Calcitonin inhibits thyrotropin-releasing hormone-induced increases in cytosolic Ca2+ in isolated rat anterior pituitary cells

Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 microM Indo-1 AM for 30 min. Indo-1-loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 microM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 microM TRH. In EGTA-pretreated AP cells, the initial EGTA-insensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system.     

Rhythmic bursts of calcium transients in acute anterior pituitary slices

Endocrine cells isolated from the anterior pituitary fire intracellular Ca2+ ([Ca2+]i) transients due to voltage-gated Ca2+ entry. However, the patterns of [Ca2+]i transients within the glandular parenchyma of the anterior pituitary are unknown. Here we describe, using real-time confocal laser microscopy, several spontaneous patterns of calcium signaling in acute pituitary slices prepared from male as well as cycling and lactating female rats. Forty percent of the cells demonstrated a spontaneous bursting mode, consisting of an active period of [Ca2+]i transients firing at a constant frequency, followed by a rest period during which cells were either silent or randomly active. The remaining recordings from endocrine cells either demonstrated random [Ca2+]i transients or were silent. These rhythmic bursts of [Ca2+]i transients, which required extracellular calcium, were detected in lactotrophs, somatotrophs, and corticotrophs within the acute slices. Of significance was the finding that the bursting mode could be adjusted by hypothalamic factors. In slices prepared from lactating rats, TRH recruited more bursting cells and finely adjusted the average duty cycle of [Ca2+]i bursts such that cells fired patterned bursts for approximately 70% of the recording period. Eighty-six percent of these cells were lactotrophs. Thus, the rhythmic [Ca2+]i bursts and their tuning by secretagogues may provide timing information that could encode for one or more cellular functions (e.g. exocytosis and/or gene expression) critical for the release of hormones by endocrine cells in the intact gland.     

Potassium depolarization elevates cytosolic free calcium concentration in rat anterior pituitary cells through 1,4-dihydropyridine-sensitive, omega-conotoxin-insensitive calcium channels

Changes in membrane potential may influence Ca2+-dependent functions through changes in cytosolic free calcium concentration [( Ca2+]i). This study characterized pharmacologically those voltage-dependent Ca2+ channels in normal rat anterior pituitary cells that are involved in the elevation of [Ca2+]i upon high potassium-induced membrane depolarization. The [Ca2+]i was monitored directly by means of the intracellularly trapped fluorescent indicator fura-2. The addition of K+ (6-100 mM) increased [Ca2+]i in a concentration-dependent manner. The fluorescent signal reached a peak within seconds and then decayed to form a new elevated plateau. K+ at the highest concentration used (100 mM) raised [Ca2+]i by about 450 nM. The K+-induced increase in [Ca2+]i was absent in a Ca2+-free medium. BAY K 8644, a 1,4-dihydropyridine Ca2+ channel agonist, also caused an increase in [Ca2+]i. The maximum response in [Ca2+]i upon stimulation with BAY K 8644 (100 nM) was about 40 nM. The half-maximally effective concentration of BAY K 8644 (100 nM) was about 20 nM. The response in [Ca2+]i upon BAY K 8644-stimulation was abolished in a Ca2+-free medium. Predepolarization with various K+ concentrations enhanced the effect of BAY K 8644 (1 microM) on [Ca2+]i. Pretreatment with BAY K 8644 (1 microM) enhanced the response in [Ca2+]i induced by K+ (25 mM). The addition of Mg2+ (30 mM) and nifedipine (1 microM) lowered the resting [Ca2+]i by about 40 and 20 nM, respectively. Mg2+, nifedipine, nimodipine, G? 5438, verapamil, and diltiazem inhibited the K+ (25 mM)-induced increase in [Ca2+]i; the order of potency (and half-maximally inhibitory concentrations) were nimodipine = G? 5438 = nifedipine (approximately 100 nM) greater than verapamil (900 nM) greater than diltiazem (greater than 10 microM) greater than Mg2+ (6 mM). Omega-Conotoxin (100 nM) did not inhibit the K+ (25 mM)-induced increase in [Ca2+]i. These data demonstrate that, over a wide range, membrane depolarization induced by high potassium concentration is indeed associated with increases in [Ca2+]i in normal rat anterior pituitary cells. This elevation of [Ca2+]i is mainly due to an influx of Ca2+ through 1,4-dihydropyridine-sensitive, omega-conotoxin-insensitive calcium channels (L-type).     

Dopamine receptors on dispersed bovine anterior pituitary cells

The dopamine antagonist [3H]-domperidone-[3H]-DOM-bound to a single class of high-affinity (Kd = 1.24 +/- 0.14 nM) and saturable receptors on dispersed bovine anterior pituitary (AP) cells. The binding of [3H]-DOM was stereoselective and reversible with agonists and antagonists. Dopamine competitions for [3H]-DOM binding modeled best for a single site consistent with an interaction with a homogeneous population of receptors. The mean number of specific binding sites labeled by [3H]-DOM was 53,000 per cell in dispersed AP cells consisting of 42% lactotrophs. Dispersed bovine AP cells attached to extracellular matrix within 3 h, and prolactin secretion from these cells was effectively inhibited by dopamine. Several observations suggested that [3H]-DOM-labeled receptors on dispersed bovine AP cells were restricted to the outer plasma membrane and not internalized. These included (1) the rapid and complete dissociation of specific [3H]-DOM binding; (2) the ability of treatment with acid or proteolytic enzymes to entirely remove specifically bound [3H]-DOM, and (3) the lack of effect of metabolic inhibitors on specific [3H]-DOM binding.     

Gonadotropin-releasing hormone-induced calcium signaling in clonal pituitary gonadotrophs.

In agonist-stimulated clonal pituitary gonadotrophs (alpha T3-1 cells), cytoplasmic calcium ([Ca2+]i) exhibited rapid and prominent peak increases, followed by lower, but sustained, elevations for up to 15 min. The [Ca2+]i response to GnRH was rapidly inhibited by prior addition of a potent GnRH antagonist. In the absence of extracellular Ca2+ the initial peak [Ca2+]i response was only slightly decreased, but the prolonged increase in [Ca2+]i was abolished, indicating that the peak is derived largely from intracellular calcium mobilization and the sustained phase from Ca2+ influx. Application of the endoplasmic reticulum Ca(2+)-ATPase blocker thapsigargin caused progressive and dose-dependent elevation of [Ca2+]i and decreased the peak amplitude of the GnRH-induced Ca2+ response. On the other hand, addition of dihydropyridine calcium channel antagonists before or after GnRH treatment prevented or terminated the plateau phase, respectively, consistent with entry of Ca2+ through L-type voltage-sensitive Ca2+ channels (VSCC) as the major Ca2+ influx pathway during GnRH action. The presence of L-type VSCC in alpha T3-1 cells was further indicated by the ability of elevated extracellular K+ levels and the dihydropyridine calcium channel agonist Bay K 8644 to elevate [Ca2+]i in an extracellular calcium-dependent manner. These actions of depolarization and Bay K 8644 were inhibited by nifedipine, with an IC50 of 10 nM. High extracellular K(+)- and GnRH-induced Ca2+ entry was also attenuated by phorbol esters and permeant diacylglycerols, indicating that protein kinase-C exerts inhibitory modulation of VSCC activity. In contrast to normal pituitary gonadotrophs, in which GnRH induces a frequency-modulated oscillatory [Ca2+]i response, single alpha T3-1 cells exhibited a nonoscillatory amplitude-modulated signal during agonist stimulation. The [Ca2+]i responses observed in alpha T3-1 gonadotrophs indicate that the immortalized cells retain functional GnRH receptors and their coupling to the Ca2+ signaling pathway. Ca2+ influx through L-type channels maintains the plateau phase of the [Ca2+]i response during agonist stimulation and is inhibited by activation of protein kinase-C.     

Spontaneous and corticotropin-releasing factor-induced cytosolic calcium transients in corticotrophs.

Spontaneous and CRF-stimulated changes in the cytosolic free calcium concentration ([Ca2+]i) were studied in two types of corticotrophs: 1) cultured human ACTH-secreting pituitary adenoma cells (hACTH cells), and 2) identified small ovoid corticotrophs cultured from normal rat pituitaries. [Ca2+]i was monitored in individual corticotrophs by dual emission microspectrofluorimetry using indo-1 as the intracellular fluorescent Ca2+ probe. In hACTH cells, [Ca2+]i measurements were carried out in combination with electrophysiological recordings obtained using whole cell patch-clamp techniques. It was shown that a single spontaneous Ca(2+)-dependent action potential led to a marked transient increase in [Ca2+]i in human tumoral corticotrophs. Spontaneous fluctuations in [Ca2+]i were also observed in unpatched corticotrophs whether derived from human pituitary tumors or normal rat tissue. Based on their striking kinetic features and their sensitivity to external Ca2+, we suggest that these spontaneous [Ca2+]i transients were the consequence of action potential firing. Under separate voltage-clamp (patch-clamp) conditions, tumor corticotrophs showed two Ca2+ current components: a low threshold, rapidly inactivating (T-type) current, and a higher threshold, slowly inactivating (L-type) current. The dihydropyridine Ca2+ channel blocker PN 200-110 (100 nM) abolished the L-type current without affecting the T-type current, while the inorganic Ca2+ channel blocker Cd2+ (200 microM) suppressed both Ca2+ currents. The Na+ channel blocker tetrodotoxin (5 microM) did not affect inward currents in tumor corticotrophs. Both L- and T-type voltage-gated Ca2+ channels were involved in controlling [Ca2+]i transients in both tumor and normal corticotrophs, inasmuch as Cd2+ (200 microM) abolished [Ca2+]i) transients, while PN 200-110 (100 nM) greatly diminished, but did not completely abolish, [Ca2+]i transients. The latter did not appear to depend on a voltage-dependent Na+ influx, since they were unaffected by tetrodotoxin (5 microM). Corticotrophs generate [Ca2+]i transients in response to the hypothalamic secretagogue CRF by acting on their membrane excitability. Indeed, we demonstrated in combined fluorescent and electrophysiological experiments that CRF (100 nM) had a coordinate action on human tumoral corticotrophs comprised of a modest depolarization and an increase in the frequency of both action potentials and subsequent [Ca2+]i transients. A coincident increase in the peak amplitude of the [Ca2+]i transient and after hyperpolarization was also observed in some CRF-stimulated cells. CRF (100 nM) evoked qualitatively similar [Ca2+]i patterns in human tumoral and normal rat corticotrophs not subjected to patch-clamping.(ABSTRACT TRUNCATED AT 400 WORDS)     

Importance of transients in cytosolic free calcium concentrations on activation of Na+/H+ exchange in GH4C1 pituitary cells.

In GH4C1 cells, TRH and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), have been shown to activate Na+/H+ exchange, probably via stimulation of protein kinase C. In the present study, the dependence of changes in intracellular pH (pHi) on transients in the cytosolic free calcium concentration [( Ca2+]i) was investigated using BCECF and fura-2, respectively. In buffer containing 0.4 mM extracellular Ca2+, both TRH and ionomycin induced rapid cytosolic alkalinization in GH4C1 cells acid loaded with nigericin. The action of ionomycin on pHi was abolished by preincubating the cells with 100 microM amiloride or by replacing extracellular Na+ with choline+, indicating that the change in pHi was probably due to activation of Na+/H+ exchange. The actions of both TRH and ionomycin on pHi were blunted in Ca2(+)-free buffer. When acid-loaded cells were stimulated first with ionomycin, to deplete intracellular Ca2+ stores, and then incubated with TRH, the TRH-induced alkalinization was blunted; thus, an increase in [Ca2+]i is needed for full activation of Na+/H+ exchange. To study further the importance of agonist-induced changes in [Ca2+]i on the activation of Na+/H+ exchange, acid-loaded cells were incubated first with TPA, and then with either TRH or ionomycin. TPA induced a rise in pHi, which was further enhanced by TRH, but not ionomycin. The actions of both TRH and ionomycin on Na+/H+ exchange were attenuated, but not abolished, in cells pretreated with TPA for 36 h. Acidification of the cytosol with nigericin increased the resting [Ca2+]i level from 125 +/- 29 to 200 +/- 25 nM (P less than 0.01). The increase in [Ca2+]i was greatly attenuated when extracellular Ca2+ was chelated with EGTA before the addition of nigericin. Both the TRH- and ionomycin-induced increases in [Ca2+]i were blunted in acid-loaded cells. We conclude that in GH4C1 cells, a transient increase in [Ca2+]i can enhance Na+/H+ exchange and cause a rise in pHi, but that to obtain full activation of exchange, protein kinase C activity must also be stimulated. Furthermore, pHi is important in maintaining an adequate store of sequestered intracellular Ca2+ and in the release of Ca2+ from that store in response to TRH and ionomycin.     

Role of cytosolic calcium in the control of cAMP content by calcium in bovine parathyroid cells

In the present studies we used the calcium (Ca2+)-sensitive dye Quin-2 to determine whether the cytosolic (Cyt) Ca2+ mediates the effects of extracellular (EC) Ca2+ on cAMP accumulation through changes in adenylate cyclase and phosphodiesterase activity in bovine parathyroid cells (bPTC). In dispersed (d) bPTC, increasing the EC Ca2+ from 0.5 to 2 mM produces a rise in the Cyt Ca2+ from 179 to 646 nM which is associated with a 52% inhibition of agonist-stimulated cAMP accumulation. Over this range of free Ca2+ adenylate cyclase activity decreased by approx. half (57%) and phosphodiesterase activity increases 2-fold (101%) suggesting that changes in the Cyt Ca2+ can account for the effects of EC Ca2+ on cAMP through changes in these enzymes. Unlike dbPTC, 4-day-old cultured bPTC show only a 23% suppression of cAMP by high EC Ca2+ and a reduced rise in the Cyt Ca2+ from 0.5 to 3 mM EC Ca2+. Although there is no reduction in the Ca2+ sensitivity of adenylate cyclase, phosphodiesterase activity shows no change at varied free Ca2+. Thus, this diminished Ca2+ sensitivity of phosphodiesterase activity, and the decreased rise in Cyt Ca2+ relative to EC Ca2+ may both contribute to the resistance to the effects of EC Ca2+ on cAMP content in cultured cells. Because in addition to Cyt Ca2+, protein kinase C may also mediate the effects of EC Ca2+ on PTH release, we studied the effects of TPA (12-alpha-tetradecanoylphorbol 13-acetate) on agonist-stimulated cAMP in dbPTC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-dependent protein phosphorylation in bovine anterior pituitary membranes and intact cells

A calmodulin-activated protein kinase has been identified in bovine anterior pituitary membranes. This enzyme phosphorylated one endogenous substrate of subunit molecular weight 53000 in the membranes. Phosphorylation of this protein was rapid, was half-maximal at 2.5 μM calcium in the presence of saturating concentrations of calmodulin (CaM), and was inhibited by trifluoperazine and thioridazine. A second protein was phosphorylated by an endogenous protein kinase in anterior pituitary membranes. Phosphorylation of this 42 000 M_r protein was reduced by calcium, was independent of exogenously added CaM, and was increased by trifluoperazine or thioridazine. The 42 000 M_r, protein may be the α-subunit of pyruvate dehydrogenase. Calcium-dependent protein phosphorylation was also observed in intact cells; the largest increases were seen in proteins of M_r 42 000, 21 000 and 17 000.     

Muscarinic regulation of basal versus thyrotropin-releasing hormone-induced prolactin secretion in rat anterior pituitary cells. differential roles of nitric oxide and intracellular calcium mobilization

Acetylcholine (ACh), synthesized in the pituitary, can act locally to modulate pituitary function. We used rat primary anterior pituitary (AP) cells to investigate how ACh affects pituitary prolactin (PRL) secretion in the presence or absence of known PRL regulators: thyrotropin-releasing hormone (TRH), 17beta-estradiol (E(2)) and triiodothyronine (T(3)). Cultured AP cells were prepared from ovariectomized rats and pretreated with diluent, 0.6 nM E(2), 10 nM T(3), or E(2) plus T(3) for 5 days, then challenged with various doses of ACh or muscarinic receptor agonists (oxotremorine or carbachol) and TRH (100 nM) for 20 min. Significant ACh (10(-5) M) suppression of both basal and TRH-induced PRL secretion was not evident in diluent-, E(2)- or T(3)-pretreated cells, but observed only in cells pretreated with both E(2) and T(3). Moreover, in E(2) plus T(3)-pretreated cells, oxotremorine and carbachol, like ACh (10(-7)-10(-5) M), suppressed both responses in a dose- related manner. Pertussis toxin (PTX; 100 ng/ml) as well as atropine (a muscarinic receptor antagonist; 1 mM) blocked these effects of cholinomimetics. ACh also inhibited both PRL responses elicited by drugs elevating intracellular cAMP (10 microM forskolin) or Ca(2+) (1 microM Bay K-8644) in a PTX-sensitive manner. ACh inhibition of basal PRL secretion was unaltered by intracellular Ca(2+) mobilization blockers, TMB-8 (100 microM) and thapsigargin (1 microM), but abrogated by the nitric oxide synthase inhibitor (300 microM L-NAME). ACh inhibition of TRH-induced PRL secretion was accentuated by TMB-8 and alleviated by thapsigargin or L-NAME. In summary, muscarinic inhibition of either basal or TRH-induced PRL secretion was augmented by E(2) and T(3), and involved the PTX-sensitive cAMP/Ca(2+) pathways. Furthermore, nitric oxide mediated the basal rather than TRH-induced PRL response to ACh, whereas the intracellular Ca(2+) mobilization concerned the TRH-induced rather than the basal PRL response to ACh. Thus, ACh synthesized in the AP appears to inhibit basal vs. TRH-induced PRL secretion via different mechanisms.     

Calcitonin inhibits basal and thyrotropin-releasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretagogue-induced increases in cytosolic Ca2+

Salmon calcitonin (sCT)-like peptide is present in the central nervous system and pituitary gland of the rat, and this peptide inhibits basal and TRH-stimulated PRL release from cultured rat anterior pituitary (AP) cells. The present studies were designed to examine further the inhibitory actions of sCT on basal and TRH-stimulated PRL release and investigated 1) the temporal dynamics of the responses, 2) the effects of sCT on PRL release induced by other secretogogues, and particularly those acting via elevations of cytosolic Ca2+, and 3) the selectivity of sCT action on basal and stimulated AP hormone release. The inhibition of basal PRL release by sCT (0.1-10 nM) was dose-dependent and was characterized by a rapid onset with a gradual recovery to normal rates of release after the period of sCT inhibition. The inhibitory effect of sCT on basal PRL release was reversed by treatment with either the Ca2+ ionophore A23187 or with the phorbol ester, phorbol myristate acetate (PMA). sCT infusion did not affect the basal release of GH, TSH, FSH, or LH by perifused AP cells. When administered in short pulses, TRH, at concentrations from 1-100 nM, elicited a dose-dependent increase in PRL release. When coadministered with short 10 nM TRH, sCT (1-100 nM) inhibited TRH-induced PRL release in a dose-dependent manner, with a maximal inhibition of 78% at a concentration of 10 nM, and an ED50 concentration of approximately 3 nM. During longer (30 min) pulses of TRH (100 nM), PRL release increased sharply over 4-fold within 2 min, followed within 12 min by a rapid decline to a level 1.5-2-fold higher than basal, and this level was maintained for the remainder of the stimulation period. sCT pretreatment inhibited the overall PRL response to TRH. In contrast to its inhibition of TRH-induced PRL release, sCT failed to prevent the stimulation of PRL release by either ionophore A23187, PMA, vasoactive intestinal peptide, or forskolin. In addition, sCT failed to block TRH-induced TSH release or GnRH-induced LH release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lysophosphatidylcholine inhibits bradykinin-induced phosphoinositide hydrolysis and calcium transients in cultured bovine aortic endothelial cells.

Vascular endothelium, which produces endothelium-derived relaxing and constricting factors, plays an important role in regulating the vascular tone. We recently demonstrated that oxidized low density lipoprotein inhibited endothelium-dependent relaxation and that lysophosphatidylcholine accumulated during the oxidative modification of low density lipoprotein was the essential substance for the inhibition of endothelium-dependent relaxation. To clarify the mechanisms of the inhibitory effect of lysophosphatidylcholine, we used a bioassay system to investigate the effect of lysophosphatidylcholine on the production and/or release of endothelium-derived relaxing factor and its effect on the cytosolic Ca2+ level ([Ca2+]i) and phosphoinositide hydrolysis in cultured bovine aortic endothelial cells. [Ca2+]i was monitored by the fura 2 method, and the accumulation of inositol phosphates in cells labeled with myo-[2-3H]inositol was measured. Bioassay experiments showed that lysophosphatidylcholine inhibited the production and/or release of endothelium-derived relaxing factor from cultured endothelial cells. Lysophosphatidylcholine (5-20 micrograms/ml) induced a biphasic increase in [Ca2+]i, which consisted of a rapid increase followed by a sustained increase, and the initial component was a result of mobilization from intracellular Ca2+ stores without detectable synthesis of inositol 1,4,5-trisphosphates. Furthermore, lysophosphatidylcholine (5-20 micrograms/ml) dose-dependently inhibited both phosphoinositide hydrolysis and the increases in [Ca2+]i evoked by bradykinin. These results indicate that the impairment of endothelium-dependent relaxation induced by lysophosphatidylcholine is due to the inhibition of phosphoinositide hydrolysis and the subsequent increases in [Ca2+]i in endothelial cells. Lysophosphatidylcholine that accumulates in oxidized low density lipoprotein and atherosclerotic arteries may play an important role in the modification of endothelial function.     

Bradykinin-induced increases in cytosolic calcium and ionic currents in cultured bovine aortic endothelial cells

The goal of the present study was to determine if voltage-sensitive calcium channels are present in bovine aortic endothelial cell plasmalemma and if they contribute to the rise in cytosolic calcium produced by bradykinin. After bradykinin (100 nM) exposure, endothelial cell associated fura-2 fluorescence peaked within 10-20 seconds and then declined to a steady level 2- to 3-fold above resting values. Pretreatment with lanthanum (20 microM) abolished the steady level produced by bradykinin but had little effect on the initial, transient rise in cytosolic calcium. Chelation of extracellular calcium with EGTA before addition of bradykinin resulted in a substantial decrease in the fura-2 transient and elimination of the long-lasting component. Nimodipine (3 microM) and nitrendipine (1 microM) were without effect on either phase of the bradykinin-induced response. Moreover, elevation of extracellular potassium failed to produce a rise in intracellular calcium. With the use of the tight seal technique to voltage clamp the cells, inwardly rectifying and calcium-activated potassium currents were found to exist in the endothelial cells. Addition of bradykinin (100 nM) elicited a calcium-activated potassium current that was eliminated in the absence of intracellular potassium. No voltage-sensitive calcium currents were activated when the cells were exposed to 10 mM or 110 mM calcium chloride in the presence or absence of bradykinin. The binding of [3H](+)PN200-110 to endothelial cell membrane preparations was 1-3 orders of magnitude lower than that observed in PC-12, GH3, or BC3H1 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of calcium ions on proopiomelanocortin mRNA levels in clonal anterior pituitary cells

The concentration of mRNA encoding proopiomelanocortin (POMC) was measured in AtT-20/D-16v cells, a clonal pituitary tumor cell line. Treatment of the cells with potassium (20 mM) or veratridine (10 microM) for 12, 24 and 48 h caused a time-dependent increase in the levels of POMC mRNA which became significant after 24 h. These effects were not seen in the presence of the sodium channel blocker tetrodotoxin (5 microM). In addition, the calcium channel blocker verapamil (10 microM) completely abolished the responses to either potassium or veratridine, whereas the calcium channel agonist Bay K 8644 (0.1 microM) potentiated the effect of potassium. Furthermore, the calcium channel blockers verapamil (10 microM) and nidefipine (1 microM) significantly decreased not only basal levels of POMC mRNA but also the increase of mRNA levels induced by corticotropin-releasing factor (CRF; 0.1 microM), 8-bromo-cAMP (1 mM) or cholera toxin (100 ng/ml). The drug-induced alterations in the mRNA POMC levels of the cells were, in each case, associated with similar alterations of immunoreactive beta-endorphin in the medium. These results indicate that membrane depolarization to activate sodium channels and calcium channels initiates an entry of calcium ions which triggers POMC gene expression in the AtT-20 cells. Moreover, calcium entry into the cells may exert a tonic stimulatory effect on POMC mRNA under basal conditions and may also contribute to the enhancing effect of CRF or cAMP on POMC mRNA in these cells.     

Characterization of anterior pituitary target cells for arginine vasopressin: including cells that store adrenocorticotropin, thyrotropin-beta, and both hormones

Arginine vasopressin (AVP) stimulates the secretion of ACTH and TSH. Recently, affinity cytochemical techniques with a potent biotinylated analog of AVP were used to identify ACTH cells as one of the target cells. Counts showed that AVP-bound cells were 10% of the population. However, if AVP bound all corticotropes and thyrotropes, one would expect AVP to bind at least 16% of the pituitary cells. Therefore, dual cytochemical labeling protocols were used to learn if thyrotropes also bound AVP (bio-AVP). Forty-eight percent of AVP target cells contained ACTH, and 42% contained TSH beta. The percentages of AVP-bound cells were increased to 12-13% of the total pituitary cells after 1-h pretreatment in 10 nM TRH or CRH. TRH and CRH together stimulated increases to 16% of the total cells. Analysis of dual labels showed that the additional AVP-bound cells stimulated by CRH or TRH stemmed from the corticotrope or thyrotrope populations, respectively. TRH stimulated an increase in the percentage of TSH cells that bound AVP from 55% to 75%. Similarly, CRH stimulated an increase in the percentage of ACTH cells that bound AVP from 61% to 79%. In addition, the populations of cells labeled for TSH beta or ACTH antigens increased by 30% after 1 h in unlabeled AVP, supporting its direct effect on these target cells. TRH stimulated a similar increment in TSH cells. CRH pretreatment had no effect on the percentages of cells labeled for TSH or ACTH. This could be the result of loss of ACTH stores needed to identify stimulated corticotropes. Finally, analysis of the total percentages of AVP-bound TSH beta or ACTH cells suggested an overlap in the population. This stimulated the application of dual labels for ACTH and TSH beta. In populations exposed to vehicle only, 1-2% of mixed pituitary cells stored both ACTH and TSH. This unique cell type also comprised 10% of a corticotrope population enriched by counterflow centrifugation. The percentage of ACTH-TSH cells in the mixed cell population was augmented to 4.8% after 1 h in AVP. It was not affected by exposure to either TRH or CRH (or both peptides). These studies demonstrate that AVP target cells include thyrotropes, corticotropes, and unique cells that store both ACTH and TSH.     

Thyrotropin-releasing hormone-induced hyperglycemia: possible involvement of cholinergic receptors in the lateral hypothalamus

The influence of the site of action of thyrotropin-releasing hormone (TRH) on the production of hyperglycemia was studied in rats by comparing the effectiveness of TRH administered by different routes. Administration of TRH (5 micrograms) into the lateral hypothalamus (LH) produced a hyperglycemia with a peak elevation of blood glucose of 140 mg/dl. Injection of 5 micrograms TRH into the ventromedial hypothalamus (VMH) produced a blood glucose elevation of 44 mg/dl, while injection of the same dose of TRH into the anterior hypothalamus (AH) produced a blood glucose elevation of 23 mg/dl. These findings indicate an LH site of action of TRH. Indeed, intra-LH administration of TRH (1-10 micrograms) caused dose-related increases in blood glucose. Administration of acetylcholine into the same site was also shown to induce hyperglycemia. The hyperglycemic effects of TRH and acetylcholine were antagonized by previous treatment of the LH site with atropine, a cholinergic receptor antagonist. Furthermore, the TRH-induced hyperglycemia was not observed or was greatly reduced in spinal rats or in adrenalectomized rats. The results indicate that TRH may act through the cholinergic receptor mechanisms within the LH region to induce hyperglycemia by promoting an increase in the sympathetic-adrenal medullary efferent activity.     

Thyrotropin-releasing hormone stimulates growth hormone release from the anterior pituitary of hypothyroid rats in vitro

We have examined the interaction of thyroid hormone and TRH on GH release from rat pituitary monolayer cultures and perifused rat pituitary fragments. TRH (10(-9) and 10(-8)M) consistently stimulated the release of TSH and PRL, but not GH, in pituitary cell cultures of euthyroid male rats. Basal and TRH-stimulated TSH secretion were significantly increased in cells from thyroidectomized rats cultured in medium supplemented with hypothyroid serum, and a dose-related stimulation of GH release by 10(-9)-10(-8) M TRH was observed. The minimum duration of hypothyroidism required to demonstrate the onset of this GH stimulatory effect of TRH was 4 weeks, a period significantly longer than that required to cause intracellular GH depletion, decreased basal secretion of GH, elevated serum TSH, or increased basal secretion of TSH by cultured cells. In vivo T4 replacement of hypothyroid rats (20 micrograms/kg, ip, daily for 4 days) restored serum TSH, intracellular GH, and basal secretion of GH and TSH to normal levels, but suppressed only slightly the stimulatory effect of TRH on GH release. The GH response to TRH was maintained for up to 10 days of T4 replacement. In vitro addition of T3 (10(-6) M) during the 4-day primary culture period significantly stimulated basal GH release, but did not affect the GH response to TRH. A GH stimulatory effect of TRH was also demonstrated in cultured adenohypophyseal cells from rats rendered hypothyroid by oral administration of methimazole for 6 weeks. TRH stimulated GH secretion in perifused [3H]leucine-prelabeled anterior pituitary fragments from euthyroid rats. A 15-min pulse of 10(-8) M TRH stimulated the release of both immunoprecipitable [3H]rat GH and [3H]rat PRL. The GH release response was markedly enhanced in pituitary fragments from hypothyroid rats, and this enhanced response was significantly suppressed by T4 replacement for 4 days. The PRL response to TRH was enhanced to a lesser extent by thyroidectomy and was not affected by T4 replacement. These data suggest the existence of TRH receptors on somatotrophs which are suppressed by normal amounts of thyroid hormones and may provide an explanation for the TRH-stimulated GH secretion observed clinically in primary hypothyroidism.     

Inhibitory effect of ergot alkaloids on nucleosides transport in cultured bovine anterior pituitary cells

Effect of ergot alkaloids on (3H)-nucleosides uptake by dispersed cells from fresh female bovine anterior pituitary glands was examined, because we had interested in the mechanism of pituitary tumor regression following bromocriptine treatment and considered nucleic acid synthesis as an important process in the metabolism of these cells. The anterior pituitary cells were implanted on culture tubes using D-valine minimal essential medium with serum to suppress the overgrowth of fibroblasts and then maintained in L-valine Dulbecco's modified Eagle medium. (3H)-Uridine uptake by these cells was suppressed by bromocriptine, at-ergocriptine or ergotamine at a concentration varing from 10(-6) M to 10(-5) M, but not 10(-5) M of lergotrile. Among these alkaloids, bromocriptine had most strong inhibitory effect and suppressed the uptake to below 25% of control value at the concentration of 10(-5) M. Bromocriptine also inhibited the uptake of (3H)-thymidine, (3H)-cytidine, (3H)-adenosine or (3H)-guanosine in the same manner as (3H)-uridine. But neither (3H)-uracil, the base of uridine, nor (3H)-galactose uptake by cells was affected by bromocriptine. The incorporation of (3H)-uridine or (3H)-thymidine into TCA-insoluble fraction of the cells was also inhibited by bromocriptine as that in the total cells. It was suspected that bromocriptine acted on distinct transport site of both ribonucleoside and deoxyribonucleoside, because a high concentration (3.3 X 10(-5) M) of radio-inactive thymidine did not modify (3H)-uridine uptake by these cells as well as inhibitory effect of bromocriptine on it. These effect of bromocriptine were not blocked by haloperidol, known as dopamine antagonist, at the same concentration as bromocriptine, and dopamine had no effect on (3H)-uridine uptake by the cells. In addition, by adding 5 X 10(-4) M of dibutyryl cyclic AMP into the medium, the effect of bromocriptine was also not affected. These data suggest that the effect of bromocriptine on nucleoside transport in anterior pituitary cells may be dependent on the other binding site than D-2 dopamine receptor in the anterior pituitary cell membrane.     

Nitric oxide donors modify free intracellular calcium levels in rat anterior pituitary cells

The effect of nitric oxide donors on intracellular calcium concentration [Ca2+]i was studied in anterior pituitary cells using ratiometric FURA 2 fluorescence measurements. Sodium nitroprusside (NP) induced a transient decrease in [Ca2+]i, after which [Ca2+]i returned to, or even increased over basal values. S-Nitroso glutathione (GSNO) induced a similar decrease. NP also inhibited high [Ca2+]i achieved by depolarization with 25 mM K+. The inhibitory effect of NP was partially blunted by pretreatment with methoxy-verapamil, and in calcium free buffer, and was not altered by thapsigargin. Interestingly, in calcium free buffer there was a significant stimulatory effect of NP, which was partially blunted by thapsigargin. We conclude that NO donors modify [Ca2+]i in anterior pituitary cells. The action is biphasic, with an initial decrease in [Ca2+]i probably related to a decrease of Ca2+ influx through VDCC, and an increase evidenced in calcium free buffer in which the inhibitory component is absent, and partially depends on thapsigargin sensitive calcium stores.     

Relationship between parathyroid hormone secretion and cytosolic calcium concentration in dispersed bovine parathyroid cells.

The parathyroid cell is unusual among exocytotic systems in that low extracellular Ca2+ concentrations stimulate, while high Ca2+ concentrations inhibit, parathyroid hormone (PTH) release, suggesting that this cell might have unique secretory mechanisms. In the present studies, we used the Ca2+-sensitive fluorescent dye QUIN -2 to examine the relationship between cytosolic Ca2+ concentration and PTH release in dispersed bovine parathyroid cells. The secretagogue dopamine, which enhances PTH release 2- to 3-fold in association with 20- to 30-fold increases in cellular cAMP, had no effect on the cytosolic Ca2+ level (261 +/- 28 vs. 236 +/- 22 nM for control cells at 1 mM extracellular Ca2+; P greater than 0.05). Dibutyryl-cAMP, which produces a comparable stimulation of PTH release, likewise did not modify the level of cytosolic Ca2+. Removal of extracellular Ca2+ produced a further decrease of the cytosolic Ca2+ to 82 +/- 10 nM. However, PTH secretion persisted at a near maximal rate despite this decrease of extracellular and cytosolic Ca2+ and was 95 +/- 2.5% of the rate of hormonal release at 0.5 mM extracellular Ca2+. In contrast, addition of the divalent cation ionophore ionomycin to parathyroid cells at 1.0 mM extracellular Ca2+ inhibited PTH secretion in association with an increase in cytosolic Ca2+ from 230 +/- 13 nM to 570 +/- 50 nM. Moreover, the magnitude of the ionomycin-induced reduction in PTH secretion (64 +/- 4% relative to the secretory rate at 0.5 mM Ca2+) was equivalent to the inhibition of PTH release caused by 1.5 mM extracellular Ca2+ (64 +/- 6%), which increased the cytosolic Ca2+ to similar levels (450 +/- 48 nM). Thus, the parathyroid cell differs from secretory cells thought to operate by stimulus-secretion coupling in the following ways: changes in PTH release can occur without detectable alterations in the cytosolic Ca2+ concentration, maximal rates of PTH secretion occur at cytosolic Ca2+ concentrations that fail to support exocytosis in other cell types, and increases in the cytosolic Ca2+ concentration due to ionomycin inhibit rather than stimulate PTH release. Therefore, the control of PTH secretion by Ca2+ and other secretagogues may involve previously undefined mechanisms whereby hormonal release is relatively independent of the cytosolic Ca2+ at low levels of this parameter and is inversely related to cytosolic Ca2+ at higher levels of intracellular Ca2+.