cytokeratin analysis via indirect immunofluorescence and receptor mediated changes in intracellular calcium and cyclic AMP were assessed and used for the identification and selection of immortalized epithelial cells. Of the more than 60 clonal cell lines, four retained moderate to high levels of acinar differentiation through >60 passages. Ultrastructurally, there were tripartate junctional complexes and moderate amounts of rough endoplasmic reticulum and secretory granules. Functional studies indicated that beta-adrenoceptors, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production in all cell lines. Alpha-adrenoceptors, muscarinic cholinoceptors, and P2U-purinoceptor agonists were effective in increasing intracellular inositol phosphate production and free calcium levels whereas substance P was ineffective. These data document the utility of the SV40 plasmid in immortalizing rat parotid and submandibular acinar cells that retain most of the features of acinar differentiation.

Salivary gland nucleotide receptors: evidence for functional expression of both P2X and P2Y subtypes.

A growing body of information now supports the suggestion that P2 receptors for extracellular nucleotides (primarily ATP) have a role in regulating salivary gland function. There is solid pharmacological and molecular evidence for the presence of P2X ligand-gated ion channel nucleotide receptors (P2X4 and P2X7/P2Z). More recently, our group and others have obtained evidence that multiple P2Y G protein-coupled nucleotide receptors (P2Y1 and P2Y2) are also expressed. Our studies have focused on defining the conditions under which P2Y receptors are expressed, the functional consequences of their activation, and the importance of co-expression of P2X and P2Y receptors. Functional and molecular approaches have been used to identify the P2 subtypes in salivary glands and in salivary cell lines. Assays include measurement of changes in [Ca2+]i, changes in transcellular short circuit current in monolayers, and RT-PCR to assess changes in receptor mRNA levels. The main observations are: (1) P2Y1 receptor activity is present in the submandibular gland (SMG) of immature rats but decreases over the first four weeks following birth, although mRNA levels remain relatively constant; (2) P2Y2 receptors are present in the cell lines and are up-regulated during short-term culture of normal parotid, sublingual, and SMG cells and following ligation of the main excretory duct of SMG; and (3) the P2X subtypes, P2X4 and P2X7, and the P2Y subtypes, P2Y1 and P2Y2, are co-expressed in salivary glands and salivary cell lines, and exhibit distinct basolateral versus apical localization in polarized cell monolayers as well as discrete patterns of intracellular signaling.

A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion.

Because of the lack of salivary gland cell lines suitable for Ussing chamber studies, a recently established rat parotid acinar cell line, Par-C10, was grown on permeable supports and evaluated for development of transcellular resistance, polarization, and changes in short-circuit current (Isc) in response to relevant receptor agonists. Par-C10 cultures reached confluence in 3-4 days and developed transcellular resistance values of >/=2,000 Omega . cm2. Morphological examination revealed that Par-C10 cells grew as polarized monolayers exhibiting tripartite junctional complexes and the acinar cell-specific characteristic of secretory canaliculi. Par-C10 Isc was increased in response to muscarinic cholinergic and alpha- and beta-adrenergic agonists on the basolateral aspect of the cultures and to ATP and UTP (through P2Y2 nucleotide receptors) applied apically. Ion replacement and inhibitor studies indicated that anion secretion was the primary factor in agonist-stimulated Isc. RT-PCR, which confirmed the presence of P2Y2 nucleotide receptor mRNA in Par-C10 cells, also revealed the presence of mRNA for the cystic fibrosis transmembrane conductance regulator and ClC-2 Cl- channel proteins. These findings establish Par-C10 cells as the first cell line of salivary gland origin useful in transcellular ion secretion studies in Ussing chambers.

Development and characterization of SV40 immortalized rat parotid acinar cell lines.

Rat parotid salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E1, and forskolin were effective activators of intracellular cyclic adenosine 3':5'-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.

Upregulation of P2Y2 nucleotide receptors in rat salivary gland cells during short-term culture.

In contrast to the widespread expression of G protein-coupled P2Y2 receptors for extracellular nucleotides in permanent cell lines of salivary gland origin, there is less evidence for robust P2Y2 receptor activity in normal rat salivary gland cells assayed immediately after isolation. We examined the effect of short-term culture (3 h to 6 days) of normal rat submandibular gland (SMG) cells on P2Y2 receptor activity and mRNA expression. Results indicate that increases in the intracellular free Ca2+ concentration in SMG cells in response to the P2Y2 receptor agonist UTP (100 microM) were detectable after 3 h in culture and that after 3 days in culture the magnitude of the response to UTP was similar to that obtained with maximal muscarinic cholinoceptor activation. The Ca2+ mobilization response exhibited the pharmacological profile (UTP = ATP > 2-methylthioadenosine 5'-triphosphate) typical of the P2Y2 receptor subtype and was accompanied by enhanced production of inositol phosphates, reflecting the activation of phospholipase C ubiquitously associated with P2Y2 receptors. The time-dependent increase in P2Y2 receptor activity was accompanied by an increase in the steady-state level of P2Y2 receptor mRNA, as assessed by reverse transcription-polymerase chain reaction. Other studies revealed that the increased P2Y2 receptor activity was independent of cell proliferation, was similar in serum-containing and defined culture media, and was blocked by inhibitors of transcription and translation. Upregulation of the P2Y2 receptor was observed in both acinar cell- and ductal cell-enriched cultures of the SMG and in cells isolated from rat parotid and sublingual glands but not in cells isolated from the pancreas. These in vitro results were complemented by in vivo studies in which P2Y2 receptor activity and mRNA levels were increased in SMG after ligation of the main excretory duct but were not increased in the contralateral, nonligated gland. These findings suggest that changes in the expression and activity of the P2Y2 receptor in salivary gland cells may be related to pathological challenges to the gland in vivo.

Identification and characterization of P2Y2 nucleotide receptors in human retinal pigment epithelial cells.

P2 nucleotide receptor expression in cultured human retinal pigment epithelial (RPE) cells was investigated using the photoaffinity ATP analog BzATP, polymerase chain reaction of reverse-transcribed RNA (RT-PCR) and fura-2 fluorescence measurement of changes in intracellular free calcium concentration ([Ca2+]i). In experiments carried out in RPE cells at passage 10-15, addition of micromolar concentrations of ATP, UTP, and ATPgammaS to RPE cells resulted in a rapid, transient 3.5-fold increase in [Ca2+]i followed by a prolonged elevation that was twofold above the original baseline. Similar results were obtained from cells at passage 2. Characteristics of nucleotide-stimulated calcium mobilization in RPE cells, including partial inhibition by pertussis toxin, suggest that a G protein-coupled receptor mediates this response. Consistent with the expression of a P2Y2 nucleotide receptor subtype in RPE cells, [alpha-32P]BzATP labeled a 53-kDa protein in plasma membranes, and RT-PCR revealed the presence of P2Y2 receptor RNA. Adenosine had no effect on [Ca2+]i in RPE cells, indicating that the A2 subtype of P1 receptor described previously in human RPE is not involved in the response to nucleotides. Together the results indicate that human RPE cells express functional P2Y2 nucleotide receptors.

Changes in P2Y1 nucleotide receptor activity during the development of rat salivary glands.

Experiments that used dispersed salivary gland cells from 1-day-old rats indicated the presence of the P2Y nucleotide receptor subtype, P2Y1, based on the agonist potency profile for mobilization of intracellular free Ca2+ [2-methylthio-ATP > ADP > adenosine 5'-O-(2-thiodiphosphate) > ATP, with UTP ineffective] and sequence analysis of reverse transcription-polymerase chain reaction (RT-PCR) products obtained with P2Y1 receptor-specific primers. P2Y1 receptor activity appears to be developmentally regulated, since Ca2+ mobilization in response to the P2Y1-selective agonist, 2-methylthio-ATP, decreased as animal age increased, with the maximal response of 129 +/- 23 nM obtained in 1-day-old animals, decreasing to 30 +/- 3 nM in 4-wk-old animals. However, the abundance of P2Y1 receptor mRNA, assessed by semiquantitative RT-PCR, did not change over this time period, suggesting that receptor activity is regulated by some mechanism other than changes in steady-state levels of P2Y1 receptor mRNA. These findings indicate that functional P2Y1 nucleotide receptors are expressed in immature salivary glands and that receptor activity decreases as the glands mature, suggesting that P2Y1 receptors may have an important role during salivary gland development.

Development and characterization of SV40 immortalized rat submandibular acinar cell lines.

Rat submandibular salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the beta-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.

A regulatory role in mammalian salivary glands for 5-hydroxytryptamine receptors coupled to increased cyclic AMP production.

Although a functional role for serotonin (5-hydroxytryptamine, 5-HT) has been defined in the salivary glands of some lower species, relatively few data supporting a role for 5-HT in the regulation of mammalian salivary glands have been presented. Our initial results from polymerase chain reaction studies in cells of mammalian submandibular gland origin using consensus sequence primers from G protein-coupled receptors suggested the presence of mRNA for a 5-HT receptor in these cells. Based on this observation, the question of a role for 5-HT in mammalian submandibular gland function was re-addressed, using isolated, perfused rat submandibular glands and dispersed-cell aggregates from this gland. In perfused glands, 5-HT decreased the rate of saliva flow initiated by acetylcholine by about 50% and increased the amount of protein in the saliva two-fold. In dispersed-cell aggregates, 5-HT elicited a concentration-dependent increase in the accumulation of adenosine 3',5' monophosphate (cyclic AMP; EC50 = 660 +/- 110 nM). In addition, functional studies, as well as radioligand binding experiments, indicated that the effects of 5-HT are independent of beta-adrenoceptors. Accumulation of cAMP in gland cells was consistent with a direct action of 5-HT on adenylyl cyclase. Similar cyclic AMP responses to 5-HT were observed in cells isolated from mouse and opossum submandibular glands and rat sublingual and parotid glands. Our findings suggest the presence of a 5-HT receptor in mammalian salivary glands coupled to the stimulation of adenylyl cyclase and, at least in rat submandibular gland, involved in modifying the volume and protein content of saliva.

Ergovaline binding and activation of D2 dopamine receptors in GH4ZR7 cells.

Ergovaline inhibition of radioligand binding to the D2 dopamine receptor and ergot alkaloid inhibition of vasoactive intestinal peptide (VIP)-stimulated cyclic AMP production in GH4ZR7 cells, stably transfected with a rat D2 dopamine receptor, were evaluated. Ergovaline inhibition of the binding of the D2-specific radioligand, [3H]YM-09151-2, exhibited a KI (inhibition constant) of 6.9 +/- 2.6 nM, whereas dopamine was much less potent (370 +/- 160 nM). Ergot alkaloids were also effective in inhibiting VIP-stimulated cyclic AMP production, with EC50 values for ergovaline, ergonovine, alpha-ergocryptine, ergotamine, and dopamine of 8 +/- 2, 47 +/- 2, 28 +/- 2, 2 +/- 1, and 8 +/- 1 nM, respectively. Inhibition of cyclic AMP production by ergovaline was blocked by the dopamine receptor antagonist, (-)-sulpiride (IC50, 300 +/- 150 nM). Our results indicate that ergot compounds, especially ergovaline, bind to D2 dopamine receptors and elicit second messenger responses similar to that of dopamine. These findings suggest that some of the deleterious effects of consumption of endophyte-infected tall fescue, which contains several ergot alkaloids including ergovaline, may be due to D2 dopamine receptor activation.