Therapy for Vitreous Seeding Caused by Retinoblastoma. A Review.

Retinoblastoma is the most common primary malignant intraocular tumor in children. Seeding, specifically the dispersion of the tumor into the adjacent compartments, represents a major parameter determining the degree of retinoblastoma according to the International Classification of Retinoblastoma. In this article we focused on vitreous seeding, one of the main limiting factors in the successful "eye preservation treatment" of retinoblastoma. This article presents an overview of the history of vitreous seeding of retinoblastoma, established treatment procedures and new-research modalities. The introduction of systemic chemotherapy in the treatment of retinoblastoma at the end of the 1990s represented a significant breakthrough, which enabled the progressive abandonment of radiotherapy with its attendant side effects. However, the attained concentrations of chemotherapeutics in the vitreous space during systemic chemotherapy are not sufficient for the treatment of vitreous seeding, and the toxic effects of systemic chemotherapy are not negligible. A significant change came with the advent of chemotherapy in situ, with the targeted administration of chemotherapeutic drugs, namely intra-arterial and intravitreal injections, contributing to the definitive eradication of external radiotherapy and a reduction of systemic chemotherapy. Although vitreous seeding remains the most common reason for the failure of intra-arterial chemotherapy, this technique has significantly influenced the original treatment regimen of children with retinoblastoma. However, intravitreal chemotherapy has made the greatest contribution to increasing the probability of preservation of the eyeball and visual functions in patients with advanced findings. Novel local drug delivery modalities, gene therapy, oncolytic viruses and immunotherapy from several ongoing preclinical and clinical trials may represent promising approaches in the treatment of vitreous retinoblastoma seeding, though no clinical trials have yet been completed for routine use.

Therapy for Vitreous Seeding Caused by Retinoblastoma. A Review.

Retinoblastoma is the most common primary malignant intraocular tumor in children. Seeding, specifically the dispersion of the tumor into the adjacent compartments, represents a  major parameter determining the degree of retinoblastoma according to the International Classification of Retinoblastoma. In this article we focused on vitreous seeding, one of the main limiting factors in the successful "eye preservation treatment" of retinoblastoma. This article presents an overview of the history of vitreous seeding of retinoblastoma, established treatment procedures and new-research modalities. The introduction of systemic chemotherapy in the treatment of retinoblastoma at the end of the 1990s represented a  significant breakthrough, which enabled the progressive abandonment of radiotherapy with its attendant side effects. However, the attained concentrations of chemotherapeutics in the vitreous space during systemic chemotherapy are not sufficient for the treatment of vitreous seeding, and the toxic effects of systemic chemotherapy are not negligible. A significant change came with the advent of chemotherapy in situ, with the targeted administration of chemotherapeutic drugs, namely intra-arterial and intravitreal injections, contributing to the definitive eradication of external radiotherapy and a reduction of systemic chemotherapy. Although vitreous seeding remains the most common reason for the failure of intra-arterial chemotherapy, this technique has significantly influenced the original treatment regimen of children with retinoblastoma. However, intravitreal chemotherapy has made the greatest contribution to increasing the probability of preservation of the eyeball and visual functions in patients with advanced findings. Novel local drug delivery modalities, gene therapy, oncolytic viruses and immunotherapy from several ongoing preclinical and clinical trials may represent promising approaches in the treatment of vitreous retinoblastoma seeding, though no clinical trials have yet been completed for routine use.

Dual effect of melatonin on gonadotropin-releasing-hormone-induced Ca(2+) signaling in neonatal rat gonadotropes.

In neonatal rat gonadotropes, melatonin inhibits gonadotropin-releasing-hormone (GnRH)-stimulated increase in intracellular Ca(2+) concentration ([Ca(2+)](i)); in cells transfected with the Mel1a melatonin receptor, however, melatonin has been shown to potentiate agonist-stimulated [Ca(2+)](i) increase. To elucidate this discrepancy, we investigated the effects of melatonin in neonatal gonadotropes over a wide range of melatonin concentrations. Nystatin perforated patch recording of Ca(2+)-dependent potassium currents was used to monitor GnRH-induced [Ca(2+)](i) changes. In 32% of cells, increasing melatonin concentrations in the range of 1 pM to 100 nM prolonged the latency of, and inhibited GnRH (10 nM)-stimulated [Ca(2+)](i) increases in a concentration-dependent manner. In the remaining 68% of cells, the Ca(2+) increase elicited by exposure to 10 nM GnRH was also inhibited by picomolar concentrations of melatonin, but at nanomolar concentrations the inhibitory effect disappeared and melatonin was only able to prolong the latency of the response. This dual effect of melatonin however was not observed in cells stimulated with lower (2 nM) GnRH concentrations; in that case, melatonin was inhibitory at all concentrations tested with an IC(50) of about 30 pM. In contrast, application of nanomolar concentrations of melatonin resulted in potentiation of the GnRH-induced Ca(2+) increase in a small population of gonadotropes which did not respond by inhibition or prolonged latency. These results indicate that in neonatal gonadotropes, melatonin has both inhibitory and potentiating effects on GnRH-stimulated [Ca(2+)](i) increases. Ranges of concentrations needed to produce either effect suggest that two distinct G proteins may be involved, as already observed in transfected cells.

In vitro entrainment of the circadian rhythm of vasopressin-releasing cells in suprachiasmatic nucleus by vasoactive intestinal polypeptide.

Mammalian circadian pacemaker is located in suprachiasmatic nuclei (SCN) of the hypothalamus. The pacemaker is entrained by light-dark cycle; the photic information is transmitted primarily via the retino-hypothalamic tract (RHT). The main neurotransmitter of the tract is glutamate. RHT fibers end on the ventrolateral part of the nucleus, where vasoactive intestinal peptide (VIP)-immunopositive neurons are localized. They send their axons into dorsomedial SCN, where most of the vasopressinergic (AVP) neurones are located. The AVP neurons retain the clock-like properties in vitro. Vasopressin release from the cultured neurons shows circadian rhythm peaking in the middle of subjective day. VIP induces phase-shifts of the rhythm, magnitude and direction of the shift depending on timing of the application. VIP applied 6-12 h before the peak of vasopressin rhythm induces advances, application 4-8 h after the peak induces delays. The lowest concentration required to induce the phase-shift is 30 nM, further increase of the concentration does not affect the magnitude of the shift. In contrast, glutamate has no effect on the phase of vasopressin rhythm, although in high concentrations it transiently stimulates vasopressin release. The data indicate that the vasopressinergic cells in the SCN contain circadian oscillators, whose rhythms run mutually synchronized in our cultures. VIP acts directly on the vasopressinergic cells to shift the phase of their pacemakers; glutamate has no such effect presumably because in vivo it acts through the VIP-ergic cells but the neuronal network is altered after the dissociation of the cells.

Different effects of melatonin pretreatment on cAMP and LH responses of the neonatal rat pituitary cells.

The rhythm of the pineal hormone melatonin transduces the effect of photoperiod on seasonal functions. Duration of the melatonin pulse provides information about season and the long melatonin pulse induces reproductive involution in the long day breeders such as photoperiodic rodents. The length of melatonin pulse thus carries photoperiodic information, which regulates the function of target cells. Therefore, we have studied the effects of melatonin pretreatment of various lengths on responsiveness of the neonatal rat pituitary cells cultured in vitro to GnRH or forskolin. In these cells, melatonin treatment inhibits the GnRH-induced LH release as well as the forskolin-induced cAMP accumulation. However, long preincubation with melatonin has a paradoxical stimulatory effect on the cellular responsiveness. When the cells are pretreated with melatonin for 16 hr or more, then rinsed thoroughly and treated with forskolin for 30 min, the increase of cAMP is potentiated. Moreover, in the melatonin-pretreated cells. the subsequent melatonin treatment inhibits the forskolin-induced cAMP accumulation relatively more than in the non-pretreated cells. Although melatonin pretreatment does not potentiate the GnRH-induced LH release, it protects the gonadotrophs against the GnRH-induced desensitization: pretreatment with GnRH for 12 hr or more renders the cells insensitive to subsequent GnRH stimulation, while after pretreatment with GnRH and melatonin, the subsequent GnRH treatment induces significant increase of LH release. These observations indicate that long pretreatment with melatonin improves responsiveness of the pituitary cells to the subsequent stimulation, but its effects on cAMP accumulation and LH release are different.

Differences in gonadotropin-releasing hormone-induced calcium signaling between melatonin-sensitive and melatonin-insensitive neonatal rat gonadotrophs.

The sensitivity of GnRH-stimulated calcium signaling to melatonin, in a subpopulation of neonatal gonadotrophs, is supposed to be attributable to melatonin receptors. However, it is not yet known whether the intracellular pathway for GnRH action in melatonin-sensitive cells is the same as in melatonin-insensitive cells. By monitoring intracellular Ca2+ changes as an outward current carried through apamin-sensitive Ca2+-activated K+ channels, we compared GnRH-induced calcium responses in these two subpopulations of neonatal gonadotrophs. GnRH induced various oscillatory, as well as nonoscillatory, responses in both cell types that was not related to melatonin sensitivity. Melatonin-sensitive GnRH-induced responses could be clearly distinguished according to the pharmacological properties of their latency. The latency increased in zero extracellular Ca2+ or with the addition of nifedipine, staurosporine, and ryanodine. This effect was only rarely observed in melatonin-insensitive cells. This indicates that there are two pathways for initiation of GnRH-induced calcium signaling in neonatal gonadotrophs. The first pathway is mediated by inositol 1,4,5,-trisphosphate production, whereas the second involves extracellular calcium entry through voltage-dependent L-type Ca2+ channels, protein kinase C activation, and Ca2+ release from a ryanodine-sensitive store, which may coactivate Ca2+ release from an inositol 1,4,5,-trisphosphate-sensitive store. Only the second mechanism is accessible to inhibition by melatonin.

Melatonin inhibits pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP accumulation and [Ca2+]i in cultured cells of neonatal rat pituitary.

The effects of melatonin on pituitary adenylyl cyclase-activating polypeptide-induced increase of cyclic AMP and [Ca2+]i were studied in neonatal rat pituitary cells. The polypeptide increased cyclic AMP accumulation. In the presence of melatonin the increase of cyclic AMP was inhibited in a dose-dependent manner, the maximal inhibition was achieved with 1-10 nM melatonin. Pituitary adenylyl cyclase-activating polypeptide also increased [Ca2+]i in 30% of the pituitary cells and melatonin inhibited the effect. Most of the cells sensitive to adenylyl cyclase-activating polypeptide (77%) were also sensitive to GnRH, suggesting they are gonadotrophs. The remaining cells were not identified. The polypeptide-induced [Ca2+]i increase was inhibited in Ca2+-free medium in 2/3 of the cells indicating that Ca2+ influx was involved. To examine causal relationship between cyclic AMP and [Ca2+]i increase, we have studied the effect of adenylyl cyclase activation by forskolin on intracellular Ca2+ concentration. Forskolin had similar effects as adenylyl cyclase-activating polypeptide: it increased [Ca2+]i in the pituitary cells and the increase was dependent on presence of Ca2+ in the medium. Melatonin inhibited the forskolin induced [Ca2+]i increase. Our observations indicate that increase of cyclic AMP stimulates Ca2+ influx in the pituitary cells of neonatal rat and that this mechanism is involved in [Ca2+]i increase induced by the pituitary adenylyl cyclase-activating polypeptide. Because melatonin inhibits increase of cyclic AMP induced by pituitary adenylyl cyclase-activating polypeptide or forskolin, the inhibitory effect of melatonin on Ca2+-influx may be mediated by the decrease of cyclic AMP concentration. This mechanism of melatonin action has not been described previously. Because melatonin inhibits the polypeptide- or forskolin-induced [Ca2+]i also in the cells not sensitive to GnRH, melatonin receptors seem to be present on both gonadotrophs and non-gonadotrophic pituitary cells.

Inhibitory effect of melatonin on GnRH-induced LH release.

Melatonin inhibits GnRH-induced release of LH and FSH from the neonatal, but not the adult, rat anterior pituitary gland. This action of melatonin is mediated by the specific high-affinity membrane-bound receptors that are absent in adult rats. The intracellular mechanism of melatonin action involves a decrease in intracellular calcium [Ca2+]i in the gonadotrophs; melatonin inhibits GnRH-induced Ca2+ release from endoplasmic reticulum as well as Ca2+ influx through voltage-sensitive channels. Melatonin also inhibits GnRH-induced accumulation of cAMP, which may result in the decreased influx of Ca2+, because cAMP, acting through protein kinase A, stimulates Ca2+ influx into the gonadotrophs. This age-dependent effect of melatonin on gonadotrophin release from the pituitary may be involved in the timing of puberty.

Mechanisms of melatonin action in the pituitary and SCN.

We have compared melatonin effects in two different cell types in order to determine general intracellular mechanisms of its action. In neonatal rat pituitary, melatonin acts via the specific membrane receptors to inhibit GnRH-induced LH release. The melatonin effect disappears in adulthood due to the disappearance of the receptors. The mechanism of the melatonin action involves inhibition of the GnRH induced increase of intracellular calcium ([Ca2+])i. Our observations indicate that melatonin has dual inhibitory effect on GnRH-induced [Ca2+]i: it inhibits mobilisation of Ca2+ from endoplasmic reticulum as well as Ca2+ influx through voltage sensitive channels. Besides, melatonin also decreases basal and GnRH- or forskolin-induced increase of cAMP concentration in the pituitary. Although cAMP is not of primary importance for regulation of LH release, the cAMP decrease may participate in the mechanism of inhibitory melatonin action on LH release. Rat suprachiasmatic nuclei (SCN) have a high density of the melatonin receptors throughout the postnatal life. Cultures of dispersed SCN cells show circadian rhythm of vasopressin (AVP) release, with several fold increase in the middle of the day and decrease during night. Melatonin inhibits the spontaneous AVP release. Melatonin also inhibits the AVP release induced by vasoactive intestinal peptide (VIP). Intracellular mechanisms of the melatonin effect may involve cAMP, because melatonin inhibits the VIP-induced increase of cAMP and increase of cAMP formation by forskolin stimulates AVP release from the cultures. On the other hand, involvement of intracellular calcium in the regulation of AVP release may not be excluded. VIP induces [Ca2+]i increase in 14% of the SCN cells and AVP release is stimulated by Ca2+ ionophore ionomycin. Our observations indicate that some of the mechanisms of melatonin action are similar in the pituitary and SCN.

Melatonin inhibits the increase of cyclic AMP in rat suprachiasmatic neurons induced by vasoactive intestinal peptide.

The effects of melatonin on basal and vasoactive intestinal peptide (VIP)-induced cAMP concentration was studied in dispersed cells of the rat suprachiasmatic nuclei (SCN). Our data indicate, that VIP induces a rapid increase of cAMP concentration in the cells followed by a slow and prolonged increase in the medium. The VIP-induced increase was dose-dependent in the range of 1-100 nM. Melatonin had no effect on basal cAMP but inhibited the cAMP increase induced by VIP in a dose-dependent manner (EC50 = 0.21 nM). Our observations indicate that melatonin acts through the inhibition of cAMP in the SCN cells similar as shown in other tissues.

Melatonin inhibits spontaneous and VIP-induced vasopressin release from suprachiasmatic neurons.

We have studied melatonin effects on vasopressin release from dispersed cells of the rat suprachiasmatic nuclei (SCN). The release follows a circadian rhythm peaking during the day and decreasing at night. Melatonin inhibits the spontaneous increase and accelerates the decrease of vasopressin release. Melatonin also inhibits vasopressin release induced by vasoactive intestinal peptide (EC50=0.4 nM). The inhibition of vasopressin release correlates with the known inhibitory effect of melatonin on spontaneous neuronal activity in SCN.

Melatonin inhibits release of luteinizing hormone (LH) via decrease of [Ca2+]i and cyclic AMP.

The role of [Ca2+]i and cAMP in transduction of the melatonin inhibitory effect on GnRH-induced LH release from neonatal rat gonadotrophs has been studied, because melatonin inhibits the increase of both intracellular messengers. Treatments increasing Ca2+ influx (S(-) Bay K8644 or KCI) or cAMP concentration (8-bromo-cAMP or 3-isobutyl-1-methylxanthine) potentiated the GnRH-induced LH release and partially diminished the inhibitory effect of melatonin. Combination of the treatments increasing cAMP and calcium concentrations blocked completely the melatonin inhibition of LH release. The combined treatment with 8-bromo-cAMP and S(-) Bay K8644 also blocked the melatonin inhibition of GnRH-induced [Ca2+]i increase in 89 % of the gonadotrophs, while any of the treatments alone blocked the melatonin effect in about 25 % of these cells. These observations suggest that a cAMP-dependent pathway is involved in regulation of Ca2+ influx by melatonin and melatonin inhibition of LH release may be mediated by the decrease of both messengers.

Melatonin inhibits GnRH-induced Ca2+ mobilization and influx through voltage-regulated channels.

In neonatal rat gonadotrophs, melatonin acts through the high-affinity membrane-bound receptors to inhibit GnRH-induced [Ca2+]i increase. GnRH increases [Ca2+]i primarily by mobilization from the inositol trisphosphate-sensitive pool followed by Ca2+ influx through the voltage-sensitive channels. Melatonin inhibits the GnRH-induced [Ca2+]i increase. When added after the GnRH-induced spike, melatonin decreases [Ca2+]i in 52% of the gonadotrophs. The effect of melatonin is dependent on extracellular Ca2+ and may be mimicked by Ca2+-free medium or verapamil. When added before GnRH, melatonin inhibits the [Ca2+]i spike. This effect of melatonin is independent of extracellular Ca2+ as it persists in Ca2+-free medium. These findings indicate that melatonin blocks Ca2+ mobilization as well as Ca2+ influx in the gonadotrophs.

Inhibitory effect of melatonin on gonadotropin-releasing hormone-induced Ca2+ oscillations in pituitary cells of newborn rats.

The effect of melatonin on the gonadotropin-releasing-hormone (GnRH)-induced oscillatory rises in intracellular calcium concentration, [Ca2+]i, was studied in cultured cells from the anterior pituitary gland of 6- to 8-day-old rats. GnRH-induced [Ca2+]i oscillations were recorded indirectly by monitoring the activity of apamin-sensitive Ca(2+)-activated K+ channels using the perforated patch-clamp technique and fast microperfusion system. Melatonin (1 nM) inhibited the initiation or attenuated the amplitude of oscillatory current responses induced by 10 nM GnRH in 72% of GnRH-sensitive cells. Analysis of the melatonin dose-inhibition relationship showed that melatonin inhibited the initiation of [Ca2+]i oscillations with IC50 = 0.35 nM. In partially inhibited cells, melatonin reduced the GnRH-induced current amplitude by 55% on the average, prolonged the delay in onset of response to GnRH and decreased the frequency of oscillations. Once initiated by GnRH, the amplitude and frequency of oscillatory currents was inhibited by melatonin after a latency of 10-30 s. These effects of melatonin were fully reversible. After pretreatment of neonatal gonadotropes with pertussis toxin, no inhibition by melatonin was observed. The inhibitory effect of melatonin on initiation, amplitude and frequency of GnRH-induced oscillatory current persisted in the absence of external Ca2+. Melatonin alone did not induce any transmembrane current or membrane potential changes. These observations suggest that melatonin reduces GnRH-induced calcium mobilization from intracellular stores.

Melatonin inhibits GnRH-induced increase of cFOS immunoreactivity in neonatal rat pituitary.

In neonatal rat gonadotrophs, melatonin inhibits several GnRH-induced effects: stimulation of LH release as well as the increase of several second messengers as cAMP, diacylglycerol and [Ca2+]i . Recently, GnRH has been shown to induce expression of immediate early genes of fos and jun family in adult rat gonadotrophs. The purpose of this study was to determine, whether melatonin inhibits the GnRH-induced induction of cFos in neonatal rat pituitary cells. The effects of GnRH and/or melatonin on cFos immunoreactivity was determined in primary cultures of neonatal rat pituitary cells attached to the coverslip. GnRH (3 nM) induced a time-dependent increase of cFos immunoreactivity in about 10 to 15% of the cultured cells. Significant increase was observed already 30 min after GnRH administration, the maximal increase occurred after about 60 min and then gradually decreased. Melatonin (100 nM) markedly attenuated the GnRH-induced increase. GnRH increased cFos immunoreactivity in the cells in a dose-dependent manner (EC50=36 pM) and melatonin (100 nM) attenuated the response at all GnRH concentrations tested. Melatonin had no effect on basal cFos immunoreactivity, but it inhibited the GnRH-induced (10 nM) increase of cFos in dose-dependent manner (EC50=12 pM). In conclusion, this is the first report showing the inhibitory effect of melatonin on gene transcriptional activity in gonadotrophs.

Mechanism of melatonin signal transduction in the neonatal rat pituitary.

Melatonin inhibits GnRH-induced LH release from anterior pituitary of the neonatal rat. It acts via specific high affinity receptors and decreases concentrations of intracellular calcium ([Ca2+]i) and cyclic AMP. To determine which of these second messengers transduces the melatonin inhibition of LH release, we have tested the effect of melatonin in the presence of specific drugs affecting either of these second messengers. Calcium channel antagonist nifedipine inhibited LH release from cultured pituitary to a similar degree as did melatonin and prevented the inhibitory effect of melatonin on LH release. Calcium channel agonist. Bay K potentiated the LH release and reduced the inhibitory effect of melatonin. This observation constitutes strong evidence that melatonin inhibits LH release via inhibition of calcium influx through voltage sensitive channels. The cyclic AMP derivative 8-bromo-cAMP potentiated GnRH-stimulation of LH release but did not prevent the melatonin-induced inhibition of the release. However, when used in combination with low concentration of Bay K, which alone reduced the melatonin effect only partially, 8-bromo-cAMP completely blocked the melatonin effect. This observation suggests that both cAMP and [Ca2+]i may be involved in the effect of melatonin on LH release.

Electrophysiological characterization of GABAA receptors in anterior pituitary cells of newborn rats.

The gamma-aminobutyric acid (GABA)-ergic communication between the CNS and the anterior pituitary gland has been documented in numerous histochemical and biochemical studies but electrophysiological studies characterizing the GABAA receptor in the anterior pituitary are still lacking. In the present report we studied the GABA-induced current responses in cultured cells from the anterior pituitary gland of 6- to 10-day-old rats using the patch-clamp technique in the whole cell configuration. Fast application of GABA (100 microM) induced membrane currents in 90% of cells in 2-day-old cultures. The EC50 for GABA was 22.9 microM and the Hill coefficient was 1.8. The responses to GABA (10 microM) were inhibited by bicuculline (2 microM) to 14%, by picrotoxin (5 microM) to 21% and by zinc (10 microM) to 33%. Inhibition to 56% was observed with 6 microM strychnine. The GABA responses were sensitive to diazepam and pentobarbital. Half-maximal potentiation of responses to GABA (10 microM) was found with 1.0 microM diazepam and with 14.4 microM pentobarbital. The maximal potentiation of GABA responses was 222% for diazepam and 195% for pentobarbital. Pentobarbital (100 microM) did not induce any response in anterior pituitary cells in the absence of GABA. The application of GABA at concentrations 10 microM or higher, induced membrane currents that desensitized. Desensitization proceeded as a biexponential process with estimated fast and slow time constants which decreased with concentration. The responses to GABA (300 microM) desensitized to 93% with time constants of 1.4 and 5.3 s. Half-maximal desensitization was found with 13.4 microM GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Melatonin inhibition of GnRH-induced LH release from neonatal rat gonadotroph: involvement of Ca2+ not cAMP.

Melatonin inhibits gonadotropin-releasing hormone-induced release of luteinizing hormone (LH) from the neonatal rat gonadotrophs. The second messenger involved is not known, although there are several candidates, including adenosine 3',5'-cyclic monophosphate (cAMP) and intracellular free Ca2+. The present study addresses the question of which second messenger mediates melatonin inhibition of LH release. We found that the effect of melatonin was not prevented by cAMP protagonists, including 8-bromo-cAMP, dibutyryl cAMP, 3-isobutyl-1-methylxanthine, and forskolin. However, treatments that enhanced Ca2+ influx masked the effects of melatonin, and treatments that blocked Ca2+ influx mimicked the effects of melatonin. Moreover, melatonin decreased K(+)-induced LH release, which is dependent on Ca2+ influx but did not block release of LH due to thapsigargin-induced mobilization of Ca2+ from intracellular stores. These findings indicate that melatonin inhibits gonadotropin-releasing hormone-induced LH release, primarily through an action involving inhibition of Ca2+ influx, and that cAMP does not seem to be involved in this effect of melatonin.

Vasoactive intestinal peptide elevates pinealocyte intracellular calcium concentrations by enhancing influx: evidence for involvement of a cyclic GMP-dependent mechanism.

Vasoactive intestinal peptide (VIP) receptor density is high in the pineal gland, which receives VIP innervation and responds to VIP with a relatively small increase in cAMP and cGMP levels. In the present study, we show that VIP (5-200 nM) treatment increased the intracellular calcium concentration ([Ca2+]i) in 64% of isolated individual pinealocytes; in comparison, norepinephrine (NE) elevated [Ca2+]i in 93% of the cells and produced more robust responses. Analysis of the role of second messengers indicated that [Ca2+]i was strongly elevated by cGMP analogs, but not by cAMP analogs. The nitric oxide-releasing agent S-nitro-N-acetylpenicillamine and 2,2-diethyl-1-nitroxyhydraxine also elevated [Ca2+]i. Investigation of the mechanisms revealed that responses to VIP or 8-bromo-cGMP involved Ca2+ influx, as did the plateau component of the response to NE; the large rapid component of the response to NE, however, appeared to reflect release from intracellular stores. Pharmacological studies indicated that the VIP-induced Ca2+ influx was mediated by a retinal rod-type cyclic nucleotidegated cation channel, expression of which was confirmed by reverse transcription-polymerase chain reaction analysis. These observations indicate that fundamentally different mechanisms generate the responses to NE and VIP. The dominant effect of VIP causing transient elevation of [Ca2+]i appears to be through cGMP gating aI-cis-diltiazem-sensitive rod-type cyclic nucleotide-gated cation channel. In contrast, the dominant effect of NE on [Ca2+]i is due to enhanced Ca2+ release from intracellular stores; the plateau component is due to influx through aI-cis-diltiazem-insensitive channel.

Melatonin inhibits increase of intracellular calcium and cyclic AMP in neonatal rat pituitary via independent pathways.

In neonatal rat pituitary, melatonin inhibits GnRH-induced increase of cAMP and [Ca2+]i. Both effects are transduced by specific high-affinity melatonin receptors coupled with pertussis toxin-sensitive G-protein. We have attempted to determine whether melatonin acts via independent pathways on both messengers or whether the indole directly inhibits only one of the messengers and the second is decreased as a secondary consequence. Melatonin inhibition of cAMP accumulation was not prevented by agents known to block melatonin effect on [Ca2+]i such as Na(+)- or Ca2(+)-free medium, Bay K, nifedipine, KCl or gramicidin. Melatonin effect on [Ca2+]i was not prevented by forskolin or 8-bromo-cAMP. We therefore conclude that melatonin inhibits cAMP accumulation and [Ca2+]i increase independently by separate pathways.