Biomedical subjects

J M Camden

Publications and source records attributed to J M Camden.

9 recordsLinked to original sources

PubMed1997

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.

Animals↗

PubMed1997

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.

Adenosine Triphosphate↗

PubMed1996

Potentiation of regulatory volume decrease by P2U purinoceptors in HSG-PA cells.

HSG-PA human salivary gland duct cells exhibit progressively increased regulatory volume decrease (RVD) in response to decreased medium osmolarity. The P2U purinoceptor agonist UTP causes a potentiation of RVD, the extent of which is most pronounced in 220 mosM medium and is least apparent in 180 mosM medium. We examined the underlying mechanisms for this effect. Exposure of HSG-PA cells to UTP promotes Ca2+ mobilization, hyperpolarization, and net K+ efflux, suggesting the participation of Ca(2+)-activated K+ channels in RVD. To delineate the anion counterpart of K+ movement during RVD, cell swelling in the presence of gramicidin, which abolishes the membrane potential, was measured. In response to a sudden dilution in hypotonic media, gramicidin-treated cells swelled immediately, followed by a "secondary swelling" in 180 but not in 220 mosM medium. The results suggest that in 180 mosM cells perform spontaneous RVD mediated by increased anion conductance. In 220 mosM medium in which RVD is minimal, the increase in anion conductance is marginal. In our model of RVD in which cells were challenged by UTP, the ensuing hyperpolarization provides the driving force for net Cl- efflux, which is confirmed by tracer flux studies during purinoceptor-activated RVD. Thus RVD, which has long been regarded as a self-sufficient cellular program, appears to be subject to extracellular control in HSG-PA cells through receptor-mediated processes.

Biological Transport↗

PubMed1996

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.

Animals↗

PubMed1995

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.

Adenosine Monophosphate↗

PubMed1992

Regulation of secretion by vasoactive intestinal peptide in isolated perfused rat submandibular glands.

The isolated, perfused gland was used to examine the regulation of saliva volume and protein content by vasoactive intestinal peptide (VIP). In the absence of other secretagogues, VIP produced a modest, sustained saliva flow with a biphasic dose-response curve in which saliva volume was greatest at 1 nM VIP (28.5 +/- 3.8 microliters in the first 5 min, n = 4) but reduced at lower and higher concentrations. The protein concentration in saliva released in response to VIP (0.86 +/- 0.13 micrograms/microliters) was substantially higher than with 30 nM acetylcholine (0.06 +/- 0.02 micrograms/microliters) or 1 nM substance P (0.30 +/- 0.05 micrograms/microliters). During the first 5 min of stimulation, VIP and substance P were synergistic in terms of volume and protein content whereas inclusion of VIP did not increase acetylcholine-stimulated flow in the first 5 min but produced a higher sustained flow over the next hour. After stimulation with acetylcholine, subsequent addition of VIP transiently enhanced saliva volume and protein content in a monophasic, dose-dependent manner with effects at 1 pM VIP and higher. The responses were different for VIP compared with other cAMP-mobilizing agents and the involvement of multiple VIP receptor subtypes was suggested from experiments in which a VIP antagonist blocked the VIP enhancement of saliva volume but not the increase in protein.

Acetylcholine↗

PubMed1992

Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL.

The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.

Adenylyl Cyclases↗

PubMed1990

The influence of vasoactive intestinal peptide receptors in dispersed acini from rat submandibular gland on cyclic AMP production and mucin release.

Vasoactive intestinal peptide has been identified as an important regulator of submandibular salivary gland function, consistent with its localization with acetylcholine in parasympathetic neurones innervating this gland. Enzymatically dispersed acini from rat submandibular gland are a useful system in which to study gland regulation at the cellular level. Here, three aspects of vasoactive intestinal peptide interactions with acini were examined: inhibition of binding of [125I]-vasoactive intestinal peptide, stimulation of cyclic AMP production and enhancement of mucin release. Vasoactive intestinal peptide and peptide histidineisoleucineamide inhibited [125I]-vasoactive intestinal peptide binding to intact acini, with IC50 values of 16 +/- 3 and 46 +/- 17 nM, respectively. This rank order of potency agrees with that observed previously in assays using rat submandibular gland membranes and is similar to values obtained in assays measuring increases in cyclic AMP production in which the ED50 values for vasoactive intestinal peptide and peptide histidineisoleucineamide were 3.1 +/- 1.8 and 29 +/- 13 nM, respectively. Although vasoactive intestinal peptide stimulation of cyclic AMP production was only about 10% of that seen in response to isoproterenol, the levels of mucin release induced by the two agents were more similar. The ED50 for vasoactive intestinal peptide-stimulated mucin release was 0.12 +/- 0.05 nM, thus suggesting an activation anomaly in the vasoactive intestinal peptide receptor-coupled signal transduction pathway at a point between cyclic AMP production and mucin release.

Animals↗

PubMed1990

Regulation of the neurotensin receptor and intracellular calcium mobilization in HT29 cells.

Regulation of the neurotensin receptor-inositol phosphate-intracellular Ca2+ ((Ca2+]i) pathway was studied in HT29 cells. Preincubation with neurotensin or phorbol 12-myristate, 13-acetate decreased the number of cell surface neurotensin receptors and neurotensin-induced increases of inositol trisphosphates and [Ca2+]i. The phorbol 12-myristate, 13-acetate-(43 +/- 1% at 1 microM) but not the neurotensin (65 +/- 9% at 10 nM)-induced decrease in receptors was blocked by staurosporine. The decrease in cell surface receptors was accompanied by a 55 +/- 7% shift of specifically bound 125I-neurotensin from the plasma membrane fraction to the light vesicle fraction of sucrose density gradients if a 37 degrees C incubation step was included. The time course for desensitization of [Ca2+]i mobilization was more rapid (maximal at approximately 1 min) than for loss of receptors (maximal at 45 min). After a 5-min exposure to neurotensin, the cell surface receptor number rapidly returned to control levels in the absence of agonist, but [Ca2+]i sensitivity to neurotensin recovered only partially. Incubation with carbachol, ATP or phorbol 12-myristate, 13-acetate desensitized the subsequent [Ca2+]i response to neurotensin. These results demonstrate a polyphosphoinositide-[Ca2+]i pathway in HT29 cells stimulable by neurotensin and other agents. The results from pre-incubation studies indicate that the neurotensin receptor-signaling pathway is homologously and heterologously regulated. Finally, differences in time courses for loss and recovery of cell surface receptors and desensitization of the [Ca2+]i response, as well as the lack of effect on 125I-neurotensin binding of other agonists that desensitize the neurotensin [Ca2+]i response, suggest that receptor internalization alone does not account for desensitization of the system.

Binding Sites↗