An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion

The pulsatile secretion of gonadotropin-releasing hormone (GnRH) from normal and immortalized hypothalamic GnRH neurons is highly calcium-dependent and is stimulated by cAMP. It is also influenced by agonist activation of the endogenous GnRH receptor (GnRH-R), which couples to G(q/11) as indicated by release of membrane-bound alpha(q/11) subunits and increased inositol phosphate/Ca(2+) signaling. Conversely, GnRH antagonists increase membrane-associated alpha(q/11) subunits and abolish pulsatile GnRH secretion. GnRH also stimulates cAMP production but at high concentrations has a pertussis toxin-sensitive inhibitory effect, indicative of receptor coupling to G(i). Coupling of the agonist-activated GnRH-R to both G(s) and G(i) proteins was demonstrated by the ability of nanomolar GnRH concentrations to reduce membrane-associated alpha(s) and alpha(i3) levels and of higher concentrations to diminish alpha(i3) levels. Conversely, alpha(i3) was increased during GnRH antagonist and pertussis toxin treatment, with concomitant loss of pulsatile GnRH secretion. In cholera toxin-treated GnRH neurons, decreases in alpha(s) immunoreactivity and increases in cAMP production paralleled the responses to nanomolar GnRH concentrations. Treatment with cholera toxin and 8-bromo-cAMP amplified episodic GnRH pulses but did not affect their frequency. These findings suggest that an agonist concentration-dependent switch in coupling of the GnRH-R between specific G proteins modulates neuronal Ca(2+) signaling via G(s)-cAMP stimulatory and G(i)-cAMP inhibitory mechanisms. Activation of G(i) may also inhibit GnRH neuronal function and episodic secretion by regulating membrane ion currents. This autocrine mechanism could serve as a timer to determine the frequency of pulsatile GnRH release by regulating Ca(2+)- and cAMP-dependent signaling and GnRH neuronal firing.     

Differential actions of endothelin and gonadotropin-releasing hormone in pituitary gonadotrophs.

Endothelin (ET) and GnRH act through specific receptors to promote Ca2+ mobilization and influx pathways in pituitary gonadotrophs. In the present study cytoplasmic calcium ([Ca2+]i) and secretory responses to these two agonists are compared. In single gonadotrophs, low concentrations of both agonists cause oscillatory [Ca2+]i responses after a latent period. Such responses usually consist of discrete transients arising from the normal resting level, but are sometimes super-imposed on an elevated basal calcium level. At high doses, ET-1 and GnRH induce biphasic responses, composed of a spike phase followed by a plateau that often shows high frequency and low amplitude Ca2+ transients. The duration of the latent period and the frequency of the subsequent oscillations are correlated, and both are dependent on agonist concentration. The frequencies and amplitudes of Ca2+ spiking are also interrelated; increases in frequency are followed by more rapid decreases in the amplitude of the Ca2+ transients. After K(+)-induced depolarization, gonadotrophs retain their oscillatory Ca2+ responses to ET-1 and GnRH, with the same frequency as controls. Activation of protein kinase-C by phorbol esters does not alter the frequency of ET-induced Ca2+ transients, but significantly reduces their amplitudes. In contrast, treatment with nanomolar concentrations of thapsigargin converts ET-induced oscillations into a biphasic response, suggesting that Ca(2+)-ATPase in the endoplasmic reticulum participates in the oscillatory mechanism. The two agonists differ in their threshold doses and concentration dependence, ET being significantly less potent than GnRH. Also, gonadotrophs stimulated by ET-1 exhibit different post-treatment responsiveness than those exposed to GnRH. While GnRH-treated cells recover their full [Ca2+]i and secretory responses within 30 min as well as normal [Ca2+]i and secretory responses to ET-1, endothelin-treated cells are refractory to further stimulation with ET and exhibit either attenuated or enhanced Ca2+ and LH responses to GnRH, depending on the duration of exposure to ET-1 and the subsequent recovery period. These data indicate that both receptors use the same mechanism(s) for Ca2+ release, but have different capacities to generate, maintain, and reinitiate the Ca2+ signal.     

Role of arachidonic acid in the regulation of adrenocorticotropin release from rat anterior pituitary cell cultures

In addition to cAMP-dependent mechanisms, stimulation of pituitary ACTH secretion by various stimuli, including CRF, may involve phospholipid and arachidonic acid turnover. To determine the role of phospholipase A2 activation in corticotroph function, we studied the effect of exogenous arachidonic acid, phospholipase A2, and the phospholipase A2 activator melittin on ACTH release in cultured rat anterior pituitary cells. Incubation with 1-100 micron arachidonic acid, 0.01-1 micron melittin, 0.1-10 U/ml phospholipase A2, and 0.01-10 nM CRF caused dose-dependent increases in ACTH release to 8.1 +/- 1.1- (+/- SE), 16.2 +/- 0.9-, 13.6 +/- 1.2-, and 2.9 +/- 0.3-fold; respectively. The participation of the major pathways of arachidonic acid metabolism in the control of ACTH release was analyzed in cells treated with nordihydroguaiaretic acid, a lipoxygenase inhibitor; indomethacin, a cycloxygenase inhibitor; and 5,8,11,14-eicosatetraynoic acid, an inhibitor of both pathways. The effects of arachidonic acid, melittin, and CRF were partially blocked by 10 micron nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid, but were significantly enhanced by 10 micron indomethacin. These results suggest that arachidonic acid is mainly metabolized through the lipoxygenase pathway to a stimulatory metabolite and, to a lesser extent, through the cycloxygenase pathway to an inhibitory metabolite. Arachidonic acid release from anterior pituitary cells labeled with [3H]arachidonic was analyzed during cell column perifusion and stimulation by CRF and other secretagogues. Two-minute pulses of CRF (10 nM), vasopressin (10 nM) and phorbol 12-myristate 13-acetate (100 nM) caused immediate 1.5- to 2-fold increases in [3H]arachidonic acid release, and melittin (100 nM) caused a 5-fold increase in [3H]arachidonic acid release. The ability of both exogenously added and endogenously generated arachidonic acid to stimulate ACTH secretion, together with the stimulation of arachidonic acid release by ACTH secretagogues and the attenuation of stimulated ACTH release by lipoxygenase blockers, indicate that lipoxygenase products of arachidonic acid metabolism participate in the control of ACTH secretion.     

Angiotensin II receptor-mediated calcium influx in bovine adrenal glomerulosa cells.

The cytoplasmic calcium ([Ca2+]i) response to angiotensin II (AII) in bovine adrenal glomerulosa cells is characterized by an initial transient peak due to intracellular Ca2+ mobilization, followed by a sustained plateau phase that is dependent on Ca2+ entry from the extracellular fluid. In Fura-2 loaded cells, the calcium channel antagonists, nifedipine (1 microM) and verapamil (20 microM), only partially reduced the cytosolic calcium profile induced by AII. The dihydropyridine agonist, Bay K 8644, caused a moderate increase in [Ca2+]i when added in concentrations of 50-100 nM, but did not enhance the AII-induced rise in [Ca2+]i. These results indicate that most of the AII-stimulated Ca2+ influx is through channels that are insensitive to dihydropyridines and verapamil. In contrast, the inorganic Ca2+ channel blocker, LaCl3 (10 microM), inhibited the AII-induced plateau phase by more than 50%. The AII-induced Ca2+ signal was not affected by treatment with pertussis toxin (100-300 ng/ml for 12 h). The prior addition of specific AII-antagonists (DuP 753, a nonpeptide antagonist, and three peptide analogs, [Sar1,Thr8]AII, [Sar1,Ala8]AII, and [Sar1,Ile8]AII) completely inhibited the AII-induced Ca2+ signal. However, addition of up to 25,000 molar excess of these antagonists at intervals from 10 sec to 5 min after AII caused progressively less attenuation of the sustained Ca2+ signal. After 5 min, addition of antagonists did not inhibit the agonist-induced Ca2+ response for up to 20 min. The progressive loss of ability of the antagonists to inhibit the sustained elevation of [Ca2+]i could reflect prolonged activation of the receptor or of a subsequent process that maintains Ca2+ influx despite receptor blockade. It is possible that sequestration and/or endocytosis of the AII-receptor complex is accompanied by continued generation of intracellular signals that contribute to the maintenance of the [Ca2+]i response.     

Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor.

The coupling of agonist-activated seven transmembrane domain receptors to G proteins is known to involve the amino-terminal region of their third cytoplasmic loop. Analysis of the amino acids in this region of the rat type in angiotensin (AT1a) receptor identified Leu-222 as an essential residue in receptor activation by the physiological agonist, angiotensin II (Ang II). Nonpolar replacements for Leu-222 yielded functionally intact AT1 receptors, while polar or charged residues caused progressive impairment of Ang II-induced inositol phosphate generation. The decrease in agonist-induced signal generation was associated with a parallel reduction of receptor internalization, and was most pronounced for the Lys-222 mutant receptor. Although this mutant showed normal binding of the peptide antagonist, [Sar1,Ile6]Ang II, its affinity for Ang II was markedly reduced, consistent with its inability to adopt the high-affinity conformation. A search revealed that many Gq-coupled receptors contain an apolar amino acid (frequently leucine) in the position corresponding to Leu-222 of the AT1 receptor. These findings suggest that such a conserved apolar residue in the third intracellular loop is a crucial element in the agonist-induced activation of the AT1 and possibly many other G protein-coupled receptors.     

Interactions in the aetiology,presentation and management of synchronous and metachronous adenocarcinoma of the prostate and rectum

Adenocarcinoma of the prostate and rectum are common male pelvic cancers and may present synchronously or metachronously and, due to their anatomic proximity. The treatment of rectal or prostate cancer (in particular surgery and/or radiotherapy) may alter the presentation, incidence and management should a metachronous tumour develop. This review focuses on the interaction between prostatic and rectal cancer diagnosis and management. We have restricted the scope of this large topic to general considerations, management of rectal cancer after prostate cancer treatment and vice versa, management of synchronous disease and cancer follow-up issues.     

Molecular characterization of human factor XSan Antonio

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.