Effects of cholinergic and α-adrenergic agonists on the monovalent ion content of rat submandibular gland acinar cells studied by X-ray microanalysis

 The effects of cholinergic and α-adrenergic stimulation (in vivo and in vitro) on the monovalent ion content of rat submandibular gland acinar cells were evaluated at the subcellular level by X-ray microanalysis. Fragments of glands or enzymatically dispersed acini were slam-frozen and cut into ultrathin cryosections. Spectra were collected from secretory granules, nucleus, the basal cytoplasm containing endoplasmic reticulum and the apical cytoplasm identified between secretory granules. No significant changes in Na and Cl content were observed after the isolation of acini, but the K concentration decreased compared with cells from in situ glands. The Cl and K content in all four compartments studied decreased significantly after cholinergic stimulation both in vivo and in vitro but in a more restricted fashion after α-adrenergic stimulation. Our findings indicate that: (1) the physiological mechanisms regulating the monovalent ion composition of submandibular cells are relatively well preserved in isolated acinar cells; (2) the results from in vivo experiments are in good agreement with those from in vitro experiments; and (3) the effects of cholinergic and α-adrenergic stimulation on the K⁺ and Cl^–efflux at the subcellular level are similar but the response is generally less with α-adrenergic stimulation. Accepted: 24 April 1997     

Protein production and release by dispersed rat submandibular gland cells in vitro after adrenergic stimulation

Enzymatically dispersed cell aggregates were prepared from rat submandibular glands. Cells were responsive to α- and β-adrenergic agonists, as measured by net K⁺ release and radiolabeled protein secretion, respectively. Protein production by submandibular gland cells was constant during the 90 min experimental period. Specific agonist and antagonist experiments demonstrated that both α- and β-adrenergic receptor stimulation were required for maximum secretion of newly synthesized protein. Proteins were radiolabeled with [³⁵S] methionine and both soluble cell and secreted proteins examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography. A broad size range of newly synthesized proteins was detected (Mr～10⁴?5 × 10⁵). Adrenergic stimulation (1-epinephrine) specifically increased the secretion of certain radiolabeled proteins and, in addition, resulted in both cellular and secreted proteins with electrophoretic characteristics distinct from that of control preparations.     

Heterogeneity and contact-dependent regulation of amylase release by individual acinar cells

We have used a reverse hemolytic plaque assay to investigate the amylase release of single and aggregated pancreatic acinar cells. We have found that a minority of single acinar cells released detectable amounts of amylase under basal conditions and were modestly stimulated, in a dose-dependent manner, during a 30-min exposure to concentrations of carbamylcholine (CCh) ranging from 10^?8 to 10^?5 M. This stimulation was largely accounted for by the recruitment of additional secreting cells, rather than by a significant increase in their individual secretory output. We have also observed that aggregates comprising two to five acinar cells secreted more frequently and released more amylase than single acinar cells in the presence of each of the CCh concentrations tested. Under both basal conditions and following CCh stimulation, the proportion of secreting aggregates and their amylase output increased linearly with the aggregate size. Under basal conditions as well as in the presence of secretagogue concentrations in the 10^?8?10^?7 M range, individual cells contributed similarly to amylase secretion whether they were single or part of aggregates. By contrast, following stimulation by 10^?6?10^?5 M CCh, aggregated cells showed a much higher average secretion than single cells. Investigating the mechanism of this contact-dependent effect, we found that 10^?3 M heptanol did not significantly modify the secretion of single cells and markedly promoted the basal amylase release of acinar cell pairs. This effect was associated with a marked reduction in gap junctional communication between acinar cells, as evaluated by microinjection of Lucifer yellow, and was not observed during exposure to high concentrations of CCh, which also reduced junctional communication. These data show that pancreatic acinar cells are intrinsically heterogeneous in their ability to release amylase and that their basal as well as stimulated secretion are promoted by the establishment of direct intercellular contacts. Our experiments also suggest that junctional coupling contributes to the contact-dependent mechanism which enhances the recruitment of secreting cells and their individual output. These observations strengthen the view that direct interactions between acinar cells are essential in the control of pancreatic secretion. © 1994 Wiley-Liss, Inc.     

The antagonistic effect of dexamethasone and insulin on alpha-fetoprotein secretion by cultured H4-II-E-C3 cells derived from the Reuber H-35 hepatoma.

Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10^?7M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10^?8M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10^?7 to 5 × 10^?9M) and insulin (8.7 × 10^?8 to 8.7 × 10^?11M) were added simultaneously, insulin diminished the stimulatory effect of dexamethasone. When α-fetoprotein secretion was elevated by dexamethasone and the medium was replaced by media containing either insulin or no hormones, the rate of α-fetoprotein secretion diminished more rapidly with the insulin-supplemented medium. Alone or in combination, insulin and dexamethasone had little effect on albumin secretion.     

The effect of aging on prolactin secretion in rat pituitary monolayer culture.

A high basal rate of prolactin (PRL) secretion (.16±.03 μg/well/hr) was produced for over four weeks by pre-confluent male rat pituitary monolayer cell cultures. When the media was changed, a rapid release of microgram quantities of PRL occurred followed by a return to the basal PRL secretory rate by seven hours. Theophylline (3.8×10^?3M), but not dibutyrl cAMP (1×10^?3M), produced a significant (p<.02) increase in PRL secretion, and simultaneous addition of these agents potentiated the PRL secretory rate. TRH (2×10^?8M) had no effect on PRL release by six hours, whereas dopamine (4.9×10^?5M) produced a significant suppression (p<.002) of PRL secretion. In addition, the effects of theophylline, TRH, and dopamine on PRL secretion were similar in cultures of various ages. Ovine prolactin in concentrations up to 50 μg per ml produced no change in PRL secretion during 72 hours of incubation suggesting that PRL feedback control of its own secretion may be transmitted via the hypothalamus. These studies show that a high rate of PRL secretion can be maintained by pre-confluent monolayer cultures for extended periods of time, permitting repeated experimentation on the same wells.     

ACTH secretion in mouse pituitary tumor cells in culture: Inhibition of CRF-stimulated hormone release by somatostatin

Synthetic corticotropin-releasing factor (CRF) stimulates ACTH secretion in the clonal mouse pituitary cell strain AtT20/D16v (D16) in a dose-dependent manner with a half-maximal effect at 2×10^?9M. A single dose of 5×10^?9M CRF maximally stimulates the rate of ACTH secretion during the initial two hrs of treatment. During the period of maximal CRF stimulation intracellular hormone concentration declines progressively to a nadir at 4 hrs. During the ensuing 24 hrs of incubation intracellular hormone levels in CRF-stimulated cells increase gradually toward control values. Somatostatin (SRIF) inhibits the secretory response to CRF. This action of SRIF is dose-dependent with a half-maximal effect at 1×10^?9M and results in decreased maximal ACTH secretion with little effect on the ED₅₀ for CRF.     

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H-2 major histocompatibility complex

In rat lacrimal gland, cholinergic, α- or β-adrenergic or methylxanthine stimulations of protein secretion are extracellular calcium dependent. 10 μM trifluoperazine (TFP) inhibited only cholinergic and α-adrenergic stimulations. Half maximal effect was observed at 30 μM, with all inducers except norepinephrine (3 μM). 10 or 30 μM TFP also suppressed the decrease of L-[³H]leucine incorporation into protein due to carbamylcholine. 100 μM TFP inhibited protein secretion and L-[³H]leucine incorporation. 500 μM TFP promoted cell lysis. It is suggested that: (a) at 100 μM TFP, inhibition is not specific for protein secretion; (b) at 30 μM TFP, inhibition could be related to a role of calmodulin in the secretory regulation process.     

Effects of papaverine on basal and on isoproterenol-stimulated renin secretion from rat kidney slices

Papaverine inhibited the basal renin secretion of rat kidney slices incubated in a physiological salt solution at 37°C. Inhibition was concentration-dependent; secretion was 99 ± 0.2 % inhibited by 5 × 10^?4 M papaverine, and 8 × 10^?5 M was the estimated ED₅₀. In contrast, 2 × 10^?4 M IBMx (3-isobutyl-1-methyl-xanthine) increased the rate of secretion from 215 ± 17 to 366 ± 30 ng hr^?1mg^?1/20 min (p < 0.001). Isoprotenol (4 × 10^?7, 8 × 10^?7, and 5 × 10^?6 M) stimulated renin secretion in a concentration-dependent manner; the stimulatory effects were antagonized by papaverine but unaffected by IBMx. Thus, two known inhibitors of phosphodiesterase--IBMx and papaverine--produce sharply contrasting effects on basal and on isoproterenol-stimulated renin secretion from rat kidney slices.     

SECRETORY PROTEIN SYNTHESIS IN THE STIMULATED RAT PAROTID GLAND : Temporal Dissociation of the Maximal Response from Secretion

Administration of the β-adrenergic drug, isoproterenol (IPR), affects the release of 98% of stored amylase from rat parotid gland acinar cells. A period of 6 h elapses from the onset of secretion to the maximum [¹⁴C]phenylalanine (Phe) incorporation into total protein and amylase. 10 h after IPR administration the rate of [¹⁴C]Phe incorporation into total protein was no longer elevated above that of control. Incorporation into amylase, however, remained elevated above the control by 2.3 times. This latent period may reflect: (a) reduced amounts of available ATP which occurs as a result of the process of secretion as well as (b) the time required for reorganization of cellular organelles and membranes after secretion. The latent period after IPR-induced secretion appears similar to the latent period which has recently been reported to occur after physiologic release of amylase from the parotid gland during the diurnal feeding cycle of the rat. These observations support the existence of a positive feedback system operant in the parotid acinar cell linking the release of secretory proteins with their synthesis. The period of greatest protein synthesis is, however, temporally dissociated from the secretory process.     

Studies on action mechanisms of a possible false cholinergic transmitter, (2-hydroxyethyl) methyldiethylammonium

(2-Hydroxyethyl) methyldiethylammonium (DEC; Diethylcholine) was found to inhibit cholinergic fibers slowly, both in skeletal muscle (ED₅₀: 2.25 × 10^?5 M in chick biventer cervicis and 42 mg/kg in rat sciatic-gastrocnemius) and in smooth muscle preparations (ED₅₀: 7.7 × 10^?4 M in transmurally stimulated guinea-pig ileum) without having any effect on dose-response curves of acetylcholine to contract chick biventer cervicis, frog rectus abdominis and guinea-pig ileum. These results indicate that DEC acts at the prejunctional nerve fibers, but not at the postjunctional cholinergic receptor sites. DEC was acetylated efficiently both by choline acetyltransferase and by minced rat brain, suggesting that it can be acetylated to acetyl-DEC in the nerve ending. Acetyl-DEC was found to block acetylcholine actions competitively both in smooth and in skeletal muscle preparations (1 × 10^?3 ? 1 × 10^?2M) indicating that the acetylated product of DEC can serve as an antagonist at the cholinergic receptor site. It is therefore concluded that DEC is a false cholinergic transmitter.     

Actions of a nicotinic agonist,DMPP, on intestinal ion transport invitro

High-dose carbachol (10^?3 M) has previously been shown to cause NaCl absorption in short-circuited rabbit ileum. The mechanism of this effect may be norepinephrine release induced by carbachol activation of presynaptic nicotinic receptors on adrenergic neurons. Norepinephrine then interacts with postsynaptic α-adrenergic receptors on intestinal mucosal cells to stimulate neutral NaCl absorption and inhibit electrogenic bicarbonate secretion. The present paper examines the $\text{in vitro}$ intestinal ion transport effects of DMPP an agent which is more specific than carbachol on nicotinic cholinergic receptors. DMPP (10^?5 M) caused a transient increase followed by prolonged depression of the short-circuit current, increased NaCl absorption and increased tissue conductance. This effect was antagonized by hexamethonium and phentolamine. It is concluded that nicotinic cholinergic agents stimulate norepinephrine release from adrenergic nerves and effect intestinal ion transport just as norepinephrine does.     

Effects of some steroid hormones on head-to-head association in bovine spermatozoa

In the presence of 2 × 10^?6 M Ca²⁺ in Tris-buffered medium 0.5 × 10^?6 M, oestradiol-17β or corticosterone significantly increased the head-to-head association of washed bull spermatozoa; in the same concentration, testosterone and 5α-dihydrotestosterone had no significant effects, whereas progesterone significantly dissociated the associated spermatozoa. At 8 × 10^?6 M Ca²⁺ in the same medium, all five hormones increased the association to about the same level. In Tyrode solution with a Ca²⁺ concentration of 1.4 × 10^?3 M, oestradiol-17β and corticosterone acted as above, whereas progesterone and the two testosterones effected dissociation. In Tyrode solution each of the dissociating hormones was combined with oestradiol-17β. In each case a sum of the effects of the two hormones was obtained without any stimulation or inhibition. All five hormones still produced significant effects at 5 × 10^?7 M in Tyrode solution. A corresponding value for ATP was found at 1 × 10^?5 M.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

In vitro effect of cortisol and urotensin I on arginine vasotocin and isotocin secretion from pituitary cells of gilthead sea bream Sparus aurata

This study aimed at determining whether in vitro secretion of two neuropeptides, arginine vasotocin (AVT) and isotocin (IT), from pituitary cells of gilthead sea bream Sparus aurata was affected by cortisol and urotensin (UI). Pituitary cells were exposed to 1·4 × 10^?8, 1·4 × 10^?7 and 0·4 × 10^?6 M cortisol and 10^?12, 10^?10 and 10^?8 M UI for 6, 24 and 48 h, respectively. AVT and IT contents were determined in the culture media by high‐performance liquid chromatography (HPLC). An increase in AVT secretion and a decrease in IT secretion were observed at all cortisol doses. UI increased AVT secretion after 6 h of incubation at all doses. After 24 h, however, only the highest dose of UI still displayed an effect. IT secretion was not influenced by UI. It was thus demonstrated that cortisol does influence AVT and IT secretion from S. aurata pituitary cells, while UI regulates AVT secretion, as a component of hypothalamic–pituitary–interrenal (HPI) axis in this species.     

Acetylcholine potentiation of field stimulated rat vas deferens: A pre-synaptic muscarinic mechanism?

Acetylcholine (ACh) was found to markedly enhance the nerve stimulation induced twitch response of isolated, field-stimulated rat vas deferens (RVD). The ED₂₀₀ (concentration which enhances the twitch response to 200% of control) for this potentiation was 6 × 10^?6M with the maximum twitch response being increased by more than 3 fold (325 ± 30%). Carbachol (ED₂₀₀ = 8.5 × 10^?7M) showed identical results. With each drug the potentiation was competitively antagonized by atropine (10^?7?10^?5M). Physostigmine 10^?8?10^?6M) both enhanced the basal twitch response (215 ± 8% of control at 10^?5M) and the sensitivity of the RVD to ACh (ED₂₀₀ = 3.3 × 10^?7M) but not to carbachol. Atropine, on the other hand reduced the basal twitch response by 18 ± 3% at 10^?5M. Hemicholinium (10^?4M) also reduced the basal twitch responses by 23 ± 5%. ACh (10^?7M?10^?5M) did not modify the responses of unstimulated RVD to norepinephrine or KCl suggesting a pre-synaptic site of action. Taken together these results are compatible with the presence of a pre-junctional, excitatory muscarinic mechanism in the field stimulated RVD. That this cholinergic system may be of physiological significance is supported by the observations that atropine and hemicholinium depress while physostigmine enhances the twitch response in the absence of exogenous ACh.     

The secretory activity of rat nasal glands and the effect of cholinergic drugs

Summary The secretory behaviour of rat nasal glands, under normal conditions and after the application of cholinergic drugs, has been studied using morphological and radiobiochemical techniques.Autoradiography and electrophoresis provide evidence for the selective incorporation of ³H-arginine into the glycoprotein containing fraction of the nasal glandular secretion. Radiobiochemical experiments show that labelled arginine is rapidly incorporated into the acinar cells of unstimulated glands, although it takes approximately 4 h before the labelled secretory proteins leave the cells. The secretion of proteins is stimulated by the parasympathetic agonist pilocarpine, whose main action is to promote discharge. Histological sections show a depletion of secretory granules after pilocarpine treatment. The cholinergic antagonist atropine inhibits the secretion; the acinar cells are completely filled with secretory granules following this treatment. The time course of the events following atropine administration suggests that there is no feed-back system controlling glycoprotein synthesis.The techniques employed here therefore appear to be useful for studying the effects of drugs that interfere with the secretory activity of the nasal glands.     

Delayed inhibition of gap-junctional intercellular communication in the acinar cells of rat submandibular glands induced by parasympathectomy and cholinergic agonists

In this study, the effects of parasympathectomy and cholinergic agonists on gap-junctional intercellular communication and salivary secretion were investigated to clarify the involvement of salivary secretion in delayed uncoupling between acinar cells of rat submandibular glands. Gap-junctional intercellular communication was monitored as dye-coupling in the acinar cells of isolated acini by the transfer of Lucifer Yellow CH. Parasympathectomy induced dye-uncoupling in the acinar cells isolated from denervated salivary glands 12 hr after parasympathectomy-induced salivary secretion. Intraperitoneal application of carbachol (CCh), acetylcholine, pilocarpine, but not isoproterenol, stimulated salivary secretion, and then induced dye-uncoupling in the acinar cells 12 hr later. Atropine suppressed both the salivary secretion and delayed dye-uncoupling induced by parasympathectomy and CCh, when atropine was applied intraperitoneally before the induction of salivary secretion. However, atropine did not suppress the delayed dye-uncoupling by intraperitoneal application of CCh, when atropine was injected after the cessation of CCh-induced secretion. These results suggest that delayed inhibition of gap-junctional intercellular communication by parasympathectomy and cholinergic agonists in rat submandibular glands might be related to the change of secretory function after salivary secretion.     

Evidence for an intracellular somatostatin receptor in pancreas: a comparative study with reference to gastric mucosa

Specific binding sites for ¹²⁵I-Tyr₁somatostatin-14 were comparatively demonstrated in isolated rat pancreatic and gastric parietal cells. In both materials, the sites occurred mostly in cytosol, with apparent affinities of 1×10^?10M and 3×10^?11M, respectively, in pancreatic and gastric cells. Somatostatin-14 stimulated cytosolic phosphoprotein phosphatases (PPPases) in pancreas as well as in gastric mucosa with concentrations for half maximal effect consistent with binding affinities. Somatostatin 28 mimicked somatostatin 14 stimulation with a higher efficacy but an equivalent potency. Secretin and cholecystokinin C terminal octapeptide were ineffective. Furthermore, in intact isolated cells, somatostatinic stimulation of PPPases was blocked by 5×10^?4M dinitrophenol. We therefore suggest that in pancreas as in gastric mucosa, somatostatin's inhibitory effect on secretory functions could involve protein dephosphorylation mediated by an intracellular receptor.     

Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

³H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the $\text{Torpedo}$ fish. The K_d for binding to intact and solubilized rat brain membranes was 5.6 × 10^?9 M and 1.1 × 10^?8M respectively, and the binding capacity 2.0 × 10^?14 and 3.0 × 10^?13 moles /mg protein respectively. The K_d for Torpedo membranes was 3.1 × 10^?7M and the binding capacity 6.8 × 10^?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in $\text{Torpedo}$ membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with ³H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.