Regulation of TRH release by the cultured neonate rat pancreas

The TRH secretory responsiveness of the pancreatic islet cell clusters from newborn rat in organ culture was studied. Basal TRH secretion was stable over a 9-day period. The response to various secretagogues was tested on day 4. TRH secretion was stimulated by high potassium-induced depolarization and also through both cAMP and protein kinase-C dependent pathways. Like insulin, TRH release was stimulated by glucose and arginine and inhibited by somatostatin. These data suggest the existence of a common mechanism for TRH and insulin secretion by the pancreatic β-cells.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Evidence that potassium channels regulate prolactin secretion in GH4C1 cells by causing extracellular calcium influx

Tetraethylammonium (TEA), a K+ channel blocker, induced prolactin (PRL) secretion in GH4C1 cells in a dose-dependent manner when applied at a concentration from 1-20 mM. During continuous exposure to TEA, a significant increase in PRL secretion occurred by 20 min and the response was sustained until the end of a 60-min exposure. Blocking Ca2+ influx by employing a Ca(2+)-depleted medium or the Ca2+ channel blocker, nifedipine, prevented induction of PRL secretion by 20 mM TEA. Preincubation of the cells for 10 min with 20 mM TEA did not inhibit PRL secretion induced by thyrotropin-releasing hormone (TRH), phorbol 12-myristate 13-acetate (TPA) or by cell swelling produced by 30% medium hyposmolarity, but significantly depressed that induced by depolarizing 30 mM K+. BaCl2, another K+ channel blocker, had the same effect on PRL secretion as TEA. The data suggest that blocking K+ channels may cause membrane depolarization, thereby inducing Ca2+ influx which is a potent stimulus for PRL secretion in GH4C1 cells.     

Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.     

Follicle stimulating hormone and prolactin release induced by prostaglandins in rat

Ten to 60 minutes following a single i.v. injection of PGE₂ (500 μg/rat) into male rats of 30 to 35 days of age FSH concentration in the serum was raised significantly. The rise in FSH was maintained from 10 to 60 minutes after treatment, then at 90 minutes FSH had declined and was not significantly different from that of the control before treatment. Prostaglandin E₁, E₂ or F_2α (670μg/rat) significantly increased the serum prolactin level 10 to 60 minutes after a single i.v. injection in spayed rats primed with estrogen and progesterone. And, rats primed with estrogen and progesterone. And, increases in prolactin in the serum were observed with as little as 2μg of PGE₁ or E₂, and 20μg of PGF_2α. Twenty μg of PGE₂, and 200μg of PGE₁ or F_2α gave the maximum stimulation. These results indicate that release of pituitary hormones is affected by prostaglandins.Prostaglandins (PGs) are widely distributed in mammalian tissues, and they have been reported to have an almost equally wide variety of endocrine and metabolic effects. It was recently postulated that PGs may be involved in the process of ovulation because ovulation was blocked by inhibitors of PG synthesis (1–5).     

Release of calcium from endolysosomes increases calcium influx through N-type calcium channels: Evidence for acidic store-operated calcium entry in neurons

Neurons possess an elaborate system of endolysosomes. Recently, endolysosomes were found to have readily releasable stores of intracellular calcium; however, relatively little is known about how such ‘acidic calcium stores’ affect calcium signaling in neurons. Here we demonstrated in primary cultured neurons that calcium released from acidic calcium stores triggered calcium influx across the plasma membrane, a phenomenon we have termed “acidic store-operated calcium entry (aSOCE)”. aSOCE was functionally distinct from store-operated calcium release and calcium entry involving endoplasmic reticulum. aSOCE appeared to be governed by N-type calcium channels (NTCCs) because aSOCE was attenuated significantly by selectively blocking NTCCs or by siRNA knockdown of NTCCs. Furthermore, we demonstrated that NTCCs co-immunoprecipitated with the lysosome associated membrane protein 1 (LAMP1), and that aSOCE is accompanied by increased cell-surface expression levels of NTCC and LAMP1 proteins. Moreover, we demonstrated that siRNA knockdown of LAMP1 or Rab27a, both of which are key proteins involved in lysosome exocytosis, attenuated significantly aSOCE. Taken together our data suggest that aSOCE occurs in neurons, that aSOCE plays an important role in regulating the levels and actions of intraneuronal calcium, and that aSOCE is regulated at least in part by exocytotic insertion of N-type calcium channels into plasma membranes through LAMP1-dependent lysosome exocytosis.     

Further evidence that central neurotensin inhibits pituitary prolactin secretion by stimulating dopamine release from the hypothalamus

Intracerebroventricular (icv) injection of neurotensin (NT) (2 micrograms/rat) suppressed prolactin (PRL) release induced by L-5-hydroxytryptophan (1 mg/100 g body wt, iv), prostaglandin E2(1 microgram/rat, icv), and FK33-824 (10 micrograms/100 g body wt, iv), a Met5-enkephalin analog, in urethane-anesthetized or conscious rats. In contrast, NT did not suppress elevated plasma PRL levels sustained by a large dose of domperidone (10 micrograms/100 g body wt, iv), a peripheral dopamine antagonist. In in vitro experiments, NT (10(-5) M) stimulated dopamine release from perifused rat hypothalamic fragments. These results suggest that central NT inhibits PRL secretion by stimulating dopamine release from the hypothalamus into hypophysical portal blood in the rat.     

Gonadotropin-releasing hormone stimulates prolactin release from lactotrophs in photoperiodic species through a gonadotropin-independent mechanism

Previous studies have provided evidence for a paracrine interaction between pituitary gonadotrophs and lactotrophs. Here, we show that GnRH is able to stimulate prolactin (PRL) release in ovine primary pituitary cultures. This effect was observed during the breeding season (BS), but not during the nonbreeding season (NBS), and was abolished by the application of bromocriptine, a specific dopamine agonist. Interestingly, GnRH gained the ability to stimulate PRL release in NBS cultures following treatment with bromocriptine. In contrast, thyrotropin-releasing hormone, a potent secretagogue of PRL, stimulated PRL release during both the BS and NBS and significantly enhanced the PRL response to GnRH during the BS. These results provide evidence for a photoperiodically modulated functional interaction between the GnRH/gonadotropic and prolactin axes in the pituitary gland of a short day breeder. Moreover, the stimulation of PRL release by GnRH was shown not to be mediated by the gonadotropins, since immunocytochemical, Western blotting, and PCR studies failed to detect pituitary LH or FSH receptor protein and mRNA expressions. Similarly, no gonadotropin receptor expression was observed in the pituitary gland of the horse, a long day breeder. In contrast, S100 protein, a marker of folliculostellate cells, which are known to participate in paracrine mechanisms within this tissue, was detected throughout the pituitaries of both these seasonal breeders. Therefore, an alternative gonadotroph secretory product, a direct effect of GnRH on the lactotroph, or another cell type, such as the folliculostellate cell, may be involved in the PRL response to GnRH in these species.     

Inhibition of prolactin release from anterior pituitary lactotrophs in culture by sulfur-containing analogs of dopamine

The effects of permanently charged and uncharged analogs of dopamine were examined for their ability to inhibit basal prolactin release from primary cultures of rat pituitary lactotrophs. The charged quaternary trimethyldopamine and the charged dimethylsulfonium analogs were active (IC50's were 4.3 and 31 microM, respectively) while the permanently uncharged monoethylsulfide was devoid of significant activity. Dopamine and dimethyldopamine, which are able to exist in both charged and uncharged forms, are more potent (IC50's were 36 and 44 nM, respectively) but all compounds were capable of approaching the same maximum degree of prolactin release inhibition. Haloperidol, a dopamine receptor antagonist, was able to block the actions of each of the agonists. The data suggest that (a) dopamine agonists do not have to be in the uncharged form in order to activate the dopamine receptor that regulates prolactin release, (b) the uncharged monomethylsulfide analog of dopamine is incapable of activating the dopamine receptor, and (c) the nitrogen on the side chain of dopamine can be replaced by another atom and still retain prolactin release inhibiting activity.     

Selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs from rat anterior pituitary in culture

Summary Semi-thin sections of three-dimensional reaggregates from adult female rat pituitary, cultured in serum-free defined medium, were stained for prolactin, gonadotropin, thyrotropin, growth hormone and S-100, using the double immunolabelling technique. The frequency of juxtaposition between lactotrophs and gonadotrophs was enumerated and compared with the expected frequency at random distribution of polygonal cell profiles in a hexagonal configuration. The proportions of lactotrophs and gonadotrophs in the aggregate sections were determined using stereometrical analysis. The observed frequency of juxtaposition did not differ significantly from the expected frequency. Hence, no reason was found to assume a selective adhesion between lactotrophs and gonadotrophs in adult female rat pituitary reaggregates. A constant proportion of lactotrophs was found to meet the criteria of a cup-shaped morphology, and 70%±9% (mean ±S.D.) of these so-called cupshaped lactotrophs were found to be juxtaposed at their concave side to gonadotrophs. Administration of 0.01 nM 17-oestradiol to the culture medium resulted in a significant reduction of the proportion of cup-shaped lactotrophs but did not affect the selectivity of juxtaposition to gonadotrophs. The selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs may be the morphological correlate of the functional relationship between these cells, which are known to be involved in an intra-pituitary paracrine communication system.     

The control of calcium influx by cytoplasmic calcium in mammalian heart muscle

The role of Ca²⁺ in the initiation and maintenance of contraction has been extensively studies. Many of these studies have focused on how Ca²⁺ influx and efflux affect cytoplasmic Ca²⁺ (Ca_i) and, therefore, contraction in cardiac muscle. However, it has recently become apparent that Ca_i itself may play a major role in the control of Ca²⁺ influx and efflux from cardiac muscle. Here we review current ideas on the mechanisms underlying Ca²⁺ homeostasis in cardiac muscle, with specific attention to how Ca_i may control Ca²⁺ influx, both under normal and pathological conditions.     

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts

Summary Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 m) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca²⁺] _i ) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca²⁺] _i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.     

Hyperosmotic media inhibit voltage-dependent calcium influx and peptide release in Aplysia neurons

Summary The bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20–30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.This work was supported by NIH Grant NS-18492 to L.K. Kaczmarek.     

Redox and mTOR-dependent regulation of plasma lamellar calcium influx controls the senescence-associated secretory phenotype

Cellular senescence has evolved as a protective mechanism to arrest growth of cells with oncogenic potential but is accompanied by the often pathologically deleterious senescence-associated secretory phenotype (SASP). Here we demonstrate an H₂O₂-dependent functional disruption controlling senescence-associated Ca²⁺ homeostasis and the SASP. Senescent cells fail to respond to H₂O₂-dependent plasma lamellar Ca²⁺ entry when compared to pre-senescent cells. Limiting exposure to senescence-associated H₂O₂ restores H₂O₂-dependent Ca²⁺ entry as well as transient receptor potential cation channel subfamily C member 6 (TRPC6) function. SA-TRPC6 and SASP expression is blocked by restoring Ca²⁺ entry with the TRP channel antagonist SKF-96365 or by the mTOR inhibitors rapamycin and Ku0063794. Together, our findings provide compelling evidence that redox and mTOR-mediated regulation of Ca²⁺ entry through TRPC6 modulates SASP gene expression and approaches which preserve normal Ca²⁺ homeostasis may prove useful in disrupting SASP activity.Impact statementThrough its ability to evoke responses from cells in a paracrine fashion, the senescence-associated secretory phenotype (SASP) has been linked to numerous age-associated disease pathologies including tumor invasion, cardiovascular dysfunction, neuroinflammation, osteoarthritis, and renal disease. Strategies which limit the amplitude and duration of SASP serve to delay age-related degenerative decline. Here we demonstrate that the SASP regulation is linked to shifts in intracellular Ca²⁺ homeostasis and strategies which rescue redox-dependent calcium entry including enzymatic H₂O₂ scavenging, TRP modulation, or mTOR inhibition block SASP and TRPC6 gene expression. As Ca²⁺ is indispensable for secretion from both secretory and non-secretory cells, it is exciting to speculate that the expression of plasma lamellar TRP channels critical for the maintenance of intracellular Ca²⁺ homeostasis may be coordinately regulated with the SASP.     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)